CN1980687B - Albumin fusion proteins - Google Patents

Abstract

The present invention encompasses albumin fusion proteins. The invention also encompasses nucleic acid molecules encoding the albumin fusion proteins of the invention, as well as vectors containing these nucleic acids, host cells transformed with these nucleic acid vectors, and methods of using these nucleic acids, vectors, and/or host cells and producing the albumin fusion proteins of the invention. In addition, the invention also encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of using the albumin fusion proteins of the invention to treat, prevent or ameliorate diseases, disorders or conditions.

Description

albumin fusion protein

Introduction to the Sequence Listing on CD-ROM

This application refers to the "Sequence Listing" set forth below, which is provided as an electronic file on three identical compact discs (CD-R) labeled "Copy 1," "Copy 2," and "Copy 3." These CD-ROMs Each includes the file "PF612PCT SL.txt" (929,048 bytes, dated February 7, 2005), which is incorporated by reference in its entirety.

Background of the invention

The present invention generally relates to therapeutic proteins (including but not limited to at least one polypeptide, antibody, peptide or fragments and variants thereof) fused to albumin or fragments or variants of albumin. The invention encompasses polynucleotides encoding therapeutic albumin fusion proteins, therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. The invention also encompasses host cells transformed with polynucleotides encoding therapeutic albumin fusion proteins, and methods of using these polynucleotides and/or host cells to produce albumin fusion proteins of the invention.

Human serum albumin (HSA or HA), a protein of 585 amino acids in its mature form (shown in Table 1 (SEQ ID NO: 1)), is responsible for a significant proportion of serum osmolarity and also acts as an endogenous and function as a carrier of exogenous ligands. Currently, HA for clinical use is produced by extracting from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP330451 and EP361991.

Native state or recombinantly produced therapeutic proteins, such as interferons and growth hormones, are often unstable molecules that exhibit a short shelf life, especially when formulated in aqueous solutions. When formulated for administration, the instability of these molecules suggests that many must be lyophilized and kept refrigerated in storage, thus making transport and/or storage of these molecules difficult. Storage problems are particularly acute when pharmaceutical formulations must be stored and distributed outside of the hospital setting.

Practical solutions to the storage problem of unstable protein molecules have rarely been proposed. Accordingly, there is a need for stable, long-lasting, easily dispensed formulations of protein therapeutic molecules, preferably simple formulations requiring minimal post-storage handling.

Following in vivo administration, native state or recombinantly produced therapeutic proteins, such as interferon and growth hormone, exhibit transient plasma stability due to rapid clearance from the bloodstream. Therefore, the therapeutic effect provided by these proteins is also short-term. Therefore, the rapid clearance of these proteins from the blood suggests that therapeutic molecules must be administered more frequently or in higher doses in order to maintain their desired therapeutic effect in vivo. However, increasing the dosing regimen of administering a therapeutic protein often results in increased injection site reactions, side effects, and toxicity in patients. Similarly, administration of therapeutic proteins at higher doses also often results in increased toxicity and side effects in patients.

The few practical solutions that have been proposed to increase the plasma stability of therapeutic molecules, including chemical conjugation, have brought limited benefit to patients. Typically, these chemically modified therapeutic molecules are still administered on a frequent dosing regimen in most cases, maintaining severe injection site reactions, side effects, and toxicity in patients. Accordingly, there is a need for stable forms of therapeutic molecules that maintain greater plasma stability in vivo than native state or recombinantly produced therapeutic proteins alone and that can be administered less frequently, thereby reducing potential side effects to the patient.

Summary of the invention

Albumin fusion proteins comprising a Therapeutic protein (such as a polypeptide, antibody, or peptide or fragment or variant thereof) fused to albumin or a fragment (portion) or variant thereof are encompassed by the invention. The invention also encompasses polynucleosides comprising or consisting of nucleic acid molecules encoding therapeutic proteins (such as polypeptides, antibodies, or peptides or fragments or variants thereof) fused to albumin or a fragment (portion) or variant thereof acid. The invention also encompasses polynucleotides comprising or consisting of nucleotide molecules encoding a protein comprising a therapeutic protein (e.g., polypeptide, antibody, or peptide or fragment or variant thereof), the albumin or fragment (portion) or variant of albumin is sufficient to prolong the shelf life of the Therapeutic protein, increase the plasma stability of the Therapeutic protein compared to its unfused state, and/ Or in vitro and/or in vivo to stabilize the therapeutic protein and/or its activity in solution (or in a pharmaceutical composition). The present invention also encompasses albumin fusion proteins encoded by the polynucleotides of the present invention, as well as host cells transformed with the polynucleotides of the present invention, and the use of these polynucleotides and/or host cells of the present invention and the production of albumin fusion proteins of the present invention protein method.

In a preferred aspect of the present invention, albumin fusion proteins include, but are not limited to, those proteins described in Table 2 and polynucleotides encoding such proteins.

The invention also encompasses pharmaceutical formulations comprising an albumin fusion protein of the invention and a pharmaceutically acceptable diluent or carrier. These formulations may be presented in kits or containers. The kit or container can be packaged with instructions for extended shelf life of the therapeutic protein. These formulations can be used in methods for treating, preventing, ameliorating or diagnosing a disease or disease symptoms in a patient, comprising the step of administering the pharmaceutical formulation to the patient, wherein the patient is preferably a mammal, most preferably a human.

In other embodiments, the present invention encompasses methods of preventing, treating or ameliorating a disease or disorder. In a preferred embodiment, the present invention encompasses a method of treating a disease or disorder listed in the "Preferred Indication: Y" column of Table 1, comprising treating, preventing or ameliorating the disease or disorder in an amount effective for the treatment, prevention or amelioration of the disease or disorder in need of such treatment. , prophylaxis or improvement of the patient administration of the albumin fusion protein of the present invention, the albumin fusion protein comprising the same table 1 "therapeutic protein: X" column (and table 1 "preferred indications: Y" column to be listed A Therapeutic protein or portion thereof corresponds to a Therapeutic protein (or fragment or variant thereof) disclosed in Treating a disease or disorder on the same row).

In one embodiment, the albumin fusion protein described in Table 1 or 2 has an extended shelf life.

In another embodiment, the albumin fusion protein described in Table 1 or 2 is more stable than the corresponding unfused Therapeutic molecule described in Table 1.

The present invention also includes transgenic organisms modified to contain nucleic acid molecules of the present invention (including but not limited to polynucleotides described in Tables 1 and 2), preferably transgenic organisms modified to express albumin fusion proteins of the present invention.

Brief description of the drawings

Figures 1A-D show the amino acid sequence of the mature form of human albumin (SEQ ID NO: 1) and the polynucleotide encoding it (SEQ ID NO: 2). Nucleotides 1-1755 of SEQ ID NO: 2 encode the mature form of human albumin (SEQ ID NO: 1).

Figure 2 shows a restriction map of pPPC0005 cloning vector ATCC deposit PTA-3278.

Figure 3 shows a restriction map of the pSAC35 S. cerevisiae expression vector (Sleep et al., BioTechnology 8:42, 1990).

Figure 4 shows the effect of various dilutions of the IFNb albumin fusion protein encoded by DNA contained in CID 2011 and 2053 on SEAP activity in ISRE-SEAP/293F reporter cells (see Example 76). Proteins were serially diluted in DMEM/10% FBS from 5e-7 to 1e-14 g/ml and used to treat ISRE-SEAP/293F reporter cells. After 24 hours, the supernatant was removed from the reporter cells and assayed for SEAP activity. IFNb albumin fusion protein was purified from three stable clones: 293F/#2011, CHO/#2011 and NSO/#2053. A mammalian-derived IFNb, Avonex, is from Biogen and is reported to have a specific activity of 2.0e5 IU/μg.

Figure 5 compares the antiproliferative activity of IFN albumin fusion protein encoded by CID 3165 (CID 3165 protein) and recombinant protein IFNa (rIFNa) on Hs294T melanoma cells. Cells were cultured in media containing various concentrations of CID 3165 protein or rIFNa, and proliferation was measured by BrdU incorporation after 3 days of culture. CID 3165 protein caused measurable inhibition of cell proliferation at concentrations above 10 ng/ml, with 50% inhibition achieved at approximately 200 ng/ml. (■) = CID 3165 protein, (◆) = rIFNa.

Figure 6 shows the effect of various dilutions of IFNa albumin fusion protein on SEAP activity in ISRE-SEAP/293F reporter cells. One preparation (♦) fused to IFNa upstream of albumin and two different preparations (•) and (♦) fused to IFNa downstream of albumin were tested.

Figure 7 shows the effect of time and dose of IFNa albumin fusion protein (CID 2249 protein) encoded by DNA contained in construct 2249 on OAS(p41) mRNA levels in treated monkeys (see Example 78) . Each time point: the first bar is vehicle control, the second bar is intravenous injection of 30 μg/kg CID 2249 protein on the first day, the third bar is subcutaneous injection of 30 μg/kg CID 2249 protein on the first day, and the fourth bar is 300μg/kg CID 2249 protein was subcutaneously injected on the first day, and 40μg/kg recombinant IFNa was injected subcutaneously on the fifth day.

Figure 8 shows the dose-response relationship of BNP albumin fusion proteins (CID 3691 and 3618 proteins) encoded by DNA contained in constructs CID 3691 and 3618 to activate cGMP formation in NPR-A/293F reporter cells (see Examples 80 and 81). Recombinant BNP (■) and two different preparations of BNP fused upstream of albumin (□) and (•) were tested.

Figure 9 shows the effect of BNP albumin fusion protein on mean arterial pressure in spontaneously hypertensive rats (see Example 80). Vehicle (□), recombinant BNP protein (•), or BNP albumin fusion protein (◯) were delivered by tail vein injection. Systolic and diastolic blood pressures were recorded by cuff-tail.

Figure 10 shows plasma cGMP levels in 11-12 week old male C57/BL6 mice following intravenous injection of recombinant BNP protein (•) or BNP albumin fusion protein (o) (see Example 80). Tail blood was collected at several time points after iv injection to prepare plasma for determination of cGMP levels.

Figure 11 shows fasting about 8 weeks old diabetic db/db mice in a single administration of tandem GLP-1 (7-36A8G) 2x-HSA fusion (CID 3610) (◇), monomeric GLP-1 ( Blood glucose levels 24 hours after 7-36A8G)-HSA fusion (Δ), or HSA alone (•). Blood glucose levels were measured by an oral glucose tolerance test. In fasted approximately 8-week-old diabetic db/db mice, tandem GLP-1(7-36A8G)2x-HSA fusion (CID 3610) (◇) was more effective than monomeric GLP- The 1(7-36A8G)-HSA fusion (Δ) had unexpectedly more potent glucose-normalizing activity.

Detailed description of the invention

definition

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

As used herein, "polynucleotide" refers to a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising at least one Therapeutic protein X (or a fragment or variant thereof) in the same reading frame Linked at least one albumin (or fragment or variant thereof) molecule or consists of it; a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising SEQ ID NO: Y (as listed in column 6 of Table 2 described above) or a fragment or variant thereof or consist of it; a nucleic acid molecule having a nucleotide sequence comprising or consisting of the sequence shown in SEQ ID NO: X; having a nucleotide sequence encoding the following fusion protein Nucleic acid molecule, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: Z; a nucleic acid molecule with a nucleotide sequence encoding the albumin fusion protein of the present invention generated as described in Table 2 or in the examples; A nucleic acid molecule having the nucleotide sequence of a therapeutic albumin fusion protein of the invention; a nucleic acid molecule having the nucleotide sequence contained in an albumin fusion construct described in Table 2; or having an albumin fusion construct deposited with the ATCC (As described in Table 3) Nucleic acid molecule containing a nucleotide sequence.

As used herein, an "albumin fusion construct" refers to an albumin fusion construct comprising a polynucleotide encoding at least one molecule linked in the same reading frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof). A polynucleotide of albumin (or a fragment or variant thereof) or a nucleic acid molecule consisting thereof; comprising a Therapeutic protein (or A polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) linked to at least one polynucleotide of a fragment or variant thereof) or a nucleic acid molecule consisting thereof; or a nucleic acid molecule comprising the same reading frame as Nucleotides encoding at least one molecule of albumin (or a fragment or variant thereof) linked to at least one polynucleotide of at least one molecule of Therapeutic protein (or a fragment or variant thereof) or consisting of Molecules, which also comprise, for example, one or more of the following elements: (1) functional autonomously replicating vectors (including but not limited to shuttle vectors, expression vectors, integrating vectors, and/or replication systems), (2) transcription initiation region (eg a promoter region such as eg a regulatable or inducible promoter, a constitutive promoter), (3) a transcription termination region, (4) a leader sequence, and (5) a selectable marker. Polynucleotides encoding a Therapeutic protein and albumin, once part of the albumin fusion construct, can each be referred to as a "part," "region," or "module" of the albumin fusion construct.

The present invention generally relates to polynucleotides encoding albumin fusion proteins; albumin fusion proteins; and methods of using albumin fusion proteins or polynucleotides encoding albumin fusion proteins to treat, prevent or ameliorate diseases or disorders. As used herein, "albumin fusion protein" refers to a protein formed by fusing at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or a fragment or variant thereof). The albumin fusion protein of the present invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are linked by gene fusion (i.e., the albumin fusion protein is produced by translation of the following nucleic acid wherein the polynucleotide encoding a complete or partial therapeutic protein is linked in the same reading frame to the polynucleotide encoding a complete or partial albumin). The Therapeutic protein and the albumin protein, once part of the albumin fusion protein, can each be referred to as a "portion," "region," or "module" of the albumin fusion protein (e.g., "therapeutic protein portion" or "albumin fusion protein"). protein protein part"). In a highly preferred embodiment, an albumin fusion protein of the invention comprises at least one molecule of Therapeutic protein X or a fragment or variant thereof (including but not limited to a mature form of Therapeutic protein X) and albumin or a fragment or variant thereof. At least one molecule of a variant (including but not limited to the mature form of albumin).

In another preferred embodiment, the albumin fusion proteins of the invention are processed by the host cell and secreted into the surrounding culture medium. Processing of nascent albumin fusion proteins that occurs in the secretory pathway of the host for expression may include, but is not limited to, signal peptide cleavage, disulfide bond formation, proper folding, addition of carbohydrates, and processing such as, for example, N- and O -linked glycosylation), specific proteolytic cleavage, and assembly into multimeric proteins. The albumin fusion proteins of the invention are preferably in processed form. In the most preferred embodiment, a "processed form of an albumin fusion protein" refers to an albumin fusion protein product cleaved by an N-terminal signal peptide, also referred to herein as a "mature albumin fusion protein".

)。此外，通过本领域已知的和本文其它地方描述的技术有可能从保藏物重新得到指定的清蛋白融合构建物。 位于美国维吉尼亚州20110-2209马纳萨斯镇大学路10801号(10801 University Boulevard，Manassas，Virginia 20110-2209，USA)。 

保藏物是根据布达佩斯条约关于用于专利程序的国际公认的微生物保藏条款生成的。 In several instances, representative clones containing albumin fusion constructs of the invention were deposited with the American Type Culture Collection (referred to herein as

). Furthermore, it is possible to retrieve a given albumin fusion construct from a deposit by techniques known in the art and described elsewhere herein. It is located at 10801 University Road, Manassas Township, 20110-2209, Virginia, USA (10801 University Boulevard, Manassas, Virginia 20110-2209, USA).

The deposit was created under the Budapest Treaty on the Internationally Recognized Deposit of Microorganisms for the Purposes of Patent Procedure.

In one embodiment, the invention provides polynucleotides encoding albumin fusion proteins comprising or consisting of a Therapeutic protein and a serum albumin protein. In another embodiment, the invention provides an albumin fusion protein comprising or consisting of a Therapeutic protein and a serum albumin protein. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a Therapeutic protein and a serum albumin protein encoded by a polynucleotide described in Table 2. In another preferred embodiment, the present invention provides a polynucleotide encoding an albumin fusion protein whose sequence is shown in Table 2 as SEQ ID NO: Y. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin. The invention also encompasses polynucleotides encoding these albumin fusion proteins.

In additional embodiments, the invention provides albumin fusion proteins comprising or consisting of a Therapeutic protein and a biologically and/or therapeutically active fragment of serum albumin. In additional embodiments, the present invention provides albumin fusion proteins comprising or consisting of a Therapeutic protein and a biologically and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein. In another preferred embodiment, the Therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the Therapeutic protein. In alternative embodiments, the Therapeutic protein portion of the albumin fusion protein is the active form of the Therapeutic protein. The invention also encompasses polynucleotides encoding these albumin fusion proteins.

In additional embodiments, the present invention provides biologically active and/or therapeutically active fragments or variants comprising or comprising a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and serum albumin. composed of albumin fusion proteins. In a preferred embodiment, the invention provides an albumin fusion protein comprising or consisting of a mature portion of a Therapeutic protein and a mature portion of serum albumin. The invention also encompasses polynucleotides encoding these albumin fusion proteins.

therapeutic protein

As noted above, the polynucleotides of the present invention encode proteins comprising or consisting of at least one fragment or variant of a Therapeutic protein and at least one fragment or variant of human serum albumin, preferably by genetically linked to each other.

Another embodiment includes a polynucleotide encoding a protein comprising or consisting of at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, the fragment or variant being chemically conjugated. combined and connected to each other.

As used herein, "therapeutic protein" refers to a protein, polypeptide, antibody, peptide or fragment or variant thereof that has one or more therapeutic and/or biological activities. Therapeutic proteins encompassed by the present invention include, but are not limited to, proteins, polypeptides, peptides, antibodies, and biologicals. (The terms peptide, protein, and polypeptide are used interchangeably herein). It is expressly envisaged that the term "therapeutic protein" encompasses antibodies and fragments and variants thereof. Thus, a protein of the invention may comprise at least one fragment or variant of a therapeutic protein and/or at least one fragment or variant of an antibody. Furthermore, the term "therapeutic protein" can refer to an endogenous or naturally occurring associate of a Therapeutic protein.

A polypeptide exhibiting "therapeutic activity" or a protein having "therapeutic activity" is one that possesses one or more proteins associated with a Therapeutic protein, such as one or more Therapeutic proteins described herein or otherwise known in the art. A polypeptide with known biological and/or therapeutic activity. By way of non-limiting example, a "therapeutic protein" refers to a protein that can be used to treat, prevent, or ameliorate a disease, condition, or disorder. As a non-limiting example, a "therapeutic protein" can be one that specifically binds to a particular cell type (normal (eg, lymphocytes) or abnormal (eg, cancer cells)) and thus can be used to specifically target a compound (drug or cytotoxic agent) proteins to that cell type.

For example, a non-exhaustive list of "therapeutic protein" moieties that may be encompassed by albumin fusion proteins of the invention include, but are not limited to, GLP-1, GLP-2, PACAP-27, PACAP-28, VIP, CD4M33, secretin, Glucagon-like peptide, oxyntomodulin, PHM, IFNα, IFNβ, ANP, BNP, NGF, BDNF, GDNF, and somatostatin.

Interferon hybrids can also be fused to the amino or carboxyl termini of albumin to form interferon hybrid albumin fusion proteins. Interferon hybrid albumin fusion proteins can have enhanced or inhibited interferon activities, such as antiviral responses, regulation of cell growth, and modulation of immune responses (Lebleu et al., PNAS USA 73:3107-3111, 1976; Gresser et al., Nature 251:543-545, 1974; and Johnson, Texas Reports Biol Med 35:357-369, 1977). Each interferon hybrid albumin fusion protein can be used to treat, prevent, or ameliorate viral infection (eg, hepatitis (eg, HCV); or HIV), multiple sclerosis, or cancer.

In one embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon alpha hybrid (referred to herein as an alpha-alpha hybrid). For example, the alpha-alpha hybrid portion of an interferon hybrid albumin fusion protein comprises or consists of interferon alpha A fused to interferon alpha D. In another embodiment, the A/D hybrid is fused to interferon αD at a consensus BglII restriction site, wherein the N-terminal portion of the A/D hybrid corresponds to amino acids 1-62 of interferon αA and the C - The terminal part corresponds to amino acids 64-166 of interferon alpha D. For example, this A/D hybrid would contain the amino acid sequence:

CDLPQTHSLGSRRTLMLLAQMRX ₁ ISLFSCLKDRHDFGFPQEEFGNQFQK

AETIPVLHEMIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACV

MQEERVGETPLMNX ₂ DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIM

RSLSLSTNLQERLRRKE (SEQ ID NO: 99),

wherein X ₁ is R or K and X ₂ is A or V (see for example construct ID #2875). In another embodiment, the A/D hybrid is fused at a consensus PvuIII restriction site, wherein the N-terminal portion of the A/D hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to Interferon αD amino acids 93-166 correspond. For example, this A/D hybrid would contain the amino acid sequence:

CDLPQTHSLGSRRTLMLLAQMRX ₁ ISLFSCLKDRHDFGFPQEEFGNQFQK

AETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACV

MQEERVGETPLMNX ₂ DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIM

RSLSLSTNLQERLRRKE (SEQ ID NO: 100),

where X ₁ is R or K and the second X ₂ is A or V (see for example construct ID #2872). These hybrids are further described in US Patent No. 4,414,510, which is incorporated herein by reference in its entirety.

In another embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein comprises or consists of interferon alpha A fused to interferon alpha F. In another embodiment, the A/F hybrid is fused at a consensus PvuIII restriction site, wherein the N-terminal portion of the A/F hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to Amino acids 93-166 of interferon alpha F correspond. For example, this A/F hybrid would contain the amino acid sequence:

CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKA

ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDMEACVIQ

EVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSF

SLSKIFQERLRRKE (SEQ ID NO: 101),

where X is either R or K (eg see construct ID #2874). These hybrids are further described in US Patent No. 4,414,510, which is incorporated herein by reference in its entirety. In another embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein comprises or consists of interferon alpha A fused to interferon alpha B. In another embodiment, the A/B hybrid is fused at a consensus PvuIII restriction site, wherein the N-terminal portion of the A/B hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to Amino acids 93-166 of interferon alpha B correspond. For example, this A/B hybrid would contain the amino acid sequence:

CDLPQTHSLGSRRTLMLLAQMRX ₁ ISLFSCLKDRHDFGFPQEEFGNQFQK

AETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEX ₂ X ₃

X ₄ X ₅ QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIM

RSFSLSINLQKRLKSKE (SEQ ID NO: 102),

wherein X ₁ is R or K and X ₂ to X ₅ are SCVM or VLCD (eg see construct ID #2873). These hybrids are further described in US Patent No. 4,414,510, which is incorporated herein by reference in its entirety.

In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon beta-interferon alpha hybrid (referred to herein as a beta-alpha hybrid). For example, the beta-alpha hybrid portion of an interferon hybrid albumin fusion protein comprises or consists of interferon beta-1 fused to interferon alphaD (referred to herein as interferon alpha-1). In another embodiment, the β-1/αD hybrid is fused in which the N-terminal portion corresponds to amino acids 1-73 of interferon β-1 and the C-terminal portion corresponds to amino acids 74-167 of interferon αD correspond. For example, this β-1/αD hybrid would contain the amino acid sequence:

MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ

FQKEDAALTIYEMLQNIFAIFRQDSSAAWDEDLLDKFCTELYQQLNDLEA

CVMQEERVGETPLMNXDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEI

MRSLSLSTNLQERLRRKE (SEQ ID NO: 103),

where X is A or V. These hybrids are further described in US Patent No. 4,758,428, which is incorporated herein by reference in its entirety.

In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon beta hybrid (referred to herein as an alpha-beta hybrid). For example, the alpha-beta hybrid portion of an interferon hybrid albumin fusion protein comprises or consists of interferon alphaD (also known as interferon alpha-1 ) fused to interferon beta-1. In another embodiment, the αD/β-1 hybrid is fused in which the N-terminal portion corresponds to amino acids 1-73 of interferon αD and the C-terminal portion corresponds to amino acids 74-166 of interferon β-1 correspond. For example, this αD/β-1 hybrid would contain the amino acid sequence:

MCDLPETHSLDNRRTLMLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQ

KAPAISVLHELIQQIFNLFTTKDSSSTGWNETIVENLLANVYHQINHLKTV

LEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL

RNFYFINRLTGYLRN (SEQ ID NO: 104).

These hybrids are further described in US Patent No. 4,758,428, which is incorporated herein by reference in its entirety.

In additional embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein may comprise other combinations of alpha-alpha interferon hybrids, alpha-beta interferon hybrids, and beta-alpha interferon hybrids thing. In additional embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein can be modified to include mutations, substitutions, deletions, or additions to the amino acid sequence of the interferon hybrid. These modifications to interferon hybrid albumin fusion proteins can be used, for example, to increase production levels, increase stability, increase or decrease activity, or confer novel biological properties.

The invention encompasses the interferon hybrid albumin fusion proteins described above, as well as host cells and vectors containing polynucleotides encoding these polypeptides. In one embodiment, the interferon hybrid albumin fusion protein encoded by the polynucleotide described above has an extended shelf life. In another embodiment, the interferon hybrid albumin fusion protein encoded by the above-mentioned polynucleotide is compared with the corresponding unfused interferon hybrid molecule in vitro and/or in vivo in solution (or in a pharmaceutical composition) Middle) have longer serum half-life and/or more stable activity.

In another non-limiting example, "therapeutic protein" refers to a protein having biological activity, especially a biological activity useful for treating, preventing or improving diseases. A non-exhaustive list of biological activities that a therapeutic protein can have includes inhibition of HIV-1 infection of cells, stimulation of intestinal epithelial cell proliferation, reduction of intestinal epithelial cell permeability, stimulation of insulin secretion, induction of bronchodilation and vasodilation, inhibition of aldosterone and Renin secretion, regulation of blood pressure, promotion of nerve growth, enhancement of immune response, enhancement of inflammation, suppression of appetite, or as described below in the "Biological Activity" section and/or disclosed in Table 1 (second column) for the indicated therapeutic protein Any one or more biological activities.

In one embodiment, the IFN-β-HSA fusion is used to inhibit Ebola virus (Ebolavirus) and SARS virus (Toronto-2 strain, Toronto-2 strain). For example, the in vitro antiviral activity of IFN-β (CID 2053 protein) fused upstream of mature HSA against Ebola virus and SARS virus was assessed in Vero cells. These cells were used to evaluate the protective effect of the CID 2053 protein based on inhibition of cytopathic effect (CPE) and neutral red assay of cell viability. In vitro signal transduction was assessed by gene expression analysis. Additionally, the pharmacokinetics and pharmacodynamics of the CID 2053 protein were evaluated in rhesus monkeys. The results showed potent in vitro antiviral activity with a favorable safety index. The IC50 of CID 2053 protein against Ebola virus is 0.4ng/ml and the IC50 against SARS virus is 2ng/ml. Array analysis revealed that CID 2053 protein and IFN-β induced the expression of a similar set of genes and elicited the IFN-stimulated response element (ISRE) signaling pathway. In rhesus macaques administered CID 2053 protein at doses of 50 μg/kg intravenously or 300 μg/kg subcutaneously, the terminal half-life was 36-40 hours. Administration of CID 2053 protein caused a sustained increase in serum neopterin levels and expression of OAS1 mRNA.

In another embodiment, IFN-alpha-HSA fusions are used to inhibit viruses classified into groups A - Filo (Ebola), Arena (Pichende), B - Picloviruses. Viruses of Toga (VEE) or Group C - Bunya (Punto toro), Flavi (yellow fever, West Nile) factor. For example, CPEE inhibition, neutral red staining, and virus yield assays were used to assess the antiviral activity of IFN-[alpha] (CID 3165 protein) fused downstream of HSA. The pharmacokinetics and pharmacodynamics of the CID 3165 protein were evaluated in rhesus monkeys and human subjects. The results showed that antiviral activity against all RNA viruses was achieved with a favorable safety index. IC50 values in the CPE assay ranged from <0.1 ng/ml (Ponta Tolu A) to 19 ng/ml (VEE). In rhesus monkeys, the CID 3165 protein had a half-life of 90 hours and was detectable up to 14 days post-dose. In human subjects, the CID 3165 protein was safe and well tolerated. _Cmax after a single injection is dose proportional. The mean C _max in the 500 μg group was 22 ng/ml and the mean t _1/2 was 150 hours. Dosing every 2-4 weeks or longer is supported by pharmacokinetics. Antiviral responses against hepatitis C were observed in most subjects in the single injection group (120-500 μg).

(Schering Corp.，Kenilworth，N.J.)、 

(Hoffman-La Roche，Nutley，N.J.)、Beroforα干扰素(Boehringer Ingelheim Pharmaceutical，Inc.，Ridgefied，Conn.)、OMNIFERON^TM(Viragen，Inc.，Plantation，FL)、MULTIFERON^TM(Viragen，Inc.，Plantation，FL)、 

(GlaxoSmithKline，London，Great Britian)、 

(Amgen，Inc.，Thousands Oaks，CA)、 

(Sumitomo，Japan)、 

(Nautilus Biotech，France)或任何纯化的干扰素α产品或其片段或由其组成。在另外的实施方案中，IFN-α-HSA融合蛋白的干扰素α部分可通过附着化学模块而得以修饰。例如，干扰素α部分可通过聚乙二醇化得以修饰。因此，在另外的实施方案中，IFN-α-HSA融合蛋白的干扰素α部分包含干扰素α-2a、2b、或共有干扰素的聚乙二醇化形式或由其组成，并且包含但不限于商品化聚乙二醇化干扰素α，诸如例如PEG- 

(Schering Corp.，Kenilworth，N.J.)、 

(Hoffman-La Roche，Nutley，N.J.)、PEG-OMNIFERON^TM(Viragen，Inc.，Plantation，FL)或其片段。然而，在用于本文时，“IFN-α-HSA”融合物指与本领域已知的任何干扰素α蛋白质或其片段融合的HSA。 In another embodiment, IFN-[alpha]-HSA fusions are used to treat patients with chronic hepatitis C infection (HCV). Interferon alpha, also known as interferon alfa or leukocyte interferon, is the standard of care for treating patients infected with HCV. The term "interferon alpha" refers to a family of highly homologous related polypeptides possessing antiviral activity. The interferon alpha portion of the IFN-alpha-HSS fusion comprises or consists of any interferon alpha or fragment thereof known in the art. Non-limiting examples of interferon alpha contemplated by the present invention include, but are not limited to, the interferon alpha proteins disclosed in Table 1 in the Therapeutic Proteins column. In specific embodiments, the interferon alpha moiety comprises interferon alpha-2a, interferon alpha-2b, interferon alpha-2c, consensus interferon, interferon alfcon-1, interferon alpha-n1, interferon alpha- n3. Any commercialized form of interferon alpha such as for example

(Schering Corp., Kenilworth, NJ),

(Hoffman-La Roche, Nutley, NJ), Berofor alpha interferon (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefied, Conn.), OMNIFERON ^™ (Viragen, Inc., Plantation, FL), MULTIFERON ^™ (Viragen, Inc., Plantation , FL),

(GlaxoSmithKline, London, Great Britian),

(Amgen, Inc., Thousands Oaks, CA),

(Sumitomo, Japan),

(Nautilus Biotech, France) or any purified interferon alpha product or fragment thereof or consisting of it. In additional embodiments, the interferon alpha portion of the IFN-alpha-HSA fusion protein can be modified by the attachment of chemical moieties. For example, the interferon alpha portion can be modified by pegylation. Accordingly, in additional embodiments, the interferon alpha portion of the IFN-alpha-HSA fusion protein comprises or consists of a pegylated form of interferon alpha-2a, 2b, or consensus interferon, and includes, but is not limited to Commercial pegylated interferon alfa, such as e.g. PEG-

(Schering Corp., Kenilworth, NJ),

(Hoffman-La Roche, Nutley, NJ), PEG-OMNIFERON ^™ (Viragen, Inc., Plantation, FL) or fragments thereof. However, as used herein, an "IFN-alpha-HSA" fusion refers to HSA fused to any interferon alpha protein or fragment thereof known in the art.

HCV-infected patients can be divided into two categories based on whether they have previously received an interferon regimen for the treatment of HCV infection. "Treatment-naive patients" are those patients who have never received an interferon regimen. "Treatment-experienced patients" refers to those patients who have received or are currently receiving an interferon regimen. "Non-responders" refer to treatment-experienced patients who were previously treated with an interferon regimen but did not achieve the primary goals of treatment such as early viral load reduction (EVR) or end-of-treatment response (ETR). However, as used herein, "HCV patient" refers to a patient infected with HCV regardless of whether he is treatment-naive, treatment-experienced, or a non-responder.

In addition, the hepatitis C virus can be divided into four genotypes, genotype 1, 2, 3, or 4. Typically, the hepatitis C virus that infects HCV patients contains a single genotype. However, hepatitis viruses can contain a combination of two or more genotypes. In addition, the genotype of the hepatitis C virus may also be a variant of one of the known HCV genotypes. In another embodiment, the hepatitis C virus of the HCV patient is genotype 1 or a variant thereof. However, as used herein, "HCV" refers to hepatitis C virus of any genotype, or combinations or variants thereof.

Standard treatment for patients with HCV involves treatment with interferon alpha in combination with antiviral agents such as ribavirin (ribavirin). Typically, interferon alpha is administered once a day, twice a week, or once a week and ribavirin is administered once a day. However, recent studies have also used interferon alpha in combination with other antiviral agents known in the art for the treatment of HCV. Thus, in another embodiment, IFN-[alpha]-HSA fusions may be administered to HCV patients alone or in combination with an antiviral agent such as, for example, ribavirin.

As noted above, the pharmacokinetics of the CID 3165 protein support a dosing regimen every 2-4 weeks or longer. Thus, in another embodiment, HCV patients are treated by administering an IFN-α-HSA fusion once every 2-4 weeks, alone or in combination with an effective amount of an antiviral agent. In a preferred embodiment, HCV patients are treated by administering the IFN-[alpha]-HSA fusion once every 2-4 weeks in combination with an effective amount of an antiviral agent. In another preferred embodiment, the IFN-[alpha]-HSA fusion is administered to HCV patients every 4 weeks. In another preferred embodiment, the HCV patient is administered the IFN-[alpha]-HSA fusion more than once every 4 weeks. In additional embodiments, the HCV patient is administered the IFN-[alpha]-HSA fusion every 4 weeks or more, wherein the treatment further comprises administering an effective amount of an antiviral agent.

In another embodiment, the IFN-[alpha]-HSA fusion can be used as a low dose monotherapy for maintenance therapy of HCV. In another embodiment, the IFN-[alpha]-HSA fusion can be used in combination with ribavirin and one or more other antiviral agents for the treatment of HCV. Alternatively, in another embodiment, the IFN-α-HSA fusion can be used in combination with one or more antiviral agents other than ribavirin for the treatment of HCV.

In another embodiment, IFN-[alpha]-HSA fusions can be used to treat other viral infections. For example, in one embodiment, IFN-[alpha]-HSA fusions are useful in the treatment of hepatitis B (HBV). In another embodiment, IFN-[alpha]-HSA fusions are useful in the treatment of human papillomavirus (HPV). In another embodiment, IFN-α-HSA fusions are useful in the treatment of cancers, including but not limited to hairy cell leukemia, malignant melanoma, follicular lymphoma, chronic myelogenous leukemia, AIDS-related Kaposi Sarcoma, multiple myeloma, or renal cell carcinoma.

In another embodiment, GLP-1-HSS fusions are used to regulate blood glucose levels in diabetic patients. In another embodiment, tandem fusions of wild-type or mutant GLP-1 are used to regulate blood glucose levels in diabetic patients. For example, monomeric GLP-1 was assessed by an oral glucose tolerance test (administration of 1 g glucose per kg body weight by oral gavage) after subcutaneous injection of GLP-1-HSS protein in diabetic db/db mice at approximately 8 weeks of age Ability of (7-36A8G)-HSA and tandem GLP-1(7-36A8G)-HSA(CID 3610) fusions to regulate blood glucose levels. This glucose tolerance test consists of a subcutaneous injection of the GLP-1-HSA fusion, followed by 1 g of glucose per kg of body weight by oral gavage. Equimolar doses (100 and 171 nmol/kg) of monomeric or tandem GLP-1-HSA fusion proteins were administered to fasted diabetic db/db mice and oral glucose tolerance tests were performed 6 or 24 hours after a single administration . Rather surprising and unexpected, compared to the monomeric GLP-1(7-36A8G)-HSA fusion, the tandem GLP-1(7-36A8G)-HSA fusion (CID3610) was Significantly lowers blood sugar. Additionally, when assessed in diabetic db/db mice 24 hours after injection, the The difference is even more significant. As shown in Figure 11, the tandem GLP-1(7-36A8G)-HSS fusion (CID 3610) (◇) possessed unexpectedly potent normalizing activity of blood glucose, whereas single GLP-1(7-36A8G)-HSS( Δ) Fasting blood glucose levels of fusions are similar to animals apparently diabetic in nature and given HSA alone (•).

As used herein, "therapeutically active" or "activity" may refer to an activity whose effect in humans is consistent with a desired therapeutic outcome, or to a desired effect in a non-human mammal or other species or organism. Therapeutic activity can be measured in vivo or in vitro. For example, desired effects can be assayed in cell culture. Such in vitro or cell culture assays are generally available in the art for such therapeutic proteins. Examples of assays include, but are not limited to, those described in the Examples section herein or in the "Exemplary Activity Assays" column of Table 1 (column 3).

Therapeutic proteins corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention, such as cell surface and secreted proteins, are typically modified by the attachment of one or more oligosaccharide groups. This modification, known as glycosylation, can significantly affect the physical properties of proteins and can be important for protein stability, secretion, and localization. Glycosylation occurs at specific locations along the polypeptide backbone. There are generally two main types of glycosylation: those attached to serine or threonine residues characterized by O-linked oligosaccharides; those attached to Asn-X-Ser or Asn-X-Thr sequences. Paragine residues are characterized by glycosylation of N-linked oligosaccharides, where X can be any amino acid except proline. N-acetylneuraminic acid (also known as sialic acid) is usually the terminal residue of N-linked and O-linked oligosaccharides. Variables such as protein structure and cell type affect the number and nature of intrachain carbohydrate units at different glycosylation sites. Glycosylation isoforms are also often present at the same site in a given cell type.

Therapeutic proteins and analogs and variants thereof corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention may be modified such that one or more positions are altered by the host cell in which they are expressed due to manipulation of their nucleic acid sequence. glycosylation at the sites, or due to their other expression conditions. For example, glycosylation isomers can be produced by eliminating or introducing glycosylation sites, e.g., by substitution or deletion of amino acid residues, such as glutamine in place of asparagine, or by Unglycosylated recombinant proteins can be produced by expressing proteins in sylated host cells, such as E. coli or yeast deficient in glycosylation. These methods are described in more detail below and are known in the art.

Therapeutic proteins, particularly those disclosed in Table 1, and their nucleic acid and amino acid sequences are well known in the art and are available from public databases such as Chemical Abstracts Service databases (eg, CAS registry numbers), GenBank, and Access to databases such as GenSeq (eg Derwent). Exemplary therapeutic protein nucleotide sequences that can be used to derive polynucleotides of the invention are shown in Table 2, column 7 "SEQ ID NO: X". The sequence shown in SEQ ID NO: X can be a wild-type polynucleotide sequence encoding a specified therapeutic protein (such as full-length or mature), or in some cases the sequence can be a sequence of the wild-type polynucleotide sequence. Variant (such as a polynucleotide encoding a wild-type Therapeutic protein, wherein the DNA sequence of the polynucleotide has been optimized, e.g., for expression in a particular species; or a polynucleotide encoding a variant of a wild-type Therapeutic protein acid (ie site-directed mutant; allelic variant)). It is well within the ability of the skilled artisan to utilize the sequence shown in SEQ ID NO:X to derive the constructs described in the same row. For example, if SEQ ID NO: X corresponds to a full-length protein, but only a portion of the protein is used to generate a specific CID, it is within the skill of the art to amplify the specific fragment and clone it into a suitable vector according to molecular biology techniques such as PCR within.

Other Therapeutic proteins corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention include, but are not limited to, one or more of the Therapeutic proteins disclosed in the "Therapeutic Protein X" column (column 1) of Table 1 or Polypeptides or fragments or variants thereof.

Table 1 provides a non-exhaustive list of Therapeutic proteins corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention or albumin fusion proteins encoded by polynucleotides of the invention. The first column "Therapeutic Protein X" discloses the Therapeutic protein molecule, followed by possible parentheses including the scientific name and brand name of the protein comprising or consisting of the Therapeutic protein molecule or a fragment or variant thereof. As used herein, "Therapeutic Protein X" can refer to an individual Therapeutic protein molecule, or to an entire group of Therapeutic proteins related to a given Therapeutic protein molecule disclosed in this list. The "Biological Activity" column (column 2) describes the biological activity associated with the Therapeutic protein molecule. Column 3 "Exemplary Activity Assays" provides a description of assays that can be used to test the therapeutic and/or biological activity of Therapeutic Protein X or an albumin fusion protein comprising a portion of Therapeutic Protein X (or a fragment thereof) references. Each of the references cited in the "Biological Activity" column is hereby incorporated by reference in its entirety, in particular each reference described in the reference for the determination of the corresponding biological activity shown in the "Biological Activity" column of Table 1. Descriptive aspects of activity assays (see, eg, the Methods section therein). The fourth column "Preferred Indications: Y" describes diseases that can be treated, prevented, diagnosed, and/or ameliorated by Therapeutic Protein X or an albumin fusion protein comprising a portion of Therapeutic Protein X (or a fragment thereof), disorder, and/or condition. The "Construct ID" column (column 5) provides an exemplary albumin fusion protein disclosed in Table 2 encoding an albumin fusion protein comprising or consisting of the mentioned portion of Therapeutic protein X (or a fragment thereof) Links to fusion constructs.

Table 2 provides a non-exhaustive list of polynucleotides comprising or consisting of nucleic acid molecules encoding albumin fusion proteins of the invention. The first column "Fusion No." gives the fusion number for each polynucleotide. Column 2 "Construct ID" provides a unique numerical identifier for each polynucleotide of the invention. Construct IDs can be used to identify polynucleotides encoding albumin fusion proteins comprising or consisting of a Therapeutic protein moiety corresponding to a given Therapeutic protein X, given Therapeutic protein X column In the corresponding column of Table 1, the construct ID is listed in column 5. The "Construct Name" column (column 3) provides the name for a given albumin fusion construct or polynucleotide.

The fourth column "Description" of Table 2 provides a general description for a given albumin fusion construct, while the fifth column "Expression Vector" lists the nucleic acid molecule comprising or consisting of a given albumin fusion protein. A vector into which a polynucleotide is cloned. Vectors are known in the art and are available commercially or as described elsewhere. For example, as described in the Examples, a convenient cloning vector comprising (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a translation One or more of the terminators, or an "expression cassette" consisting of it, is then transferred into other vectors, such as, for example, expression vectors including, for example, yeast expression vectors or mammalian expression vectors. In one embodiment, for expression in Saccharomyces cerevisiae, an expression cassette comprising or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into DSAC35. In another embodiment, for expression in CHO cells, an expression cassette comprising or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pC4. In yet another embodiment, a polynucleotide comprising or consisting of a nucleic acid molecule encoding a Therapeutic protein portion of an albumin fusion protein is cloned into pC4:HSA. In yet another embodiment, an expression cassette comprising or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pEE12 for expression in NSO cells. Other useful cloning and/or expression vectors are known to the skilled artisan and are within the scope of the present invention.

Column 6 "SEQ ID NO: Y" provides the full-length amino acid sequence of the albumin fusion protein of the present invention. In most cases, SEQ ID NO: Y shows the unprocessed form of the encoded albumin fusion protein, in other words, SEQ ID NO: Y shows the signal sequence, HSA portion, and therapeutic portion all encoded by a particular construct. All polynucleotides encoding SEQ ID NO:Y are expressly encompassed by the present invention. When these polynucleotides are used to express the encoded protein from the cell, the cell's natural secretion and processing steps produce the protein lacking the signal sequence listed in column 4 and/or column 11 of Table 2. The specific amino acid sequences of the listed signal sequences are shown in the specification below, or are already known in the art. Thus, the most preferred embodiments of the invention include albumin fusion proteins produced by cells (which will lack the leader sequence shown in column 4 and/or column 11 of Table 2). Also most preferred are polypeptides comprising SEQ ID NO: Y but not having the particular leader sequence listed in column 4 and/or column 11 of Table 2. Compositions, including pharmaceutical compositions, comprising these two preferred embodiments are also preferred. Furthermore, replacement of the signal sequences listed in columns 4 and/or 11 of Table 2 with different signal sequences such as those described later in the specification to facilitate secretion of processed albumin fusion proteins is also entirely within within the ability of skilled technicians.

The seventh column "SEQ ID NO: X" provides the parental nucleic acid sequence from which the polynucleotide encoding the Therapeutic protein portion of a given albumin fusion protein was derived. In one embodiment, the parent nucleic acid sequence from which the polynucleotide encoding the Therapeutic protein portion of the albumin fusion protein can be derived includes the wild-type gene sequence encoding the Therapeutic protein shown in Table 1. In alternative embodiments, the parental nucleic acid sequence from which the polynucleotide encoding the Therapeutic protein portion of the albumin fusion protein can be derived includes a variant or derivative of the wild-type gene sequence encoding the Therapeutic protein shown in Table 1, such as, for example, Synthetic codon-optimized variants of the wild-type gene sequence encoding a therapeutic protein.

The eighth column "SEQ ID NO: Z" provides the predicted translation of the parental nucleic acid sequence (SEQ ID NO: X). This parental sequence can be the full-length parental protein used to derive a particular construct, the mature portion of the parental protein, a variant or fragment of the wild-type protein, or an artificial sequence that can be used to generate the construct. One of skill in the art can use this amino acid sequence shown in SEQ ID NO: Z to determine which amino acid residues of an albumin fusion protein encoded by a given construct are donated by a Therapeutic protein. Furthermore, it is well within the ability of the skilled artisan to use the sequence shown in SEQ ID NO: Z to derive the construct described in the same row. For example, if SEQ ID NO: Z corresponds to a full-length protein, and only a part of the protein is used to generate a specific CID, then relying on molecular biology techniques such as PCR to amplify the specific fragment and clone it into an appropriate vector, this is in within the technical scope of this field.

The amplification primers "SEQ ID NO: A" and "SEQ ID NO: B" provided in columns 9 and 10, respectively, are used to generate a nucleic acid molecule comprising or consisting of a Therapeutic protein portion encoding a given albumin fusion protein Exemplary primers for polynucleotides. In one embodiment of the invention, oligonucleotide primers having the sequence shown in column 9 and/or 10 (SEQ ID NO: A and/or B) are used to PCR amplify a therapeutic protein encoding an albumin fusion protein A polynucleotide of a sex protein part, wherein a nucleic acid molecule comprising or consisting of the nucleotide sequence (SEQ ID NO: X) provided in column 7 of the corresponding row is used as template DNA. PCR methods are well established in the art. Other useful primer sequences can be readily envisioned and used by one of ordinary skill in the art.

In alternative embodiments, oligonucleotides may be used in overlapping PCR reactions to generate mutations in the template DNA sequence. PCR methods are known in the art.

As shown in Table 3, certain albumin fusion constructs disclosed in this application have been deposited with the ATCC.

table 3

It is possible to retrieve a given albumin fusion construct from a depository by techniques known in the art, also described elsewhere in this application (see Example 10). ATCC is located at 10801 University Road, Manassas, Virginia 20110-2209 (10801 University Boulevard, Manassas, Virginia 20110-2209, USA). The ATCC deposit was made in accordance with the Budapest Treaty on the Internationally Recognized Deposit of Microorganisms for the Purposes of Patent Procedure.

In yet another embodiment of the invention, one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a translation terminator Items, or "expression cassettes" consisting of them, can be moved or "subcloned" from one vector into another. Fragments for subcloning can be generated by methods well known in the art, such as, for example, PCR amplification (e.g., using oligonucleotide primers having the sequence shown in SEQ ID NO: A or B) and/or restriction enzyme digestion.

In a preferred embodiment, the albumin fusion protein of the present invention has therapeutic activity and/or biological activity corresponding to the therapeutic activity and/or biological activity of the therapeutic protein, wherein the therapeutic protein corresponds to the corresponding row of Table 1 Therapeutic protein portions of listed albumin fusion proteins. In yet another preferred embodiment, the therapeutically active protein portion of the albumin fusion protein of the present invention is a fragment or variant of a protein encoded by the sequence shown in the SEQ ID NO: X column of Table 2, and has a corresponding therapeutic protein Therapeutic activity and/or biological activity.

Polypeptide and polynucleotide fragments and variants

fragment

The invention also relates to fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.

The invention also relates to polynucleotides encoding fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.

Even if deletion of one or more amino acids from the N-terminus of the protein results in modification or loss of one or more biological functions of the Therapeutic protein, albumin protein, and/or albumin fusion protein of the invention, it can still be retained Other therapeutic and/or functional activities (eg, biological activity, ability to multimerize, ability to bind ligand). For example, the ability of a polypeptide having an N-terminal deletion to induce and/or bind antibodies that recognize intact or mature forms of the polypeptide is generally retained when less than a majority of residues from the intact polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residues of the entire polypeptide retains such immunological activity can be readily determined by routine methods described herein and otherwise known in the art. It is not impossible that muteins with large deletions of N-terminal amino acid residues retain some biological or immunological activity. In fact, peptides consisting of as few as six amino acid residues can often elicit an immune response.

Thus, fragments of Therapeutic proteins that correspond to the Therapeutic protein portion of the albumin fusion proteins of the invention include full-length proteins, as well as fragments from reference polypeptides (i.e., Therapeutic proteins mentioned in Table 1, or from the polypeptides described in Table 2). A polypeptide having one or more residues deleted from the amino terminus of the amino acid sequence of the Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or an albumin fusion construct. Specifically, N-terminal deletions can be described by the general formula m to q, where q is the therapeutic protein moiety representing a reference polypeptide (such as the therapeutic protein mentioned in Table 1, or the albumin fusion protein of the present invention) , or the Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2) is a complete integer of the total number of amino acid residues in the range 2 to q minus 6 any integer. The invention also encompasses polynucleotides encoding these polypeptides.

In addition, fragments of serum albumin polypeptides corresponding to the albumin protein portion of the albumin fusion proteins of the invention include full-length proteins, as well as fragments of serum albumin polypeptides derived from reference polypeptides (i.e. A polypeptide in which one or more residues have been deleted from the amino-terminus of the amino acid sequence of the albumin fusion protein (serum albumin portion) encoded by the protein fusion construct. In a preferred embodiment, the N-terminal deletion can be described by the general formula from m to 585, wherein 585 is a whole integer representing the total number of amino acid residues of mature human serum albumin (SEQ ID NO: 1), and m defines is any integer in the range 2 to 579. The invention also encompasses polynucleotides encoding these polypeptides. In another embodiment, the N-terminal deletion can be described by the general formula m to 609, wherein 609 is a whole integer representing the total number of amino acid residues of full-length human serum albumin (SEQ ID NO: 3), and m Defined as any integer in the range 2 to 603. The invention also encompasses polynucleotides encoding these polypeptides.

Furthermore, fragments of albumin fusion proteins of the invention include full-length albumin fusion proteins, as well as albumin fusion proteins encoded from albumin fusion proteins (i.e., encoded by polynucleotides or albumin fusion constructs described in Table 2, or Albumin fusion protein having the amino acid sequence disclosed in column 6 of Table 2) has one or more residues deleted from its amino terminus. Specifically, N-terminal deletions can be described by the general formula m to q, where q is a whole integer representing the total number of amino acid residues in an albumin fusion protein, and m is defined as any integer in the range of 2 to q minus 6 . The invention also encompasses polynucleotides encoding these polypeptides.

Also as noted above, even if deletion of one or more amino acids from the N-terminus or C-terminus of a reference polypeptide (e.g., a Therapeutic protein, a serum albumin protein, or an albumin fusion protein of the invention) results in one or more of the protein's Modification or loss of various biological functions, but may still retain other functional activities (eg, biological activity, ability to multimerize, ability to bind ligands) and/or therapeutic activity. For example, the ability of a polypeptide having a C-terminal deletion to induce and/or bind antibodies recognizing the intact or mature form of the polypeptide is generally retained when less than a majority of residues from the intact or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the N-terminal and/or C-terminal residues of a reference polypeptide retains therapeutic activity can readily be determined by routine methods described herein and/or otherwise known in the art.

The present invention also provides a Therapeutic protein corresponding to the Therapeutic protein part of the albumin fusion protein of the present invention (such as the Therapeutic protein mentioned in Table 1, or the polynucleotide described in Table 2 or albumin fusion A polypeptide having one or more residues deleted from the carboxyl-terminus of the amino acid sequence of the therapeutic protein portion of the albumin fusion protein encoded by the construct. Specifically, C-terminal deletions can be described by a general formula from 1 to n, where n is any whole integer ranging from 6 to q minus 1, and q represents a reference polypeptide (e.g., a therapeutic protein as mentioned in Table 1). , or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2) is a whole integer of the total number of amino acid residues. The invention also encompasses polynucleotides encoding these polypeptides.

In addition, the present invention provides albumin proteins corresponding to the albumin protein portion of the albumin fusion proteins of the invention (such as serum albumin, or albumin fusion constructs encoded by polynucleotides or albumin fusion constructs described in Table 2). A polypeptide having one or more residues deleted from the carboxyl-terminus of the amino acid sequence of the albumin protein portion of the protein fusion protein. Specifically, the C-terminal deletion can be described by the general formula 1 to n, where n is any whole integer ranging from 6 to 584, and 584 is the amino acid representing mature human serum albumin (SEQ ID NO: 1) A complete integer minus 1 from the total number of residues. The invention also encompasses polynucleotides encoding these polypeptides. Specifically, C-terminal deletions can be described by the general formula 1 to n, where n is any whole integer ranging from 6 to 608, and 608 is the amino acid residue representing serum albumin (SEQ ID NO: 3) A complete integer minus 1 from the total. The invention also encompasses polynucleotides encoding these polypeptides.

Furthermore, the invention provides polypeptides having one or more residues deleted from the carboxyl terminus of the albumin fusion proteins of the invention. Specifically, C-terminal deletions can be described by the general formula 1 to n, where n is any whole integer ranging from 6 to q minus 1, and q is the total number of amino acid residues representing the albumin fusion protein of the invention the complete integer of . The invention also encompasses polynucleotides encoding these polypeptides.

Additionally, any of the N- or C-terminal deletions described above can be combined to generate N- and C-terminal deleted reference polypeptides. The invention also provides polypeptides having one or more amino acids deleted from both the amino and carboxyl termini, which can generally be described as having a reference polypeptide (e.g., a therapeutic protein as mentioned in Table 1, or an albumin fusion protein of the invention) or a Therapeutic protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or serum albumin (e.g., SEQ ID NO: 1), or an albumin fusion protein of the invention or an albumin protein portion encoded by a polynucleotide or an albumin fusion construct described in Table 2, or an albumin fusion protein, or an albumin protein portion encoded by a polynucleotide or an albumin fusion construct of the present invention albumin fusion protein), wherein n and m are integers as described above. The invention also encompasses polynucleotides encoding these polypeptides.

The present application also relates to a therapeutic protein portion comprising a reference polypeptide sequence listed herein (such as a therapeutic protein mentioned in Table 1, or an albumin fusion protein of the present invention, or a polynucleotide or albumin described in Table 2). The Therapeutic protein portion encoded by the protein fusion construct, or serum albumin (e.g., SEQ ID NO: 1), or the albumin protein portion of an albumin fusion protein of the invention, or the polynucleotide or albumin protein portion described in Table 2. The albumin protein portion encoded by the protein fusion construct, or an albumin fusion protein, or an albumin fusion protein encoded by a polynucleotide of the invention or an albumin fusion construct) or a fragment thereof has at least 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% identical polypeptide protein. In preferred embodiments, the present application relates to a polypeptide comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a reference polypeptide and having the N- and A protein that is a polypeptide of a C-terminal deleted amino acid sequence. The invention also encompasses polynucleotides encoding these polypeptides.

A preferred polypeptide fragment of the present invention is a fragment comprising or consisting of an amino acid sequence exhibiting therapeutic and/or functional activity (e.g., biological activity) of a therapeutic protein or serum albumin protein polypeptide sequence, wherein the amino acid sequence is A fragment of the polypeptide sequence of the Therapeutic protein or serum albumin protein.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those that exhibit an activity similar, but not necessarily identical, to that of the polypeptides of the invention. Biological activities of fragments may include improved desired activities, or reduced undesirable activities.

Variants

"Variant" refers to a polypeptide or nucleic acid that differs from a reference nucleic acid or polypeptide but retains its essential properties. Typically, a variant is closely similar overall and identical in many regions to a reference nucleic acid or polypeptide.

As used herein, "variant" refers to a sequence that differs from, respectively, a Therapeutic protein (see, e.g., the "Therapeutic" column of Table 1), an albumin protein, and/or an albumin fusion protein but retains its description elsewhere herein A Therapeutic protein portion of an albumin fusion protein of the invention, an albumin portion of an albumin fusion protein of the invention, or an albumin fusion protein of the invention that has at least one functional and/or therapeutic property known or otherwise known in the art protein. Typically, the variant is in general with the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein, the albumin protein corresponding to the albumin protein portion of the albumin fusion protein, and/or the amino acid sequence of the albumin fusion protein Very similar and identical to it in many areas. The invention also encompasses nucleic acids encoding these variants.

The invention also relates to a Therapeutic protein comprising a Therapeutic protein moiety corresponding to, for example, an albumin fusion protein of the invention (for example, the amino acid sequence of Therapeutic protein X disclosed in Table 1; amino acid sequence of a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct; or a fragment or variant thereof), an albumin protein corresponding to the albumin protein portion of an albumin fusion protein of the invention (e.g. the amino acid sequence of the albumin protein portion of an albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in Tables 1 and 2; the amino acid sequence shown in SEQ ID NO: 1; or a fragment thereof or variant), and/or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of an albumin fusion protein or the proteins it consists of. Fragments of these polypeptides (eg, fragments described herein) are also provided. The present invention also includes polypeptides encoded by polynucleotides that bind DNA to filter membranes under stringent hybridization conditions (e.g., at about 45 degrees Celsius in 6X sodium chloride/sodium citrate (SSC) Hybridization, followed by washing one or more times in 0.2X SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (for example, at about 45 degrees Celsius, in 6X sodium chloride/sodium citrate (SSC) hybridization to filter-bound DNA in a medium, followed by one or more washes in 0.1X SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F.M. et al., edited, 1989, "Current protocol in Molecular Biology", Green publishing associates, Inc. and John Wiley & Sons Inc., NewYork, 6.3.1-6.3.6 and 2.10.3) with the albumin fusion protein encoding the present invention Complementary strands of nucleic acid molecules hybridize. The invention also encompasses polynucleotides encoding these polypeptides.

A polypeptide having an amino acid sequence having at least, e.g., 95% "identity" to a query amino acid sequence refers to a polypeptide whose amino acid sequence is identical to the query sequence, except that the polypeptide sequence of interest may comprise up to five of every 100 amino acids in the query amino acid sequence Amino acid changes. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the sequence of interest may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxyl-terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, and may be interspersed individually between residues of the reference sequence or at one or more of the residues within the reference sequence. in the adjacent group.

Indeed, known computer programs can be used to routinely determine the amino acid composition of any particular polypeptide with an albumin fusion protein of the invention or a fragment thereof (e.g., the Therapeutic protein portion of an albumin fusion protein or the albumin portion of an albumin fusion protein). Whether the sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. The FASTDB computer program based on the algorithm of Brutlag et al. (Comp.App.Biosci.6:237-245, 1990) can be used to determine the best overall combination between the query sequence (sequence of the present invention) and the sequence of interest. The preferred method of matching, also known as global sequence alignment. In a sequence alignment, the query and subject sequences can be both nucleotide sequences or both amino acid sequences. The results of the global sequence alignment are expressed in percent identity. The preferred parameters for FASTDB amino acid alignment are: matrix = PAM0, k-tuple = 2, mismatch penalty = 1, join penalty = 20, randomization group length = 0, cutoff score = 1, window size = sequence Shorter of Length, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 or the length of the amino acid sequence of interest.

If the target sequence is shorter than the query sequence because of N- or C-terminal deletions rather than internal deletions, the results must be manually corrected. This is because the FASTDB program does not take into account N- and C-terminal truncations of the target sequence when calculating the percent global identity. For target sequences that are truncated at the N- and C-terminals relative to the query sequence, correct the percent identity as follows, calculating the N- and C-terminal parts of the query sequence that are not matched/aligned with the corresponding target sequence The number of residues, as a percentage of the total number of bases in the query sequence. Whether a residue is matched/aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program described above using the specified parameters to give a final percent identity score. This final percent identity score is what is used in the present invention. Only residues located at the N- and C-termini of the subject sequence that are not matched/aligned to the query sequence are considered when manually adjusting the percent identity score. That is, only query residues located outside the furthest N- and C-terminal residues of the subject sequence are considered.

For example, a 90 amino acid residue subject sequence is aligned to a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the target sequence, so the FASTDB alignment does not show matching/alignment results for the first 10 residues at the N-terminus. The 10 unpaired residues account for 10% of the sequence (number of N- and C-terminal mismatched residues/total number of residues in the query sequence) and are therefore subtracted from the percent identity score calculated by the FASTDB program. Go for 10%. If the remaining 90 residues were a perfect match, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. This time the deletion is an internal deletion, so there are no residues at the N- or C-terminus of the subject sequence that do not match/align with the query sequence. In this case, the percent identity calculated by FASTDB does not need to be manually corrected. Again, only residue positions that, according to the FASTDB alignment, were shown to lie outside the N- and C-termini of the subject sequence and not matched/aligned to the query sequence required manual correction. No other form of manual correction was performed in the present invention.

A variant typically has at least 75% (preferably at least about 80%, 90%, 95%, or 99%) sequence identity to a normal HA or therapeutic protein of the same length as it. Using the programs blastp, blastn, blastx, tblastn and tblastx modified for sequence similarity searches (Karlin et al., Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; and Altschul, J. Mol. Evol . 36:290-300, 1993, fully incorporated as a reference) using the algorithm used to analyze and determine homology or identity at the nucleotide or amino acid sequence level through BLAST (Basic Local Alignment Search Tool) .

The method utilized by the BLAST program first considers similar segments between a query sequence and database sequences, then evaluates statistical significance for all matches identified, and finally summarizes only those matches that meet a predetermined significance threshold. For a discussion of fundamental issues in similarity searching of sequence databases, see Altschul et al., Nature Genetics 6:119-129, 1994, which is incorporated by reference in its entirety. Search parameters for histograms, descriptions, alignments, expectations (ie, the statistical significance threshold for reporting sequences paired against database sequences), cutoffs, matrices, and filters were set with default settings. The default scoring matrix employed by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992, incorporated by reference in its entirety). For blastn, the scoring matrix is set by the ratio of M (reward score for matching residues) and N (penalty score for mismatching residues), where the default values of M and N are 5 and -4, respectively. Four blastn parameters can be adjusted as follows: Q=10 (gap generation penalty); R=10 (gap extension penalty); wink=1 (word samples generated at each wink ^th position along the query sequence); gapw= 16 (sets window width in which to generate gapped alignments). The equivalent blastp parameter settings are Q=9; R=2; wink=1 and gapw=32. The inter-sequence comparison program Bestfit, available from the GCG software package version 10.0, uses DNA parameters of GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), while the equivalent setting for protein comparisons is GAP=8 and LEN=2.

A polynucleotide variant of the invention may contain alterations in coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations that result in silent substitutions, additions, or deletions that do not alter the properties or activities of the encoded polypeptide. Nucleotide variants resulting from silent substitutions caused by the degeneracy of the genetic code are preferred. Moreover, it is also preferred that less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5 or 1-2 amino acids in any combination are substituted, deleted, or added polypeptide variants. Polynucleotide variants can be produced for a variety of reasons, for example to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts such as yeast or E. coli).

In a preferred embodiment, the polynucleotide of the invention encoding the albumin portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In another preferred embodiment, a polynucleotide of the invention encoding a Therapeutic protein portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In yet another preferred embodiment, the polynucleotide encoding the albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.

In an alternative embodiment, a codon-optimized polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein does not hybridize to a wild-type polynucleotide encoding a Therapeutic protein under stringent hybridization conditions described herein. In yet another embodiment, a codon-optimized polynucleotide encoding an albumin portion of an albumin fusion protein does not hybridize to a wild-type polynucleotide encoding an albumin protein under the stringent hybridization conditions described herein. In another embodiment, a codon-optimized polynucleotide encoding an albumin fusion protein does not hybridize to a wild-type polynucleotide encoding a Therapeutic protein portion or an albumin protein portion under the stringent hybridization conditions described herein.

In additional embodiments, the polynucleotide encoding the Therapeutic protein portion of the albumin fusion protein does not comprise or consist of the naturally occurring sequence of the Therapeutic protein. In yet another embodiment, the polynucleotide encoding the albumin protein portion of the albumin fusion protein does not comprise or consist of naturally occurring sequences of the albumin protein. In alternative embodiments, the polynucleotide encoding an albumin fusion protein does not comprise or consist of a naturally occurring sequence of a Therapeutic protein portion or an albumin protein portion.

A naturally occurring variant called an "allelic variant" refers to one of several alternative forms of a gene occupying a given locus on an organism's chromosome (Genesll, Lewin, B. eds., John Wiley & Sons, New York, 1985 ). These allelic variants may differ at the polynucleotide and/or polypeptide level and are encompassed by the present invention. Alternatively, non-naturally occurring variants may be prepared by mutagenesis or direct synthesis techniques.

Using known methods of protein engineering and recombinant DNA technology, variants may be produced to improve or alter the characteristics of the polypeptides of the invention. For example, one or more amino acids may be deleted from the N- or C-terminus of a polypeptide of the invention without substantial loss of biological function. For example, Ron et al. (J. Biol. Chem. 268:2984-2988, 1993) reported variant KGF proteins with heparin-binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma exhibited up to ten-fold higher activity after deletion of 8-10 amino acid residues from the carboxyl terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216, 1988).

Furthermore, there is ample evidence that variants generally retain biological activity similar to that of the naturally occurring protein. For example, Gayle and colleagues (J. Biol. Chem. 268:22105-22111, 1993) performed extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate more than 3,500 unique IL-1a mutants, each with an average of 2.5 amino acid changes over the full length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that "most molecules can be altered with little effect on [binding or biological activity]." In fact, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with activities significantly different from wild-type.

Furthermore, even though deletion of one or more amino acids from the N- or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, when removing a majority of residues from the N-terminus or C-terminus less than the secreted form, it will be possible to retain the ability of the deletion variant to induce and/or bind antibodies that recognize the secreted form. Whether a particular polypeptide lacking the N-terminal or C-terminal residues of a protein retains such immunogenic activity can be readily determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention also includes polypeptide variants having functional activity (eg, biological activity and/or therapeutic activity). In one embodiment, the invention provides albumin fusion protein variants having functional activity (e.g., biological and/or therapeutic activity) corresponding to one or more biological and/or therapeutic activities of a Therapeutic protein , wherein the Therapeutic protein corresponds to the Therapeutic protein portion of an albumin fusion protein. In another embodiment, the invention provides albumin fusion protein variants having functional activity (e.g., biological and/or therapeutic activity) corresponding to one or more biological and/or therapeutic activities of a Therapeutic protein , wherein the Therapeutic protein corresponds to the Therapeutic protein portion of an albumin fusion protein. Such variants include deletions, insertions, inversions, duplications, and substitutions that have little effect on activity, selected according to general rules known in the art. The invention also encompasses polynucleotides encoding such variants.

In preferred embodiments, the variants of the invention have conservative substitutions. "Conservative substitution" refers to exchange within a group, such as substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; substitution of hydroxyl residues Ser and Thr; substitution of acidic residues Asp and Glu; substitution of amide residues Asn and Substitution of Gln; substitution of basic residues Lys, Arg, and His; substitution of aromatic residues Phe, Tyr, and Trp; and substitution of small amino acids Ala, Ser, Thr, Met, and Gly.

For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247:1306-1310, 1990, in which the authors state that studying amino acid sequences There are two main strategies for tolerance to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during evolution. Conserved amino acids can be identified by comparing the amino acid sequences of different species. These conserved amino acids may be important for protein function. In contrast, substituted amino acid positions that have been tolerated by natural selection indicate that these positions are not critical for protein function. Thus, positions that tolerate amino acid substitutions can be modified while still maintaining the biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions in cloned genes to identify regions critical for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (introducing single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085, 1989. The mutant molecules so produced can then be tested for biological activity.

As the authors describe, these two strategies have revealed that proteins are surprisingly tolerant to amino acid substitutions. The authors also indicate which amino acid changes are likely to be tolerated at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of proteins) amino acid residues require non-polar side chains, whereas few features of surface side chains are usually conserved. Moreover, conservative amino acid substitutions tolerated involve substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; substitution of hydroxyl residues Ser and Thr; substitution of acidic residues Asp and Glu; substitution of amide residues Asn and Gln substitutions; substitutions of basic residues Lys, Arg, and His; substitutions of aromatic residues Phe, Tyr, and Trp; and substitutions of small amino acids Ala, Ser, Thr, Met, and Gly. In addition to conservative amino acid substitutions, variants of the invention include (i) the mentioned polypeptides containing one or more non-conservative amino acid residues, wherein the substituted amino acid residues may or may not be encoded by the genetic code, or ( ii) a polypeptide that contains one or more substituted amino acid residues with substituents, or (iii) has been fused or chemically conjugated to another compound such as a compound that improves the stability and/or solubility of the polypeptide (e.g. polyethylene glycol) or (iv) polypeptides containing additional amino acids such as, for example, IgG Fc region fusion peptides. Such variant polypeptides are considered to be within the purview of those skilled in the art in light of the teachings herein.

For example, polypeptide variants containing amino acid substitutions for charged amino acids with other charged or neutral amino acids can result in proteins with improved properties, such as less aggregation. Aggregation of pharmaceutical agents not only reduces activity but also increases clearance due to the immunogenicity of the aggregates. See Pinckard et al., Clin. Exp. Immunol. 2:331-340, 1967; Robbins et al., Diabetes 36:838-845, 1987; Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377, 1993.

In a specific embodiment, the polypeptide of the present invention comprises or consists of a fragment or variant of the amino acid sequence of an albumin fusion protein, a therapeutic protein and/or an amino acid sequence of human serum albumin, wherein the fragment or variant is the same as that of the reference Amino acid sequences have 1-5, 5-10, 5-25, 5-50, 10-50, or 50-150 amino acid residue additions, substitutions, and/or deletions compared to the amino acid sequences. In preferred embodiments, amino acid substitutions are conservative. The invention also encompasses nucleic acids encoding these polypeptides.

The polypeptide of the present invention may be composed of amino acids linked together by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides can be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. This modification is well described in basic textbooks and more detailed monographs, as well as in the long research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It is understood that the same type of modification may be present at several positions in a given polypeptide to the same or to varying degrees. Furthermore, a given polypeptide may contain many types of modifications. Polypeptides may be branched, eg due to ubiquitination, and they may also be circular with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes, or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, lipids or lipid Covalent attachment of phosphatidylinositol derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, pyroglutamic acid Formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, PEGylation, proteolytic processing, phosphorylation, isoprene Cylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See e.g. "PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES", 2nd Edition, T.E. Creighton, W.H. Freeman and Company, New York, 1993; "POST-TRANSLATIONALCOVALENT MODIFICATION OF PROTEINS", edited by B.C. Johnson, Academic Press, New York, pp. 1- 12 pages, 1983; Seifter et al., Meth. Enzymol. 182:626-646, 1990; Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62, 1992).

functional activity

A "functionally active polypeptide" refers to a polypeptide capable of exhibiting one or more known functional activities associated with a full-length, preprotein, and/or mature form of a Therapeutic protein. Such functional activities include, but are not limited to, biological activity, antigenicity (the ability to bind (or compete with the polypeptide) for anti-polypeptide antibodies), immunogenicity (the ability to produce antibodies that bind to a particular polypeptide of the invention), and The ability of the inventive polypeptide to form multimers, and the ability to bind to receptors or ligands of the polypeptide.

A "polypeptide having biological activity" refers to a polypeptide that exhibits an activity similar, but not necessarily identical, to that of a Therapeutic protein of the invention, including mature forms, as measured by a specific biological assay, with or without dose dependence. Where a dose dependence does exist, the dose dependence for a given activity need not be the same as that of a polypeptide, but only substantially similar (i.e., a candidate polypeptide will exhibit greater Greater activity or no more than about 25-fold less activity, preferably no more than about 10-fold less activity, most preferably no more than about 3-fold less activity).

In preferred embodiments, albumin fusion proteins of the invention possess at least one biological and/or therapeutic activity associated with the Therapeutic protein moiety (or fragment or variant thereof) not fused to albumin.

In additional preferred embodiments, albumin fusion proteins of the invention have increased plasma stability compared to the Therapeutic protein moiety (or fragment or variant thereof) in the fused state. Plasma stability of albumin fusion proteins or unfused Therapeutic protein portions (or fragments or variants thereof) of the invention can be determined using or routinely adapting assays known in the art.

Functional activity (eg, biological activity) of albumin fusion proteins of the invention can be determined using or routinely adapting assays known in the art, as well as the assays described herein. Additionally, one skilled in the art can routinely assay for the activity of fragments of a Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein using the assays referenced in the corresponding row of Table 1 (e.g., column 3 of Table 1). . Additionally, the activity of fragments of the albumin protein corresponding to the albumin protein portion of an albumin fusion protein can be routinely assayed by one of skill in the art using assays known in the art and/or described in the Examples section below.

For example, in one embodiment of determining the ability of an albumin fusion protein to bind or compete with a Therapeutic protein for binding to anti-Therapeutic polypeptide antibodies and/or anti-albumin antibodies, various immunoassays known in the art can be used, including But not limited to using methods such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich immunoassay", immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay (eg Utilizing colloidal gold, enzymes, or radioisotope labels), Western blot, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay), complement fixation assay, immunofluorescence assay, protein A assay Competitive and non-competitive assay systems for techniques such as immunoelectrophoretic assays. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting the binding of the secondary antibody or reagent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. Many methods for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In a preferred embodiment, binding partners (e.g., receptors or ligands) of a Therapeutic protein are identified, for example, by methods well known in the art such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography and affinity chromatography. Binding of an albumin fusion protein comprising the Therapeutic protein as the Therapeutic protein portion of the fusion to the binding partner can be determined by blotting. See generally Phizicky et al., Microbiol. Rev. 59:94-123,1995. In another embodiment, the physiologically relevant ability of an albumin fusion protein to bind a substrate of a Therapeutic polypeptide corresponding to the Therapeutic protein portion of the fusion can be routinely assayed using techniques known in the art.

In an alternative embodiment of assessing the multimerization ability of albumin fusion proteins, multimerization can be determined, for example, by methods well known in the art such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. Association with other components. See generally Phizicky et al., supra.

In preferred embodiments, albumin fusion proteins comprising all or part of an antibody that binds a Therapeutic protein have at least one protein that is associated with the antibody (or fragment or variant thereof) that binds a Therapeutic protein when it is not fused to albumin. Biological and/or therapeutic activity (eg, specific binding to a polypeptide or epitope). In other preferred embodiments, the biological and/or therapeutic activity of the albumin fusion protein comprising all or part of an antibody that binds a Therapeutic protein is a function of the polypeptide to which the antibody that binds a Therapeutic protein specifically binds. Inhibition (ie, antagonism) or activation (ie, agonism) of multiple biological and/or therapeutic activities.

Albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be characterized in a variety of ways. In particular, albumin fusion protein-specific binding can be assayed for albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein using the techniques described herein or routinely adapting techniques known in the art. The ability of the same antigen to which an antibody specifically binds to a Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein.

Assays for the ability of an albumin fusion protein (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to (specifically) bind a particular protein or epitope can be performed in solution (e.g., Houghten, Bio/Techniques 13:412 -421, 1992), on beads (e.g. Lam, Nature 354:82-84, 1991), on chips (e.g. Fodor, Nature 364:555-556, 1993), on bacteria (e.g. U.S. Pat. No. 5,223,409) , on spores (e.g. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g. Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869, 1992), or on phage (e.g. Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al., Proc.Natl.Acad.Sci.USA 87:6378-6382, 1990; and Felici, J.Mol . Biol. 222:301-310, 1991). (These references are incorporated herein by reference in their entirety). Albumin fusion proteins comprising at least a fragment or variant of a therapeutic antibody can also be assayed for specificity and affinity for a particular protein or epitope using or routinely adapting techniques described herein or otherwise known in the art.

Albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be assayed for sequence/structural conservation with other antigens (e.g., with the molecule to which the antibody specifically binds) using any method known in the art. Molecules wherein the antibody binds a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention) cross-reactivity.

Immunoassays that can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, methods such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoassay Competitiveness of Precipitation Assay, Precipitin Reaction, Gel Diffusion Precipitin Reaction, Immunodiffusion Assay, Agglutination Assay, Complement Fixation Assay, Immunoradiometric Assay, Fluorescence Immunoassay, and Protein A Immunoassay and noncompetitive assay systems, to mention only a few here. Such assays are routine and well known in the art (see for example Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated in its entirety This article is used as a reference). Exemplary immunoassays are briefly described below (but not intended to be limiting).

Immunoprecipitation protocols typically include lysis buffers such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate at pH 7.2, 1% Trasylol) to dissolve the cell population, add the albumin fusion protein of the present invention (such as comprising at least one fragment or variant of an antibody that binds a Therapeutic protein), incubated at 40° C. for a period of time (e.g., 1 to 4 hours), adding, for example, sepharose beads conjugated to an anti-albumin antibody, to the cell lysate, and at Incubate at 40°C for approximately one hour or more, wash beads in lysis buffer, and resuspend beads in SDS/sample buffer. The ability of albumin fusion proteins to immunoprecipitate specific antigens can be assessed using, for example, Western blot analysis. Those skilled in the art understand that parameters can be modified to increase binding of albumin fusion proteins to antigen and to reduce background (eg pre-clearing cell lysates with sepharose beads). For more discussion of assay-free precipitation protocols see, e.g., Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, p. 10.16.1.

Western blot analysis typically involves preparing the protein sample, electrophoresis of the protein sample in a polyacrylamide gel (e.g. 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitric acid Cellulose, PVDF or nylon, blocking film in blocking solution (such as PBS containing 3% BSA or skimmed milk), washing film in washing buffer (such as PBS-Tween 20), the albumin fusion protein of the present invention (diluted in blocking buffer) applied to the membrane, washed in washing buffer, applied diluted in blocking buffer with an enzyme substrate (such as horseradish peroxidase or alkaline phosphatase) or a radioactive molecule ( For example ^32P or ^125I ) conjugated secondary antibody (which recognizes albumin fusion protein, eg anti-human serum albumin antibody), the membrane is washed in wash buffer and the presence of antigen detected. Those skilled in the art understand that parameters can be modified to increase detected signal and reduce background noise. See, eg, Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, p. 10.8.1 for more discussion of Western blotting protocols.

ELISA involves preparing the antigen, coating the wells of a 96-well microtiter plate with the antigen, washing away unbound antigen from the wells, and adding to the wells a combination of a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase). ) conjugated albumin fusion protein of the invention (eg, comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and incubating for a period of time to wash away unbound or non-specifically bound albumin fusion protein, and The presence of an albumin fusion protein that specifically binds to the antigen coating the wells is detected. In an ELISA, the albumin fusion protein need not be conjugated to a detectable compound; instead a secondary antibody (which recognizes the albumin fusion protein) conjugated to the detectable compound can be added to the wells. Alternatively, the wells may be coated with an albumin fusion protein instead of the antigen. In such cases, the detectable molecule may be an antigen conjugated to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). Those skilled in the art understand that the detection signal can be increased by modifying parameters, as well as other ELISA variations known in the art. For more discussion of ELISA see Ausubel et al., Ed., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, p. 11.2.1.

Binding affinities of albumin fusion proteins to proteins, antigens, or epitopes and off-rates of albumin fusion protein-protein/antigen/epitope interactions can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay, which involves combining a labeled antigen (eg, ^{3H or125I} ) with an albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen. Incubate, and detect antibody binding to labeled antigen. The affinities and binding off-rates of albumin fusion proteins for specific proteins, antigens, or epitopes can be determined from Scatchard plot analysis data. Competition with a second protein that binds to the same protein, antigen, or epitope as the albumin fusion protein can also be determined using radioimmunoassays. In this case, the protein, antigen, or epitope is incubated with an albumin fusion protein conjugated to a labeled compound (eg, ³ H or ¹²⁵ I) in the presence of increasing amounts of an unlabeled second protein , the second protein binds to the same protein, antigen, or epitope as the albumin fusion protein of the present invention.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rate of albumin fusion proteins of the present invention to proteins, antigens, or epitopes. BIAcore kinetic analysis includes analyzing the binding and dissociation of albumin fusion proteins or specific polypeptides, antigens or epitopes to chips immobilized on the surface of specific polypeptides, antigens or epitopes or albumin fusion proteins, respectively.

Antibodies that bind a Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein can also be described or specified in terms of their binding affinity for a given protein or antigen, preferably the antigen to which they specifically bind. Preferred binding affinities include binding affinities with a dissociation constant or Kd of less than 5×10 ⁻² M, 10 ⁻² M, 5×10 ⁻³ M, 10 ⁻³ M, 5×10 ⁻⁴ M, 10 ⁻⁴ . More preferred binding affinities include dissociation constants or Kd less than 5×10 ⁻⁵ M, 10 ⁻⁵ M, 5×10 ⁻⁶ M, 10 ⁻⁶ M, 5×10 ⁻⁷ M, 10 ⁷ M, 5×10 ^-8 M or 10 ^-8 M binding affinity. Even more preferred binding affinities include dissociation constants or Kds less than 5×10 ⁻⁹ M, 10 ⁻⁹ M, 5×10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5×10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 Binding affinity of ×10 ⁻¹² M, 10 ⁻¹² M, 5×10 ⁻¹³ M, 10 ⁻¹³ M, 5×10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5×10 ⁻¹⁵ M or 10 ⁻¹⁵ M. In preferred embodiments, the albumin fusion protein comprising at least a fragment of an antibody that binds a Therapeutic protein comprises at least a fragment of an antibody that binds a Therapeutic protein, taking into account the potency of the albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the potency of the corresponding antibody or variant albumin fusion proteins have an affinity for a given protein or epitope that is similar to the affinity of a corresponding antibody (not fused to albumin) that binds a Therapeutic protein. Additionally, assays described herein (see Examples and Table 1) and otherwise known in the art can be used routinely to measure albumin fusion proteins and fragments, variants and derivatives thereof eliciting therapeutic proteins with albumin fusion proteins Biological activity and/or therapeutic activity (either in vitro or in vivo) capacity associated with the portion and/or albumin portion. Other methods are known to the skilled artisan and are within the scope of the present invention.

Albumin

As mentioned above, the albumin fusion protein of the present invention comprises at least one fragment or variant of therapeutic protein and at least one fragment or variant of human serum albumin, the two are connected to each other, preferably by gene fusion.

Additional embodiments comprise at least one fragment or variant of a Therapeutic protein and at least one fragment or variant of human serum albumin linked to each other by chemical conjugation.

The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof) as well as albumin (and fragments and variants thereof) from other species.

As used herein, "albumin" refers to the albumin protein or amino acid sequence, or the collection of albumin fragments or variants that possess one or more functional activities (eg, biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments thereof (see for example EP201 239, EP322094, WO97/24445, WO95/23857), especially the human albumin as shown in Figure 1 and SEQ ID NO:1 The mature form, or albumin or fragments thereof from other vertebrates, or analogs or variants of these molecules or fragments thereof.

In a preferred embodiment, the human serum albumin protein used in the albumin fusion protein of the present invention contains one or two of the following two sets of point mutation combinations: numbering refers to SEQ ID NO: 1, Leu-407 is mutated to Ala, Leu-408 is mutated to Val, Val-409 is mutated to Ala, and Arg-410 is mutated to Ala; or Arg-410 is mutated to A, Lys-413 is mutated to Gln, and Lys-414 is mutated to Gln (for example, see International Publication No. WO 95/23857, incorporated herein by reference in its entirety). In an even more preferred embodiment, albumin fusion proteins of the invention containing one or both of the above two sets of point mutations have improved stability/resistance to proteolytic cleavage by yeast Yap3p, allowing Increased production of recombinant albumin fusion protein expressed in cells.

As used herein, a portion of albumin sufficient to prolong the therapeutic activity, plasma stability or shelf life of a Therapeutic protein refers to a portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, thus the albumin fusion protein. Fractions of albumin in which the shelf-life or plasma stability of the Therapeutic protein moiety is extended or extended compared to the shelf-life or plasma stability of the non-fused state. The albumin portion of the albumin fusion protein may comprise the full-length HA sequence as described above, or may comprise one or more fragments thereof capable of stabilizing or prolonging therapeutic activity. Such fragments may be 10 or more amino acids in length, or may include about 15, 20, 25, 30, 50 or more contiguous amino acids from the HA sequence, or may include part or all of a particular domain of HA . For example, one or more HA fragments spanning the first two immunoglobulin-like domains can be utilized. In preferred embodiments, the HA fragment is a mature form of HA.

The albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA. The Therapeutic protein portion of an albumin fusion protein of the invention can also be a variant of a Therapeutic protein described herein. The term "variant" includes conservative or non-conservative insertions, deletions and substitutions, wherein such changes do not substantially alter one or more of albumin's oncotic, useful ligand binding and non-immunogenic properties species, or an active site or domain that confers therapeutic activity on a therapeutic protein.

In particular, albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin, such as the fragments disclosed in EP322094 (i.e. HA(Pn), where n is 369 to 419) . Albumin may be derived from any vertebrate, especially any mammal such as human, bovine, ovine or porcine. Non-mammalian albumins include, but are not limited to, chicken and salmon. The albumin portion of the albumin fusion protein can be from a different animal than the Therapeutic protein portion.

Generally, HA fragments or variants are at least 100 amino acids in length, preferably at least 150 amino acids in length. The HA variant may consist of or comprise at least one complete domain of HA, e.g. domain 1 (amino acids 1-194 of SEQ ID NO: 1), domain 2 (amino acids 195-387 of SEQ ID NO: 1), Domain 3 (amino acids 388-585 of SEQ ID NO: 1), Domains 1 and 2 (1-387 of SEQ ID NO: 1), Domains 2 and 3 (195-585 of SEQ ID NO: 1), or domains 1 and 3 (amino acids 1-194 of SEQ ID NO: 1 and amino acids 388-585 of SEQ ID NO: 1). Each domain itself consists of two homologous subdomains, namely 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585, while the intersubdomain flexible linker region contains residues Base Lys106 to Glu119, Glu292 to Val315 and Glu492 to Ala511.

Preferably, the albumin portion of the albumin fusion protein of the invention comprises at least one subdomain or domain of HA or a conservative modification thereof. If the fusion is based on subdomains, some or all of the adjacent linkers are preferably used to link the Therapeutic protein moieties.

An antibody that specifically binds a Therapeutic protein is also a Therapeutic protein

Albumin fusion proteins comprising at least a fragment or variant of an antibody that specifically binds a Therapeutic protein disclosed in Table 1 are also encompassed by the invention. It is expressly contemplated that the term "therapeutic protein" encompasses antibodies that bind a Therapeutic protein (such as described in column 1 of Table 1) and fragments and variants thereof. Thus, an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein and/or at least a fragment or variant of an antibody that binds a Therapeutic protein.

Antibody structure and background

The basic antibody structural unit is known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines the constant region, which is primarily responsible for effector functions. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally Fundamental Immunology, Chapters 3-5 (Paul, W., ed., 4th ed., Raven Press, N.Y., 1998) (which is incorporated herein in its entirety for all purposes). The variable regions of each light chain/heavy chain pair form the antibody combining site.

Thus, intact IgG antibodies have two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are identical.

All chains display the same overall structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDR region is usually the part of the antibody that contacts the antigen and determines its specificity. The CDRs of the heavy and light chains from each pair are aligned by the framework regions, enabling binding to specific epitopes. From N-terminus to C-terminus, both light and heavy chain variable regions comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable region is linked to a heavy or light chain constant region. The amino acid assignment of each domain conforms to Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md., 1987 and 1991; Chothia and Lesk, J.Mol.Biol.196:901-917, 1987; Definition by Chothia et al., Nature 342:878-883, 1989.

As used herein, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen binding site that specifically binds an antigen (e.g., molecules containing one or more CDR regions of an antibody) . Antibodies that may correspond to a Therapeutic protein portion of an albumin fusion protein include, but are not limited to, monoclonal, multispecific, human, humanized, or chimeric antibodies, single chain antibodies (e.g., single chain Fv), Fab fragments, F(ab') fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies specific for antibodies of the invention), and epitope-binding fragments of any of the above (e.g., VH domain, VL domain, or one or more CDR regions).

Antibodies that bind therapeutic proteins

Albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (such as disclosed in Table 1 ) or a fragment or variant thereof are encompassed by the invention.

Antibodies that bind a Therapeutic protein (or fragment or variant thereof) can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (eg, mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. Most preferably, the antibodies are human antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include transgenic mice (xenomics) or other organisms that have been genetically engineered to produce human antibodies from human immunoglobulin repertoires. Antibodies isolated from the body.

Antibody molecules that bind a Therapeutic protein and can correspond to the Therapeutic protein portion of an albumin fusion protein of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses. In preferred embodiments, the antibody molecule that binds a Therapeutic protein and may correspond to the Therapeutic protein portion of the albumin fusion protein is IgGl. In other preferred embodiments, the immunoglobulin molecule that binds a Therapeutic protein and may correspond to the Therapeutic protein portion of the albumin fusion protein is IgG2. In other preferred embodiments, the immunoglobulin molecule that binds a Therapeutic protein and may correspond to the Therapeutic protein portion of the albumin fusion protein is IgG4.

Most preferably, the antibody that binds a Therapeutic protein and may correspond to the Therapeutic protein portion of an albumin fusion protein is a human antigen-binding antibody fragment of the invention, including but not limited to Fab, Fab' and F(ab')2, Fd, single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdfv), and fragments comprising VL or VH domains. Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable region alone, or together with all or part of the following: hinge region, CH1, CH2 and CH3 domains.

Antibodies that bind a Therapeutic protein and can correspond to the Therapeutic protein portion of an albumin fusion protein can be monospecific, bispecific, trispecific, or higher multispecific antibodies. Multispecific antibodies can be specific for different epitopes of a Therapeutic protein, or can be specific for both the Therapeutic protein as well as a heterologous epitope such as a heterologous polypeptide or a solid support. See, eg, PCT Publications WO93/17715; WO92/08802; WO91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69, 1991; Kostelny et al., J. Immunol. 148:1547-1553, 1992.

Antibodies that bind to a Therapeutic protein (or a fragment or variant thereof) can be bispecific or bifunctional, meaning that the antibody is an artificially constructed protein with two different pairs of heavy/light chains and two different binding sites. Hybrid antibody. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, eg, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321, 1990; Kostelny et al., J. Immunol. 148:1547-1553, 1992. Alternatively, bispecific antibodies can be identified as "diabodies" (Holliger et al., "'Diabodies': small bivalent and bispecific antibody fragments", PNAS USA 90:6444-6448, 1993) or "Janusins" (Traunecker et al., "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells", EMBO J 10:3655-3659, 1991; and Traunecker et al., "Janusin: new molecular design for bispecific reagents", Int. J. Cancer Suppl 7:51- 52, 1992) form.

The invention also provides albumin fusion proteins comprising fragments or variants (including derivatives) of the antibodies described herein or otherwise known in the art. Mutations can be introduced into a nucleotide sequence encoding a molecule of the invention using standard techniques known to those skilled in the art, including, for example, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutagenesis. Preferably, the variant (including derivatives) encodes fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer Less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions. In a specific embodiment, the variant encodes a VHCDR3 substitution. In a preferred embodiment, variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues.

Antibodies that bind a Therapeutic protein and may correspond to a Therapeutic protein portion of an albumin fusion protein may be described or illustrated in terms of the epitope or portion of Therapeutic protein that it recognizes or specifically binds. Antibodies that specifically bind a Therapeutic protein or a particular epitope of a Therapeutic protein can also be excluded. Thus, the invention encompasses antibodies that specifically bind a Therapeutic protein and allows for the exclusion of such antibodies. In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein binds to the same epitope as the unfused fragment or variant of the antibody itself.

Antibodies that bind a Therapeutic protein and may correspond to a Therapeutic protein portion of an albumin fusion protein may also be described or illustrated in terms of their cross-reactivity. Antibodies that do not bind any other analogs, orthologs, or homologues of Therapeutic proteins are also included. The binding has at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% sequence identity with the Therapeutic protein Antibodies to polypeptides (calculated using methods known in the art and described herein) are also included in the invention. In specific embodiments, antibodies that bind a Therapeutic protein and may correspond to a Therapeutic protein portion of an albumin fusion protein cross-react with the murine, rat and/or rabbit homologues of the human protein and their corresponding epitopes. Does not bind to a Therapeutic protein having less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% sequence identity Antibodies to polypeptides (calculated using methods known in the art and described herein) are also included in the invention. In a specific embodiment, the above-mentioned cross-reactivity relates to any single specific antigenic or immunogenic polypeptide disclosed herein, or 2, 3, 4, 5 or more specific antigenic and/or immunogenic polypeptides Combination of original peptides. In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein has similar or substantially the same cross-reactivity profile as the fragment or variant of that particular antibody itself.

Also included in the invention are antibodies that bind a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions (as described herein) to a polynucleotide encoding a Therapeutic protein. Antibodies that bind a Therapeutic protein and may correspond to the Therapeutic protein moiety of an albumin fusion protein of the invention may also be described or illustrated in terms of their binding affinity for a polypeptide of the invention. Preferred binding affinities include binding affinities with a dissociation constant or Kd of less than 5×10 ⁻² M, 10 ⁻² M, 5×10 ⁻³ M, 10 ⁻³ M, 5×10 ⁻⁴ M, 10 ⁻⁴ . More preferred binding affinities include dissociation constants or Kd less than 5×10 ⁻⁵ M, 10 ⁻⁵ M, 5×10 ⁻⁶ M, 10 ⁻⁶ M, 5×10 ⁻⁷ M, 10 ⁷ M, 5×10 ^-8 M or 10 ^-8 M binding affinity. Even more preferred binding affinities include dissociation constants or Kds less than 5×10 ⁻⁹ M, 10 ⁻⁹ M, 5×10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5×10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 Binding affinity of ×10 ⁻¹² M, 10 ⁻¹² M, 5×10 ⁻¹³ M, 10 ⁻¹³ M, 5×10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5×10 ⁻¹⁵ M or 10 ⁻¹⁵ M. In preferred embodiments, the albumin fusion protein comprising at least a fragment of an antibody that binds a Therapeutic protein comprises at least a fragment of an antibody that binds a Therapeutic protein, taking into account the potency of the albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the potency of the corresponding antibody or variant albumin fusion proteins have an affinity for a given protein or epitope that is similar to the affinity of a corresponding antibody (not fused to albumin) that binds a Therapeutic protein.

The invention also provides antibodies that competitively inhibit binding of the antibody to an epitope of a Therapeutic protein, as determined by any method known in the art for determining competitive binding, such as the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein competitively inhibits binding of the secondary antibody to an epitope of the Therapeutic protein. In other preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein competitively inhibit at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, At least 70%, at least 60%, or at least 50% binding of the secondary antibody to an epitope of the Therapeutic protein.

Antibodies that bind to a Therapeutic protein and can correspond to the Therapeutic protein portion of the albumin fusion proteins of the invention can function as agonists or antagonists of the Therapeutic protein. For example, the invention includes antibodies that partially or completely disrupt the receptor/ligand interaction with the polypeptides of the invention. The present invention features not only receptor-specific antibodies, but also ligand-specific antibodies. The invention also features receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (ie, signaling) can be determined using techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting phosphorylation (eg, tyrosine or serine/threonine) of the receptor or its substrates by immunoprecipitation followed by Western blot analysis (eg, as described above). In specific embodiments, there is provided an inhibition of ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% relative to the activity in the absence of the antibody , at least 60%, or at least 50% antibodies. In a preferred embodiment, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein has the property of preventing ligand binding and/or receptor activation from an unfused fragment of an antibody that binds a Therapeutic protein. or variants with similar or substantially similar characteristics.

The invention also features receptor-specific antibodies that prevent both ligand binding and receptor activation, and antibodies that recognize receptor-ligand complexes, and preferably do not specifically recognize unbound receptors or unbound receptors. Ligand. Likewise, the invention includes neutralizing antibodies that bind a ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand thereby preventing activation of the receptor but do not prevent binding of the ligand to the receptor. The invention also includes antibodies that activate the receptor. These antibodies may act as receptor agonists, ie potentiate or activate all or a subset of the biological activities of ligand-mediated receptor activation, for example by inducing dimerization of the receptor. Antibodies can be designated as agonists, antagonists or inverse agonists of a biological activity including a particular biological activity of a Therapeutic protein (such as disclosed in Table 1). The antibody agonists described above can be produced using methods known in the art. See, eg, PCT Publication WO96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988, 1988; Chen et al., Cancer Res. 58(16):3668-3678, 1998; Harrop et al. People, J. Immunol.161(4):1786-1794, 1998; Zhu et al., Cancer Res.58(15):3209-3214, 1998; Yoon et al., J.Immunol.160(7):3170- 3179,1998; Prat et al., J.Cell.Sci.111(Pt2):237-147,1998; Pitard et al., J.Immunol.Methods 205(2):177-190,1997; Liautard et al., Cytokine9 (4): 233-241, 1997; Carlson et al., J. Biol. Chem. 271(17): 11295-11301, 1997; Taryman et al., Neuron 14(4): 755-762, 1995; Muller et al. , Structure 6(9): 1153-1167, 1998; Bartunek et al., Cytokine 8(1): 14-20, 1996 (both of which are incorporated herein by reference in their entirety). In preferred embodiments, the albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein has a similar or substantially the same agonist or antagonist as the unfused fragment or variant of an antibody that binds a Therapeutic protein. Agent properties.

For example, antibodies that bind to a Therapeutic protein and that may correspond to the Therapeutic protein portion of the albumin fusion proteins of the invention can be used to purify, detect, and target Therapeutic proteins, including in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies can be used in immunoassays that qualitatively and quantitatively measure the levels of therapeutic proteins in biological samples. See, eg, Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, 2nd ed., 1988, which is incorporated herein by reference in its entirety. Likewise, for example, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be utilized to purify, detect, and target a Therapeutic protein, including in vitro and in vivo diagnostic and therapeutic methods.

Antibodies that bind a Therapeutic protein and may correspond to the Therapeutic protein portion of an albumin fusion protein include modified derivatives, ie, by covalently attaching any type of molecule to the antibody. By way of example and not limitation, antibody derivatives include, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, interaction with cellular ligands or other Modified antibodies such as protein linkage. Any of a variety of chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives may contain one or more non-classical amino acids. Albumin fusion proteins of the invention may also be modified as described above.

Methods of generating antibodies that bind therapeutic proteins

血蓝蛋白、二硝基酚、和潜在有用的人佐剂诸如BCG(卡介苗)和小棒杆菌。这种佐剂也是本领域众所周知的。 Antibodies that bind a Therapeutic protein and may correspond to the Therapeutic protein portion of an albumin fusion protein of the invention can be generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest can be generated by various procedures well known in the art. For example, a therapeutic protein can be administered to various host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. Depending on the host species, various adjuvants can be utilized to enhance the immune response, including but not limited to Freund's (complete and incomplete) adjuvants, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, Pluronic polyols , polyanions, peptides, oil emulsions, keyhole

Hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, hybridoma technology can be utilized, including those known in the art and such as Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd Edition, 1988; Hammering et al., in Monoclonal Antibodies and T-Cell Hybridomas , pp. 563-681, Elsevier, N.Y., 1981, which is incorporated by reference in its entirety, to produce monoclonal antibodies by the hybridoma technique. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and does not refer to the method by which it is produced.

Methods for generating and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a Therapeutic protein or fragment or variant thereof, an albumin fusion protein, cells expressing such a Therapeutic protein or a fragment or variant thereof, or an albumin fusion protein. Once an immune response is detected, eg, antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The spleen cells are then fused with any suitable myeloma cells, eg cells from the cell line SP20 available from the ATCC, by well known techniques. Hybridomas were selected and cloned by limiting dilution. Hybridoma clones are then assayed by methods known in the art to select for cells that secrete antibodies that bind a polypeptide of the invention. Immunization of mice with positive hybridoma clones produces ascites fluid that often contains high levels of antibodies.

Accordingly, the present invention provides methods for producing monoclonal antibodies, as well as antibodies produced by a method comprising the steps of culturing antibody-secreting hybridoma cells, wherein preferably, the hybridomas are obtained by immunizing small cells immunized with an antigen of the present invention. The splenocytes isolated from the mouse are fused with myeloma cells, and the resulting hybridomas are screened for hybridoma clones that secrete antibodies that can bind to the polypeptide of the present invention.

Another well-known method for generating both polyclonal and monoclonal human B cell lines is transformation with Epstein-Barr virus (EBV). Protocols for generating EBV-transformed B cell lines are generally known in the art, such as, for example, the protocol outlined in Coligan et al., eds., "Current Protocols in Immunology", 1994, John Wiley & Sons, NY, chapter 7.22, which is incorporated herein in its entirety as refer to. The source of B cells for transformation is typically human peripheral blood, but B cells for transformation can also be derived from other sources including, but not limited to, lymph nodes, tonsils, spleen, tumor tissue, and infected tissues. Before EBV transformation, the tissue is usually made into a single-cell suspension. Additionally, steps may be taken to physically remove or inactivate T cells (eg, treatment with cyclosporin A) in samples containing B cells, since T cells from individuals who are seropositive for anti-EBV antibodies can suppress EBV-induced B Cell immortalization.

Typically, samples containing human B cells are inoculated with EBV and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC number VR-1492). Physical signs of EBV transformation are usually visible towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells can appear large, clear, hairy, and tend to aggregate in tight cell clusters. Initially, EBV lines were usually polyclonal. However, after prolonged cell culture time, EBV lines can become monoclonal or polyclonal due to the selective growth of specific B cell clones. Alternatively, polyclonal EBV transformants can be subcloned (eg, by limiting dilution culture) or fused to an appropriate fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g. SP2/0, X63-Ag8.653), heteromyeloma (heteromyeloma) cell lines (human x mouse; e.g. SPAM-8, SBC- H20 and CB-F7) and human cell lines (such as GM1500, SKO-007, RPMI 8226 and KR4). Accordingly, the present invention also provides methods of producing polyclonal or monoclonal human antibodies directed against the polypeptides of the present invention or fragments thereof, comprising EBV transformation of human B cells.

Antibody fragments that recognize specific epitopes can be prepared by known techniques. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments) . The F(ab')2 fragment contains the variable region, the constant region of the light chain and the CH1 domain of the heavy chain.

For example, antibodies that bind a Therapeutic protein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In a specific embodiment, such phage can be used to display antigen binding domains expressed from repertoire or combinatorial antibody repertoires (eg, human or murine). Phage expressing an antigen-binding domain that binds an antigen of interest can be selected or identified with antigen, eg, labeled antigen or antigen bound or captured on a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13, in which the binding domain expressed by phage with Fab, Fv, or disulfide bond-stabilized Fv antibody domains is associated with phage gene III or gene VIII Protein recombinant fusion. Examples of phage display methods that can be used to generate antibodies that bind a Therapeutic protein include Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur.J.Immunol.24:952-958, 1994; Persic et al., Gene 187:9-18, 1997; Burton et al., Advances in Immunology 57:191-280, 1994; PCT Application No. PCT/ GB91/01134; PCT publications WO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982; WO95/20401; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;

Following phage selection, antibody coding regions can be isolated from phage and used to generate whole antibodies, including human antibodies or any other desired antigen-binding fragment, and can be expressed in any desired host, as described in the above references , including mammalian cells, insect cells, plant cells, yeast, and bacteria, such as those described in detail below. For example, using methods known in the art, such as PCT publication WO92/22324; Mullinax et al., BioTechniques 12(6):864-869, 1992; Sawai et al., AJRI 34:26-34, 1995; and Better et al. Techniques for recombinant production of Fab, Fab' and F(ab')2 fragments may also be employed as disclosed in Human, Science 240: 1041-1043, 1988 (said reference is incorporated by reference in its entirety).

Examples of techniques that can be used to generate single chain Fv and antibodies include U.S. Patents 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88, 1991; Shu et al., PNAS 90:7995-7999, 1993; and Skerra et al. Man, Science 240:1038-1040, 1988 as described. For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized or human antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods 125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567 and 4,816397, They are incorporated herein by reference in their entirety. A humanized antibody is an antibody molecule from a non-human species that binds the desired antigen, having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Framework residues in the human framework regions are often substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, binding to the antigen. These framework substitutions can be identified using methods well known in the art, such as modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and comparing sequences to identify unusual framework residues at particular positions. (See, eg, Queen et al., US Patent No. 5,585,089; Riechmann et al., Nature 332:323, 1988, which are hereby incorporated by reference in their entirety). Antibodies can be humanized using a variety of methods known in the art including, for example, CDR grafting (EP239,400; PCT Publication WO91/09967; US Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (resurfacing) (EP592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498,1991; Studnicka et al., Protein Engineering 7(6):805-814,1994; Roguska et al., PNAS 91:969-973, 1994) and chain shuffling (US Patent No. 5,565,332).

Fully human antibodies are particularly desirable for treating human patients. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, as described above. See also U.S. Patent Nos. 4,444,887 and 4,716,111; PCT Publications WO98/46645, WO98/50433, WO98/24893, WO98/16654, WO96/34096, WO96/33735, and WO91/10741, each of which is incorporated herein by reference in its entirety .

Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but instead express human immunoglobulin genes. For example, human immunoglobulin heavy and light chain gene complexes can be introduced into mouse embryonic stem cells either randomly or by homologous recombination. Alternatively, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Mouse immunoglobulin heavy and light chains are rendered non-functional separately or simultaneously by introduction of human immunoglobulin loci by homologous recombination. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized with the antigen of choice, eg, all or part of a polypeptide of the invention, in the normal manner. Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. Transgenic mice harbor human immunoglobulin transgenes that rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using this technique, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. An overview of this technique for generating human antibodies can be found in Lonberg and Huszar, Int. Rev. Immunol. 13:65-93,1995. A detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies can be found in PCT Publications WO98/24893; WO92/01047; WO96/34096; WO96/33735; 0598877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; Alternatively, companies such as Abgenix (Freemont, CA) and Genpharm (San Jose, CA) can be hired to provide human antibodies to selected antigens using techniques similar to those described above.

Fully human antibodies that recognize selected epitopes can be generated using a technique known as "directed selection." In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Bio/technology 12:899-903, 1988).

Polynucleotides Encoding Antibodies

The invention also provides polynucleotides comprising nucleotide sequences encoding antibodies and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or low stringency hybridization conditions, such as those defined above, to polynucleotides encoding antibodies, preferably antibodies that specifically bind to a Therapeutic protein, more preferably to an antibody having the " Antibodies to polypeptides of the amino acid sequence of "Therapeutic Protein X" disclosed in the "SEQ ID NO: Z" column.

Obtaining a polynucleotide and determining the nucleotide sequence of a polynucleotide can be performed by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g. as described in Kutmeier et al., BioTechniques 17:242, 1994), Briefly, it involves synthesizing overlapping oligonucleotides containing partial sequences encoding antibodies, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be generated from nucleic acid from an appropriate source. If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the immunoglobulin-encoding nucleic acid can be synthesized chemically or using synthetic primers that hybridize to the 3' and 5' ends of the sequence Obtained by PCR amplification from an appropriate source (e.g., an antibody cDNA library, or from any tissue or cell expressing the antibody, such as a cDNA library generated by hybridoma cells selected to express the antibody or nucleic acid isolated therefrom, preferably polyA+RNA) Or identified by cloning with oligonucleotide probes specific for particular gene sequences, eg, cDNA clones from cDNA libraries encoding antibodies. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art (see Example 65).

Once the nucleotide sequence and corresponding amino acid sequence of the antibody have been determined, the nucleotides of the antibody can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, Sambrook et al., 1990, "Molecular Cloning, A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, "Current Protocols in Molecular Biology", John Wiley & Sons, NY, which are hereby incorporated by reference in their entirety), to generate antibodies with different amino acid sequences, for example by making amino acid substitutions, deletions and/or insertions.

In a specific embodiment, the heavy and/or light chains can be examined by methods well known in the art, e.g., by comparison with known amino acid sequences of other heavy and light chain variable regions to determine sequence hypervariable regions. The amino acid sequences of the variable regions were used to identify the sequences of the complementarity determining regions (CDRs). Non-human antibodies can be humanized by inserting one or more CDRs into framework regions, such as those of a human, using conventional recombinant DNA techniques, as described above. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see for example Chothia et al., J. Mol. Biol. 278:457-479, 1998 for a list of human framework regions). Preferably, the polynucleotide produced by combining the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as described above, one or more amino acid substitutions may be made within the framework regions, preferably, the amino acid substitutions improve binding of the antibody to its antigen. In addition, amino acid substitutions or deletions of one or more variable region cysteine residues that participate in intrachain disulfide bonds can be made in this way to generate antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotides are encompassed by the invention and are within the skill of the art.

Alternatively, techniques developed for the generation of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985), that is, genes from a mouse antibody molecule with appropriate antigen specificity are spliced together with genes from a human antibody molecule with appropriate biological activity. As noted above, chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from murine mAbs and human immunoglobulin constant regions, eg, humanized antibodies.

Alternatively, techniques described for the preparation of single-chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423-42, 1988; Huston et al., Proc. and Ward et al., Nature 334:544-54, 1989) are suitable for generating single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through an amino acid bridge to generate a single-chain polypeptide. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041, 1988).

Recombinant expression of antibodies

Recombinant expression of antibodies or fragments, derivatives or analogs thereof (eg heavy or light chains of antibodies or single chain antibodies) requires construction of expression vectors containing polynucleotides encoding the antibodies. Once a polynucleotide encoding an antibody molecule of the invention or an antibody heavy or light chain or portion thereof (preferably containing a heavy or light chain variable region) is obtained, it can be produced by recombinant DNA techniques using techniques well known in the art. A vector for the production of antibody molecules. Thus, described herein are methods of making proteins by expressing a polynucleotide comprising a nucleotide sequence encoding an antibody. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention, or its heavy or light chain, or heavy or light chain variable region, operably linked to a promoter. Such vectors may include nucleotide sequences encoding constant regions of antibody molecules (see, e.g., PCT Publication WO86/05807; PCT Publication WO89/01036; and U.S. Patent No. 5,122,464), and for expression of the entire heavy or light chain, may The variable regions of the antibodies were cloned into this vector.

The expression vector is transferred into host cells by conventional techniques, and the transfected cells are cultured by conventional methods to produce antibodies. Accordingly, the invention includes host cells comprising a polynucleotide encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a single chain antibody, operably linked to a heterologous promoter. In a preferred embodiment for expressing diabodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell to express the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems can be utilized to express the antibody molecules of the invention. Such a host-expression system represents a vehicle by which the coding sequence of interest can be produced and subsequently purified, as well as a cell which can express the antibody molecule of the invention in situ after transformation or transfection with the appropriate nucleotide coding sequence. They include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; those transformed with recombinant yeast expression vectors containing antibody coding sequences Yeast (such as Saccharomyces, Pichia); insect cell systems infected with recombinant viral expression vectors (such as baculovirus) containing antibody coding sequences; Viruses, CaMV; Tobacco Mosaic Virus, TMV) infection or plant cell systems transformed by recombinant plasmid expression vectors (such as Ti plasmids) containing antibody coding sequences; ) or a mammalian cell system (eg COS, CHO, BHK, 293, 3T3 cells) of a recombinant expression construct of a promoter of a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter). Preferably, bacterial cells, such as E. coli, more preferably eukaryotic cells are used to express the recombinant antibody molecule, especially for the expression of the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) together with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are efficient antibody expression systems (Foecking et al., Gene 45:101, 1986; Cockett et al., Bio/Technology 8:2, 1990).

In bacterial systems, various expression vectors can be conveniently selected according to the intended use of the expressed antibody molecule. For example, when preparing large quantities of such proteins to generate pharmaceutical compositions of antibody molecules, vectors directing high-level expression of the easily purified fusion protein product may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791, 1983), in which antibody coding sequences can be ligated individually into the vector in the same reading frame as the lacZ coding region to create a fusion protein; pIN vector (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109, 1985; Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509, 1989) and the like. A pGEX vector can also be used to express a foreign polypeptide that forms a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and readily purified from lysed cells by adsorption and binding to the matrix glutathione-agarose beads followed by elution in the presence of free glutathione come out. The pGEX vector is designed to include a thrombin or Factor Xa protease cleavage site, allowing the release of the cloned target gene product from the GST moiety.

In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) was used as a vector to express foreign genes. The virus grows in the cells of the fall armyworm (Spondoptera frugiperda). Antibody coding sequences can be cloned individually into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems are available. If an adenovirus is used as the expression vector, the antibody coding sequence of interest can be linked to an adenovirus transcriptional/translational control complex such as the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertions in nonessential regions of the viral genome (eg, the El or E3 regions) will result in recombinant viruses that are viable and capable of expressing the antibody molecule in an infected host. (See eg Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:355-359, 1984). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544, 1987).

In addition, host cell strains can be selected that regulate the expression of the inserted sequence, or modify and process the gene product in the particular manner desired. Such modification (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with the correct cellular machinery for processing primary transcripts, glycosylated and phosphorylated gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, particularly breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20, and T47D, and normal breast cells Lines such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered to stably express antibody molecules. Rather than utilizing expression vectors containing viral origins of replication, hosts can be transformed with DNA and selectable markers under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) cell. After the introduction of exogenous DNA, the engineered cells can be allowed to grow for 1-2 days in a nourishing medium before switching to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be conveniently used to engineer cell lines expressing antibody molecules. This engineered cell line is particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

A number of selection systems are available including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine, which can be used in tk-, hgprt- or aprt-cells, respectively. Phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202, 1992) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes. Moreover, antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357, 1980; O'Hare et al., Proc. .Natl.Acad.Sci.USA 78:1527,1981); Confer mycophenolic acid resistant gpt (Mulligan and Berg, Proc.Natl.Acad.Sci.USA 78:2072,1981); Confer aminoglucoside G- 41-resistant neo (Clinical Pharmacy 12: 488-505; Wu and Wu, Biotherapy 3: 87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596, 1993; Mulligan, Science 260: 926-932,1993; and Morgan and Anderson, Ann.Rev.Biochem.62:191-217,1993; May, TIB TECH 11(5):155-215,1993); and hygromycin-resistant conferring (Santerre et al., Gene 30:147, 1984). Methods of recombinant DNA technology generally known in the art are routinely used to select desired recombinant clones and are described, for example, in Ausubel et al., eds., "Current Protocols in Molecular Biology", John Wiley & Sons, NY, 1993; Kriegler, "Gene Transfer and Expression, A Laboratory Manual", Stockton Press, NY, 1990; and Dracopoli et al., eds., "Current Protocols in Human Genetics", Chapters 12 and 13, John Wiley & Sons, NY, 1994; Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981, which is incorporated herein by reference in its entirety.

The expression level of antibody molecules can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3, Academic Press, New York, 1987 ). When the marker in the vector system expressing the antibody is amplifiable, increasing the level of the inhibitor in the host cell culture medium will increase the copy number of the marker gene. Because the amplified region is associated with the antibody gene, antibody production will also be increased (Crouse et al., Mol. Cell. Biol. 3:257, 1983).

Vectors utilizing glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase-based vectors is the availability of glutamine synthase negative cell lines (eg, murine myeloma cell line, NSO). Glutamine synthase expression systems can also function in glutamine synthase expressing cells, such as Chinese hamster ovary (CHO) cells, by providing additional inhibitors that prevent endogenous genes from functioning. Glutamine synthase expression systems and components thereof are described in detail in PCT publications WO87/04462; WO86/05807; WO89/01036; WO89/10404 and WO91/06657, which are incorporated herein by reference in their entirety. Additionally, glutamine synthase expression vectors that may be used in accordance with the present invention are commercially available from suppliers including Lonza Biologics (Portsmouth, NH). Bebbington et al., Bioltechnology 10: 169, 1992 and Biblia and Robinson, Biolechnol. Prog. 11: 1, 1995 describe the expression and production of monoclonal antibodies in murine myeloma cells using the GS expression system, which are incorporated herein in their entirety This article is for reference.

Host cells can be co-transfected with two expression vectors of the invention, wherein the first vector encodes a heavy chain-derived polypeptide and the second vector encodes a light chain-derived polypeptide. The two vectors may contain the same selectable marker allowing equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of both heavy and light chain polypeptides may also be used. In these cases, the light chain should be placed ahead of the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52, 1986; Kohler, Proc. Natl. Acad. Sci. USA 77:2197, 1980). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced by animal, chemical synthesis or recombinant expression, it can be purified using any method known in the art for the purification of immunoglobulin molecules, for example by chromatography (e.g. ion exchange, affinity especially Affinity for specific antigens followed by protein A, and sizing (column chromatography), centrifugation, differential solubility, or any other standard technique used to purify proteins. In addition, antibodies or fragments thereof that bind to a Therapeutic protein and may correspond to the Therapeutic protein portion of the albumin fusion proteins of the invention can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

Antibody modification

Antibodies that bind a Therapeutic protein, or a fragment or variant thereof, can be fused to a marker sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311 ), many of which are commercially available. Hexahistidine provides for convenient purification of fusion proteins as described, for example, in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin tag (also called "HA tag") corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984) and " flag" tag.

The invention also encompasses antibodies or fragments thereof conjugated to diagnostic or therapeutic agents. Antibodies can be used diagnostically, eg, to monitor the development or progression of tumors as part of a clinical testing procedure, eg, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive substances, positron emitting metals using various positron emission tomography, and nonradioactive paramagnetic metal ions. A detectable substance may be coupled or conjugated to the antibody (or fragment thereof) either directly or indirectly through an intermediate such as, for example, a linker known in the art, using techniques known in the art. Metal ions that can be conjugated to antibodies for use as diagnostic agents in accordance with the present invention can be found in, eg, US Pat. No. 4,741,900. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material Examples include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111I or 99Tc. Other examples of detectable substances are described elsewhere herein.

In addition, antibodies of the invention may be conjugated to therapeutic moieties, such as cytotoxins, eg cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, eg alpha-emitters such as eg 213Bi. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide (etoposide), teniposide (podophyllothiophene Glycoside, tenoposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxin diketone, mitoxantrone, mitoxantrone, actinomycin D, 1 - Dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil diazepam), alkylating agents (e.g., dichloromethyldiethyl Ammonium (nitrogen mustard), thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, chain Ureacin, mitomycin C, and cis-dichlorodiamminoplatinum(II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunorubicin) and doxorubicin) , antibiotics (such as dactinomycin (formerly known as actinomycin), bleomycin, shimmermycin, and anthranimycin (AMC)) and antimitotic agents (such as vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response and the therapeutic or drug moiety is not considered limited to classical chemotherapeutics. For example, a drug moiety can be a protein or polypeptide with a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet Derived growth factor, tissue plasminogen activator, apoptotic agent, such as TNF-α, TNF-β, AIM I (see International Publication No. WO97/33899), AIM II (see International Publication No. WO97/34911), Fas Ligands (Takahashi et al., Int. Immunol. 6:1567-1574, 1994), VEGI (see International Publication No. WO99/23105), thrombotic or anti-angiogenic agents, such as angiostatin or endostatin; or Biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colonies stimulatory factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other growth factors.

Antibodies can also be attached to solid supports, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating therapeutic moieties to antibodies are well known. See, eg, Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., eds., pp. 243-56, Alan R. Liss, Inc., 1985; Hellstrom et al. , "Antibodies For Drug Delivery," in Controlled Drug Delivery, 2nd ed., Robinson et al., eds., pp. 623-53, Marcel Dekker, Inc., 1987; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in "Monoclonal Antibodies'84: Biological And Clinical Applications", Pinchera et al., eds., pp. 475-506, 1985; "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", In "Monoclonal Antibodies For Cancer Detection And Therapy", Baldwin et al., eds., pp. 303-16, Academic Press, 1985; and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.Rev.62:119- 58, 1982.

Alternatively, the antibody can be conjugated to a secondary antibody to form an antibody heteroconjugate as described by Segal in US Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Antibodies, administered alone or in conjunction with cytotoxic factors and/or cytokines, conjugated or not conjugated to a therapeutic moiety, can be used as therapeutic agents.

Antibody-albumin fusion

Antibodies that bind a Therapeutic protein and may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, antibodies that bind a Therapeutic protein disclosed in the "Therapeutic Protein X" column of Table 1, or fragments or variants thereof .

In specific embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of a VH domain. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of 1, 2, or 3 VHCDRs. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of VHCDR1. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of VHCDR2. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of VHCDR3.

In specific embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of a VL domain. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of 1, 2, or 3 VLCDRs. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of VLCDR1. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to a Therapeutic protein portion of an albumin fusion protein comprises or consists of VLCDR2. In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of VLCDR3.

In other embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises 1, 2, 3, 4, 5, or 6 VH and/or or VL CDR or consist thereof.

In a preferred embodiment, the fragment or variant of an antibody that immunospecifically binds to a Therapeutic protein and corresponds to the Therapeutic protein portion of an albumin fusion protein comprises or consists of a scFv comprising an expression such as (Gly ₄ Ser) ₃ (SEQ ID NO: 4) isopeptide linker-linked therapeutic antibody VL domain and therapeutic antibody VH domain.

Immunophenotyping

Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or a fragment or variant thereof) can be used for immunophenotyping of cell lines and biological samples. Therapeutic proteins of the invention are useful as cell-specific markers, or more specifically, cellular markers that are differentially expressed at different stages of differentiation and/or maturation of a particular cell type. A monoclonal antibody (or at least an albumin fusion protein comprising a fragment or variant of an antibody that binds a Therapeutic protein) directed against a particular epitope or combination of epitopes will allow screening of cell populations expressing the marker. Monoclonal antibodies (or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) can be used to screen for marker-expressing cell populations using a variety of techniques, including magnetic testing using antibody-coated magnetic beads. Separation, "panning" with antibodies attached to solid substrates (ie, plates), and flow cytometry (see, eg, US Patent 5,985,660; and Morrison et al., Cell 96:737-49, 1999).

These techniques allow screening of specific cell populations such as hematological malignancies (ie mild sequelae (MRD) in acute leukemia patients) and "non-self" cells in transplantation to prevent graft versus host disease (GVHD). Alternatively, these techniques allow screening for hematopoietic stem and progenitor cells capable of proliferation and/or differentiation, such as can be found in human umbilical cord blood.

Identification of antibodies that bind Therapeutic proteins and albumin fusion proteins comprising fragments or variants of the antibodies that bind Therapeutic proteins

Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or a fragment or variant thereof) can be identified in a number of ways. Specifically, specific binding can be determined for albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein using the techniques described herein or routinely adapting techniques known in the art. The ability of the antibody to bind a Therapeutic protein corresponding to an antibody that binds the Therapeutic protein portion of an albumin fusion protein, for the same antigen.

Determination of the ability of an antibody of the invention or at least a fragment or variant of an albumin fusion protein of the invention comprising an antibody that binds a Therapeutic protein (or a fragment or variant thereof) to (specifically) bind a particular protein or epitope can be In solution (such as Houghten, Bio/Techniques 13:412-421, 1992), on beads (such as Lam, Nature 354:82-84, 1991), on a chip (such as Fodor, Nature 364:555-556, 1993), on bacteria (such as U.S. Patent No. 5,223,409), on spores (such as Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (such as Cull et al., Proc.Natl.Acad.Sci.USA 89:1865 -1869,1992), or on bacteriophage (such as Scott and Smith, Science 249:386-390,1990; Devlin, Science 249:404-406,1990; Cwirla et al., Proc.Natl.Acad.Sci.USA 87 : 6378-6382, 1990; and Felici, J. Mol. Biol. 222: 301-310, 1991) (each of these references is incorporated herein by reference in its entirety). Antibodies of the invention or albumin fusions of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or a fragment or variant thereof) may also be fused using or routinely adapting techniques described herein or otherwise known in the art. Proteins measure their specificity and affinity for specific proteins or epitopes.

An albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be determined to have sequence/structural conservation with other antigens (e.g., with a molecule specifically bound by an antibody) by any method known in the art Molecules that bind to the cross-reactivity of a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention).

Immunoassays that can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, methods such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoassay Competitiveness and Competitiveness of Technologies such as Precipitation Assay, Precipitin Reaction, Gel Diffusion Precipitin Reaction, Immunodiffusion Assay, Agglutination Assay, Complement Fixation Assay, Immunoradiometric Assay, Fluorescence Immunoassay and Protein A Immunoassay Noncompetitive assay systems, to name a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein in its entirety as refer to). Exemplary immunoassays are briefly described below (but not intended to be limiting).

Immunoprecipitation protocols usually include RIPA buffer (1% NP-40 or TritonX-100, 1% desarcosin) supplemented with protein phosphatase and/or protease inhibitors (such as EDTA, PMSF, aprotinin, sodium vanadate). Sodium Oxycholate, 0.1% SDS, 0.15M NaCl, 0.01M Sodium Phosphate (pH7.2, 1% Trasylol) and other lysis buffers to lyse the cell population, add the antibody of the present invention to the cell lysate or at least contain the binding therapeutic The albumin fusion protein of the present invention that is a fragment or variant of an antibody to the protein (or its fragment or variant) is incubated at 40°C for a period of time (for example, 1-4 hours), and protein A and/or protein A are added to the cell lysate G Sepharose beads (or in the case of albumin fusion proteins comprising at least a fragment or variant of an antibody that binds Therapeutic protein, beads coated with an appropriate anti-idiotypic or anti-albumin antibody), incubated at 40°C For about 1 hour or more, wash the beads in lysis buffer and resuspend the beads in SDS/sample buffer. The ability of an antibody or albumin fusion protein of the invention to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art will be aware of parameters that can be modified to increase binding of the antibody or albumin fusion protein to the antigen and reduce background (eg, preclearing cell lysates with agarose beads). For further discussion of immunoprecipitation protocols see, eg, Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, 10.16.1.

Western blot analysis typically involves preparing a protein sample, electrophoresis of the protein sample in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE, depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to On a membrane such as digested fiber, PVDF or nylon, block the membrane in a blocking solution (such as PBS containing 3% BSA or skimmed milk), wash the membrane in a washing buffer (such as PBS-Tween 20), and apply the antibody of the present invention or albumin fusion protein (diluted in blocking buffer) is applied to the membrane, the membrane is washed in wash buffer, and an enzyme substrate (e.g. horseradish peroxidase or alkali) conjugated to it is applied diluted in blocking buffer. phosphatase) or radioactive molecules (such as ³² P or ¹²⁵ I) that recognize albumin fusion proteins, such as anti-human serum albumin antibodies, the membrane is washed in wash buffer, and the presence of the antigen is detected. Those skilled in the art will be aware of parameters that can be modified to increase the detected signal and reduce background noise. For further discussion of Western blotting protocols see, eg, Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, 10.8.1.

ELISA involves preparing the antigen, coating the wells of a 96-well microtiter plate with the antigen, washing away the antigen not bound to the wells, adding to the wells conjugated enzyme substrates such as horseradish peroxidase or alkaline phosphate enzyme) and other detectable compounds of the present invention or albumin fusion protein (at least comprising fragments or variants of antibodies that bind to therapeutic proteins) and incubated for a period of time to wash away unbound or non-specifically bound albumin fusion protein, and The presence of antibodies or albumin fusion proteins that specifically bind to the antigen coated wells is detected. In an ELISA, the antibody or albumin fusion protein does not have to be conjugated to a detectable compound; instead, a secondary antibody (which recognizes the antibody or albumin fusion protein, respectively) to which the detectable compound will be conjugated can be added to the wells. In addition, instead of coating the wells with antigen, antibodies or albumin fusion proteins can be coated onto the wells. In this case, the detectable molecule may be an antigen conjugated to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). Those skilled in the art will be aware of parameters that can be modified to increase the signal detected, as well as other variations of ELISA known in the art. For further discussion of ELISA see, eg, Ausubel et al., eds., 1994, "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York, 11.2.1.

Binding affinities of albumin fusion proteins to proteins, antigens or epitopes and off-rates of antibody or albumin fusion protein-protein/antigen/epitope interactions can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay comprising incubating labeled antigen (such ^{as3H or125I} ) with an antibody or albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, And detect antibodies that bind to the labeled antigen. The data from the Scatchert plot analysis can be used to determine the affinity and binding and dissociation rate of the antibody or albumin fusion protein of the present invention to a specific protein, antigen or epitope. Radioimmunoassays can also be used to determine the competition of a second protein for binding to the same protein, antigen or epitope as the antibody or albumin fusion protein. In this case, the protein, antigen or epitope is combined with a labeled compound (e.g. ³ H or ¹²⁵ I) antibodies of the present invention or albumin fusion protein are incubated together.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the on and off rates of binding of an antibody or albumin fusion protein of the invention to a protein, antigen or epitope. BIAcore kinetic analysis includes analyzing the binding and dissociation of antibodies, albumin fusion proteins or specific polypeptides, antigens or epitopes to chips, where specific polypeptides, antigens or epitopes, antibodies or albumin fusion proteins are respectively immobilized on the surface of the chip.

therapeutic use

The present invention also contemplates antibody-based therapy comprising administering to an animal, preferably a mammal, most preferably a human patient, an antibody of the invention or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein , for treating one or more of the disclosed diseases, disorders or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof described herein), antibodies encoding the antibodies of the invention (including fragments, analogs and derivatives thereof described herein, and anti-idiotypic Antibodies), albumin fusion proteins of the invention comprising at least fragments or variants of antibodies that bind Therapeutic proteins, and nucleic acids encoding such albumin fusion proteins. Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein are useful in the treatment, inhibition or prevention of diseases, disorders or conditions associated with aberrant expression and/or activity of Therapeutic proteins , including but not limited to any one or more of the diseases, disorders or conditions described herein. Treatment and/or prevention of diseases, disorders or conditions associated with aberrant expression and/or activity of therapeutic proteins includes, but is not limited to, alleviation of symptoms associated with those diseases, disorders or conditions. An antibody of the invention or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in a pharmaceutically acceptable composition as known in the art or described herein.

In a particular and preferred embodiment, the invention is directed to antibody-based therapy comprising administering to an animal an antibody of the invention or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, Preferably a mammalian, most preferably a human patient, for the treatment of one or more diseases, disorders or conditions including, but not limited to: nervous disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions, and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies against full-length proteins expressed on the surface of mammalian cells; antibodies against epitopes of therapeutic proteins) and antibodies encoding the invention (including those described herein) fragments, analogs and derivatives thereof and anti-idiosyncratic antibodies). The antibodies of the invention are useful for treating, inhibiting or preventing diseases, disorders or conditions associated with aberrant expression and/or activity of therapeutic proteins, including but not limited to any one or more of the diseases, disorders or conditions described herein. The treatment and/or prevention of diseases, disorders or conditions associated with aberrant expression and/or activity of a Therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. An antibody of the invention or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in a pharmaceutically acceptable composition as known in the art or described herein.

A summary of the ways in which an antibody of the invention or at least a fragment or variant of an albumin fusion protein of the invention comprising an antibody that binds a Therapeutic protein can be used therapeutically includes local or systemic binding of a Therapeutic protein in vivo, or via antibody Direct cytotoxicity, such as that mediated by complement (CDC) or effector cells (ADCC). Some of these methods are described in detail below. Using the teachings provided herein, one of ordinary skill in the art will know how to use an antibody of the invention, or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, for diagnostic, monitoring, or therapeutic purposes, while No need to over-experiment.

Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may advantageously be combined with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors such as IL-2 , IL-3 and IL-7) in combination, for example to help increase the number or activity of effector cells interacting with the antibody.

Antibodies of the invention, or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, may be administered alone or in combination with other types of therapy (e.g., radiotherapy, chemotherapy, hormonal therapy, immunotherapy, and antineoplastic agents) Combined administration. In general, administration of products of species origin or species reactivity (in the case of antibodies) of the same species as the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments, derivatives, analogs or nucleic acids are administered to human patients for treatment or prophylaxis.

High affinity and/or potent inhibitory and/or neutralizing antibodies, fragments or regions thereof (or albumin fusion proteins associated with such antibodies) directed against a Therapeutic protein are preferably used against the polynucleotides of the invention or polypeptides including fragments thereof for immunoassays and treatment of disorders related thereto. Such antibodies, fragments or regions preferably have an affinity for the polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include dissociation constants or Kds of less than ^5x10-2 M, ^10-2 M, ^5x10-3 M, ^10-3 M, ^5x10-4 M, ^10-4 M. More preferred binding affinities include dissociation constants or Kd less than 5×10 ⁻⁵ M, 10 ⁻⁵ M, 5×10 −6 M, 10 ⁻⁶ M, 5×10 ⁻⁷ M, ^{10 −7 M} , 5× Those of 10 ^-8 M or 10 ^-8 M. Even more preferred binding affinities include dissociation constants or Kds less than 5×10 ⁻⁹ M, 10 ⁻⁹ M, 5×10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5×10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 Those of × ^10-12 M, ^10-12 M, 5× ^10-13 M, ^10-13 M, 5× ^10-14 M, ^10-14 M, 5× ^10-15 M or ^10-15 M.

gene therapy

In a specific embodiment, nucleic acid comprising a sequence encoding an antibody that binds a Therapeutic protein, or at least an albumin fusion protein comprising a fragment or variant of an antibody that binds a Therapeutic protein is administered to treat, inhibit Or to prevent a disease or disorder associated with aberrant expression and/or activity of a therapeutic protein. Gene therapy refers to treatment by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates the therapeutic effect.

Any method of gene therapy available in the art may be used in accordance with the present invention. Exemplary methods are described in more detail elsewhere in this application.

Evidence of therapeutic or prophylactic activity

Prior to use in humans, the compounds or pharmaceutical compositions of the invention are preferably tested in vitro and then in vivo for the desired therapeutic or prophylactic activity. For example, in vitro assays for demonstrating the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of compounds or compositions on cell lines and/or tissue samples can be assayed using techniques known to those skilled in the art, including but not limited to rosetting assays and cell lysis assays. In accordance with the present invention, in vitro assays that can be used to determine whether to administer a particular compound are outlined, including in vitro cell culture assays in which a patient's tissue sample is cultured and exposed to or otherwise administered a compound, and the effect of such compound on the tissue is observed Sample effect.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention. In a preferred embodiment, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, most preferably a human.

When the compound comprises a nucleic acid or an immunoglobulin, the formulations and methods of administration that may be used are as described above; other suitable formulations and routes of administration may be selected from those described below.

A variety of delivery systems are known and can be used to administer the compounds of the invention, e.g., encapsulation within liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987), construction of nucleic acids as part of retroviral or other vectors, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Compounds or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in combination with other biologically active agents. Combined administration. It can be administered systemically or locally. In addition, it may be desirable to introduce a pharmaceutical compound or composition of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as Omayer ( Ommaya) receptacle. Pulmonary administration may also be used, for example by use of an inhaler or nebulizer, as well as dosage forms containing aerosols.

In a particular embodiment, it may be desirable to administer a pharmaceutical compound or composition of the invention topically to the area in need of treatment; this may be through, for example, but not limited to, local infusion during surgery, topical application such as in conjunction with a post-operative wound dressing , by injection, via a catheter, by means of a suppository, or via an implant which is a porous, non-porous or gel-like substance comprising a membrane such as a sialastic membrane or fibers. Preferably, when administering proteins of the invention, including antibodies, care must be taken to use materials that do not absorb proteins.

In another embodiment, the compound or composition may be delivered in a vesicle, particularly a liposome (see Langer, Science 249:1527-1533, 1990; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, eds. Lopez-Berestein and Fidler, Liss, New York, pp.353-365, 1989; Lopez-Berestein, op. cit., pp. 317-327; see mainly ibid).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Seflon, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). In another embodiment, polymeric materials can be used (see "Medical Applications of Controlled Release", Langer and Wise eds., CRC Press., Boca Raton, Florida, 1974; "Controlled Drug Bioavailability, Drug Product Design and Performance", Smolen and Ball eds., Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 71:105, 1989). In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target, such as the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2 , pp.115-138, 1984).

For a discussion of other controlled release systems see Langer's review (Science 249:1527-1533, 1990).

In one embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of the protein it encodes by constructing it as part of a suitable nucleic acid expression vector and administering it into Intracellularly, for example by use of retroviral vectors (see U.S. Patent No. 4,980,286), or by direct injection, or by use of particle bombardment (e.g., gene gun; Biolistic, Dupont), or with lipid or cell surface receptors or transfection It is coated with an agent, or it is administered by linking a homeobox-like peptide known to enter the nucleus (see, eg, Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868, 1991) and the like. Alternatively, the nucleic acid can be introduced into the cell and incorporated into host cell DNA by homologous recombination for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopoeia or other recognized pharmacopoeia for use in animals, more particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin , Propylene, Ethylene Glycol, Water, Ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release dosage forms and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral dosage forms can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a patient. The dosage form should suit the mode of administration.

In a preferred embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are presented separately or mixed in unit dosage form, like a lyophilized powder or a water-free concentrate in a sealed container, such as an ampoule or sachette labeled with the quantity of active agent. When the composition is to be administered by infusion, it can be dispensed from an infusion bottle filled with sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations, such as those derived from sodium hydroxide, potassium, ammonium, calcium, iron , isopropylamine, triethylamine, 3-ethylaminoethanol, histidine, procaine and the like.

The amount of a compound of the invention effective in the treatment, inhibition and prevention of diseases or disorders associated with aberrant expression and/or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help determine optimal dosage ranges. The precise dosage to be employed in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dose administered to a patient is typically 0.1 mg/kg to 100 mg/kg of patient body weight. Preferably, the dose administered to the patient is 0.1 mg/kg-20 mg/kg of the patient's body weight, more preferably 1 mg/kg-10 mg/kg of the patient's body weight. In general, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower doses of human antibodies and less frequent dosing are often possible. In addition, the dose and frequency of administration of antibodies of the invention can be reduced by enhancing antibody uptake and tissue penetration (eg, into the brain) through modifications such as, for example, lipidation.

Diagnostics and Imaging

Labeled antibodies that bind a Therapeutic protein (or fragments or variants thereof) and derivatives and analogs thereof (including albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) can be used for diagnostic purposes to Detecting, diagnosing or monitoring diseases, disorders and/or conditions associated with aberrant expression and/or activity of therapeutic proteins. The present invention provides a method for detecting abnormal expression of a therapeutic protein, comprising (a) measuring the expression of the therapeutic protein in individual cells or body fluids using one or more antibodies specific to the target polypeptide, and (b) measuring the expression level of the gene An increase or decrease in the expression level of the therapeutic protein measured thereby compared to the standard expression level is indicative of aberrant expression.

The invention provides diagnostic assays for diagnosing a disorder comprising (a) the use of one or more antibodies specific for a Therapeutic protein or an albumin fusion comprising at least a fragment or variant of an antibody specific for a Therapeutic protein Protein measures the expression of a therapeutic protein in an individual's cells or body fluids and (b) compares the gene expression level to a standard gene expression level, whereby an increase or decrease in the measured therapeutic protein expression level compared to the standard expression level is indicative of specific disorder. In the case of cancer, the presence of relatively high numbers of transcripts in individual biopsies may indicate a predisposition to develop disease, or may provide a means for detecting disease before actual clinical symptoms appear. A more definitive diagnosis of this type may allow medical personnel to take preventive measures or attack treatment earlier, thereby preventing the development or further progression of the cancer.

Antibodies of the invention or albumin fusion proteins comprising at least fragments or variants of antibodies specific for a Therapeutic protein can be used to determine protein levels in biological samples using classical immunohistological methods known to those skilled in the art (e.g. See Jalkanen et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen et al., J. Cell. Biol. 105:3087-3096, 1987). Other antibody-based methods that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioactive isotopes such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium ( 121In) and technetium (99Tc); luminescent labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin.

One aspect of the invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of therapeutic proteins in animals, preferably mammals, most preferably humans. In one embodiment, the diagnosis comprises: a) administering (e.g. parenterally, subcutaneously or intraperitoneally) to a subject an effective amount of a labeled molecule that specifically binds a polypeptide of interest; b) waiting for a period of time after administration to allowing the preferential concentration of the labeled molecule in the subject at the site of expression of the therapeutic protein (and clearing of unbound labeled molecule to background levels); c) determining the background level; and d) detecting the labeled molecule in the subject, the labeled molecule exceeding A detection result of a background level indicates that the subject has a particular disease or disorder associated with aberrant expression of a therapeutic protein. Background levels can be determined in a variety of ways, including comparison of the amount of detected labeled molecule to a standard previously determined for a particular system.

As will be understood in the art, the size of the subject and the imaging system used will determine the amount of imaging modules required to produce a diagnostic image. In the case of radioisotope modules, for human subjects, the amount of injected radioactivity is typically in the range of about 5 to 20 millicuries99mTc. The labeled antibody, antibody fragment, or albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein will then preferentially accumulate at the site of the cell that contains the particular Therapeutic protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments", Chapter 13, Tumor Imaging: The Radiochemical Detection of Cancer, eds. S.W. Burchiel and B.A. Rhodes, Masson Publishing Inc., 1982.

Depending on a number of variables, including the type of marker used and the mode of administration, the time interval following administration to allow for preferential concentration of the marker molecule at the site in the subject and clearance of unbound marker molecule to background levels is 6-48 hours or 6-48 hours. 24 hours or 6-12 hours. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, the monitoring of the disease or disorder is performed by repeating the method used to diagnose the disease or disorder, eg, 1 month after the initial diagnosis, 6 months after the initial diagnosis, 1 year after the initial diagnosis, etc.

The presence of the marker molecule in the patient can be detected using methods known in the art for in vivo scanning. These methods depend on the type of marker used. A skilled artisan will be able to determine suitable methods for detecting a particular marker. Methods and devices that may be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography.

In a specific embodiment, the molecule is labeled with a radioisotope and detected in the patient using a radioresponsive surgical instrument (Thurston et al., US Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence-responsive scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic marker and detected in the patient using magnetic resonance imaging (MRI). Antibodies that specifically detect albumin fusion proteins rather than albumin or Therapeutic protein alone are a preferred embodiment. These can be used to detect the albumin fusion proteins described throughout the specification.

Reagent test kit

The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises antibodies, preferably purified antibodies, in one or more containers. In a specific embodiment, a kit of the invention contains a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody included in the kit. Preferably, the kit of the present invention further comprises a control antibody, which does not react with the polypeptide of interest. In another specific embodiment, the kit of the invention comprises means for detecting the binding of the antibody to the polypeptide of interest (e.g. the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound , or a secondary antibody that recognizes the primary antibody can be conjugated to a detectable substrate).

In another specific embodiment of the invention, the kit is a diagnostic kit for screening serum containing antibodies specific for proliferative and/or cancerous polynucleotides and polypeptides. Such kits may include control antibodies that do not react with the polypeptide of interest. Such kits may include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. In addition, such kits include means for detecting binding of the antibody to the antigen (eg, the antibody can be conjugated to a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). In specific embodiments, kits may include recombinantly produced or chemically synthesized polypeptide antigens. The polypeptide antigens of the kit can also be attached to a solid support.

In a more specific embodiment, the detection means of the above kit includes a solid support to which the polypeptide antigen is attached. Such kits may also include anti-human antibodies to which no reporter label is attached. In this embodiment, the binding of the antibody to the polypeptide antigen can be detected by the antibody labeled with the reporter.

In another embodiment, the invention includes a diagnostic kit for screening sera containing antigens of polypeptides of the invention. A diagnostic kit includes a substantially isolated antibody specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detection means of the kit may include a second labeled monoclonal antibody. Alternatively, or additionally, the detection means may comprise a labeled competing antigen.

In one diagnostic format, test serum is reacted with a solid phase reagent obtained by the method of the invention having surface-bound antigens. After the specific antigen antibody is combined with the reagent and the unbound serum components are removed by washing, the reagent is reacted with the anti-human antibody labeled with the reporter molecule so that the reporter molecule is in a certain proportion of the amount of anti-antigen antibody bound to the solid support. Binding reagents. The reagent is washed again to remove unbound labeled antibody and the amount of reporter molecule associated with the reagent is determined. Typically, the reporter molecule is an enzyme, which is detected by incubating the solid phase in the presence of an appropriate fluorescent, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

The solid surface reagents in the above assays are prepared by known techniques for attaching proteinaceous materials to solid support materials such as polymeric beads, dipsticks, 96-well plates or filter materials. These attachment methods typically involve non-specific adsorption of proteins to the support, or covalent attachment of proteins to chemically reactive groups on the solid support, usually through free amine groups, such as activated carboxyl, hydroxyl, or aldehyde groups. Alternatively, streptavidin-coated plates can be used in conjunction with biotinylated antigen.

Thus, the present invention provides assay systems or kits for performing this diagnostic method. Kits typically include a carrier with surface-bound recombinant antigen and a reporter-labeled anti-human antibody for detection of surface-bound anti-antigen antibody.

albumin fusion protein

The present invention mainly relates to albumin fusion proteins and methods for treating, preventing or improving diseases or disorders. As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a Therapeutic protein (or a fragment or variant thereof). The albumin fusion proteins of the present invention comprise at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, preferably by genetic fusion (i.e., the albumin fusion protein is composed of a protein in which the whole or part of the Therapeutic protein is encoded. Polynucleotides in the same reading frame and polynucleotides encoding complete or partial albumin are translated into each other) to each other. The Therapeutic protein and albumin, once part of an albumin fusion protein, can be referred to as a "portion," "region," or "module" of an albumin fusion protein.

In a preferred embodiment, the present invention provides an albumin fusion protein encoded by a polynucleotide described in Table 1 or Table 2 or an albumin fusion construct. The invention also encompasses polynucleotides encoding these albumin fusion proteins.

Preferred albumin fusion proteins of the invention include, but are not limited to, albumin fusion proteins encoded by a nucleic acid molecule comprising a Therapeutic protein (or fragment or variant thereof) in the same reading frame as encoding at least one molecule A polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) linked to or consisting of at least one polynucleotide; said nuclide molecule comprises in the same reading frame as Polynucleoside encoding at least one molecule of albumin (or a fragment or variant thereof) linked to at least one polynucleotide of the therapeutic protein (or its fragment or variant) produced as described in Table 1, Table 2 or the Examples or consisting of an acid; or the nucleic acid molecule comprises albumin encoding at least one molecule ( or a fragment or variant thereof), further comprising, for example, one or more of the following elements: (1) functional autonomously replicating vectors (including but not limited to shuttle vectors, expression vectors, integrating vectors, and/or replication system), (2) transcription initiation region (e.g. promoter region, such as e.g. regulatable or inducible promoter, constitutive promoter), (3) transcription termination region, (4) leader sequence, and (5) select markers.

In one embodiment, the invention provides an albumin fusion protein comprising or consisting of a Therapeutic protein (eg, as described in Table 1) and a serum albumin protein. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of a biologically and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of a biologically and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.

In other embodiments, the invention provides albumin fusion proteins comprising or consisting of a Therapeutic protein and a biologically and/or therapeutically active fragment of serum albumin. In other embodiments, the invention provides albumin fusion proteins comprising or consisting of a Therapeutic protein and biologically and/or therapeutically active variants of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein.

In other embodiments, the invention provides serum albumin comprising or consisting of a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. protein fusion protein. In a preferred embodiment, the invention provides an albumin fusion protein comprising or consisting of a mature portion of a Therapeutic protein and a mature portion of serum albumin.

Preferably, the albumin fusion protein comprises HA as the N-terminal part and a Therapeutic protein as the C-terminal part. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion and a Therapeutic protein as the N-terminal portion can also be used.

In other embodiments, albumin fusion proteins have a Therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the Therapeutic protein fused at the N-terminus and C-terminus are the same Therapeutic protein. In another preferred embodiment, the Therapeutic proteins fused at the N-terminus and C-terminus are different Therapeutic proteins. In another preferred embodiment, Therapeutic proteins fused at the N-terminus and C-terminus are useful for the treatment or prevention of the same or related diseases, disorders or conditions (such as listed in the "Preferred Indications Y" column of Table 1) different therapeutic proteins. In another preferred embodiment, Therapeutic proteins fused at the N-terminus and C-terminus are useful for the treatment, amelioration or prevention of the known in the art that usually occur simultaneously, concurrently or sequentially in a patient or usually in association with each other in a patient. A different therapeutic protein for a disease or disorder (such as listed in the "Preferred Indication Y" column of Table 1).

The albumin fusion proteins of the invention encompass compounds containing 1, 2, 3 fused to the N- or C-terminus of the albumin fusion proteins of the invention and/or fused to the N- and/or C-terminus of albumin or variants thereof. , 4 or more molecules of the specified Therapeutic Protein X or a variant thereof. A molecule of a given Therapeutic Protein X, or variant thereof, can be in any number of orientations, including but not limited to a "head-to-head" orientation (e.g., one in which the N-terminus of one Therapeutic Protein X molecule is fused to another Therapeutic Protein X molecule N-terminus), or a "head-to-tail" orientation (eg, where the C-terminus of one Therapeutic Protein X molecule is fused to the N-terminus of another Therapeutic Protein X molecule).

In one embodiment, 1, 2, 3 or more Therapeutic Protein X polypeptides (or fragments or variants thereof) in a tandem orientation are fused to the N- or C-terminus of an albumin fusion protein of the invention, and/or Fused to the N- and/or C-terminus of albumin or a variant thereof.

The albumin fusion protein of the present invention also encompasses 1,2, 3, 4 or more molecules of a given Therapeutic protein X or a variant thereof wherein the molecules are linked by a peptide linker. Examples include those peptide linkers described in US Patent No. 5,073,627 (incorporated herein by reference). Albumin fusion proteins comprising multiple Therapeutic Protein X polypeptides separated by peptide linkers can be produced using conventional recombinant DNA techniques. Linkers are especially important when fusing small peptides to large HAS molecules. The peptide itself may serve as a linker by fusion of tandem copies of the peptide, or other known linkers may be employed. Constructs incorporating linkers are described in Table 2 or are evident upon inspection of SEQ ID NO:Y.

In addition, the albumin fusion protein of the present invention can also be obtained by fusing Therapeutic protein X or its variants to albumin or its variants in such a way that allows the formation of intramolecular and/or intermolecular multimer forms N-terminal and/or C-terminal produced. In one embodiment of the invention, albumin fusion proteins may be in monomeric or multimeric form (ie dimers, trimers, tetramers and higher polymers). In another embodiment of the invention, the Therapeutic protein portion of the albumin fusion protein can be in monomeric or multimeric form (ie, dimers, trimers, tetramers and higher polymers). In a specific embodiment, the Therapeutic protein portion of the albumin fusion protein is in multimeric form (i.e., dimers, trimers, tetramers, and higher), while the albumin protein portion is monomeric. body form.

In addition to albumin fusion proteins in which the albumin moiety is fused to the N-terminus and/or C-terminus of the Therapeutic protein moiety, the albumin fusion proteins of the invention can also be obtained by combining the Therapeutic protein or the peptide of interest (such as in Table 1). A disclosed Therapeutic protein X, or an antibody that binds a Therapeutic protein or a fragment or variant thereof) is generated by insertion into the internal region of HA. For example, in the protein sequence of the HA molecule there are many loops or turns stabilized by disulfide bonds between the end and the start of the α-helix. According to the crystal structure of HA (PDB designators 1AO6, 1BJ5, 1BKE, 1BMO, 1E7E to 1E71 and 1UOR), these rings are mostly located away from the main body of the molecule. These loops can be used for insertion or internal fusion of therapeutically active peptides, especially those requiring secondary structure for function, or therapeutic proteins, to essentially generate albumin molecules with specific biological activities.

The loops in which peptides or polypeptides can be inserted into the human albumin structure to produce the albumin fusion protein of the present invention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486 and Lys560-Thr566. In a more preferred embodiment, the peptide or polypeptide is inserted into the loops Val54-Asn61, Gln170-Ala176 and/or Lys560-Thr566 of mature human albumin (SEQ ID NO: 1).

The peptide to be inserted may be derived from a phage-displayed or synthetic peptide library screened for a specific biological activity or from an active portion of a molecule with the desired function. In addition, random peptide libraries can be generated within specific loops, or by inserting random peptides into specific loops of HA molecules, where all possible amino acid combinations are represented.

Such libraries can be generated on HA or HA domain fragments by one of the following methods:

(a) Randomly mutating amino acids in one or more peptide loops of HA or HA domain fragments. One, more or all residues within the loop may be mutated in this manner;

(b) random peptide substitutions or insertions of length _Xn (where X is an amino acid and n is the number of residues) in one or more loops of the HA or HA domain fragment (i.e., an internal fusion);

(c) N-terminal, C-terminal, or N and C-terminal peptide/protein fusions other than (a) and/or (b).

HA or HA domain fragments can also be made multifunctional by grafting peptides derived from different screenings of different loops against different targets into the same HA or HA domain fragment.

In a preferred embodiment, the peptide inserted into the loop of human serum albumin is a peptide fragment or peptide variant of a Therapeutic protein disclosed in Table 1 . More specifically, the invention encompasses a loop comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least Albumin fusion proteins of peptide fragments or peptide variants of 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids. The invention also encompasses fusions comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, Albumin fusion proteins of peptide fragments or peptide variants of at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids. The invention also encompasses fusions comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least Albumin fusion proteins of peptide fragments or peptide variants of 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids. For example, short peptides described in Tables 1 and 2 (eg, therapeutic agent Y) can be inserted into the loop of albumin.

In general, albumin fusion proteins of the invention may have one HA-derived domain and one Therapeutic protein-derived domain. However, multiple regions of each protein can be used to construct albumin fusion proteins of the invention. Similarly, more than one therapeutic protein can be used to construct albumin fusion proteins of the invention. For example, a Therapeutic protein can be fused to both the N- and C-termini of HA. In such a configuration, the Therapeutic protein moieties can be the same or different Therapeutic protein molecules. The structure of the bifunctional albumin fusion protein can be expressed as: X-HA-Y or Y-HA-X.

For example, anti-BLyS ^™ scFv-HA-IFNα-2b fusions can be prepared to modulate the immune response to IFNα-2b by anti-BLyS ^™ scFv. Alternatively, bi-(or even multi)functional agents of HA-fusions can be prepared, such as HA-IFNα-2b fusions, and mixed with HA-anti-BLyS ^™ scFv fusions or other HA-- fusion.

Bi- or multifunctional albumin fusion proteins that target the therapeutic protein portion of the fusion to a target organ or cell type can also be prepared via a protein or peptide located at the other end of the HA.

As an alternative to fusions of known therapeutic molecules, peptides, typically six, 8, 12, 20 or 25 or Xn (where X is an amino acid (aa) and n is the number of residues) random amino acids, where all possible combinations of amino acids are represented. A particular advantage of this approach is that the peptides can be selected in situ on the HA molecule, so the properties of the peptides are as selected, without potential changes as would be the case if the peptides were derivatized by any other method and then attached to HA.

In addition, albumin fusion proteins of the invention may contain linker peptides between the fusion moieties, thereby providing greater physical separation between the modules and thereby maximizing the accessibility of the Therapeutic protein moiety, e.g., to its associated receptor combined. Linker peptides can be composed of amino acids, making them flexible or more rigid.

The linker sequence can be cleaved by protease or chemically to generate the growth hormone-associated portion. Preferably, the protease is naturally produced by the host, such as S. cerevisiae protease kex2 or an equivalent protease.

Thus, as described above, albumin fusion proteins of the invention may have the general formula: R1-L-R2; R2-L-R1 or R1-L-R2-L-R1, wherein R1 is at least one therapeutic protein , peptide or polypeptide sequence, and not necessarily the same Therapeutic protein, L is a linker, and R2 is a serum albumin sequence.

In preferred embodiments, an albumin fusion protein of the invention comprising a Therapeutic protein has a higher plasma stability than the plasma stability of the same Therapeutic protein not fused to albumin. Plasma stability generally refers to the time interval between when a therapeutic protein is administered in vivo and enters the bloodstream, and when the therapeutic protein is degraded and cleared from the bloodstream into an organ such as the kidney or liver, and finally cleared from the body. Plasma stability was calculated from the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

In preferred embodiments, albumin fusion proteins of the invention comprising a Therapeutic protein have an extended shelf life compared to the shelf life of the same Therapeutic protein not fused to albumin. Shelf life generally refers to the period of time during which the therapeutic activity of a therapeutic protein in solution or in some other depot formulation remains stable without undue loss of therapeutic activity. Many therapeutic proteins are highly variable in their unfused state. As described below, the shelf life of these therapeutic proteins is often significantly increased upon incorporation into the albumin fusion proteins of the invention.

"Extended" shelf-life albumin fusion proteins of the invention exhibit greater therapeutic activity relative to standards subjected to the same storage and handling conditions. Standards can be unfused full-length therapeutic proteins. When the Therapeutic protein portion of an albumin fusion protein is an analog, variant, or other alteration of the protein or does not comprise the entire sequence, the prolongation of therapeutic activity may be associated with the analog, variant, altered peptide, or not. The unfused equivalents of the complete sequences were compared. As an example, an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity or greater than about 105%, 110%, or greater than the therapeutic activity of a standard subject to the same storage and handling conditions when compared at a given time point. 120%, 130%, 150%, or 200%.

Shelf life can also be assessed based on residual therapeutic activity after storage, normalized to the therapeutic activity at the beginning of storage. Albumin fusion proteins of the invention with extended shelf life exhibit extended therapeutic activity, retaining greater than about 50%, about 60%, 70%, 80%, or 90% or more.

Expression of fusion protein

Albumin fusion proteins of the invention can be produced as recombinant molecules secreted by yeast, microorganisms such as bacteria, or human or animal cell lines. Optionally, the polypeptide is secreted by the host cell.

A particular embodiment of the invention includes DNA constructs encoding signal sequences effective for directing secretion in yeast, in particular yeast-derived signal sequences (especially homologous to yeast hosts), and the first aspect of the invention Fusion molecules without yeast-derived pro sequences between the signal and mature polypeptides.

The S. cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.

Conjugates of the type prepared by Poznansky et al. (FEBS Lett. 239: 18, 1988) in which separately prepared polypeptides are linked by chemical crosslinking are not envisaged.

The invention also includes cells, preferably yeast cells, transformed to express an albumin fusion protein of the invention. In addition to the transformed host cells themselves, the invention also encompasses cultures of those cells in nutrient media, preferably monoclonal (clonally homogeneous) cultures or cultures derived from monoclonal cultures. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if the cells have been removed by filtration or centrifugation. Many expression systems are known and available, including bacteria (such as Escherichia coli (E. coli) and Bacillus subtilis (Bacillus subtilis)), yeast (such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Kluyvern Yeast (Kluyveromyces lactis and Pichia pastoris), filamentous fungi (such as Aspergillus), plant cells, animal cells and insect cells.

Preferred yeast strains for the production of albumin fusion proteins are D88, DXY1 and BXP10. D88[leu2-3, leu2-122, can1, pra1, ubc4] is a derivative of the parental strain AH22his ⁺ (also known as DB1; see eg Sleep et al., Biotechnology 8:42-46, 1990). This strain contains a leu2 mutation that allows auxotrophic selection of 2 μm-based plasmids containing the LEU2 gene. D88 also exhibited derepression of PRB1 upon glucose excess. The PRB1 promoter is normally under the control of two checkpoints that monitor glucose levels and growth phases. In wild-type yeast, this promoter is activated after glucose depletion and entry into stationary phase. Strain D88 exhibited repression by glucose but maintained induction after entering stationary phase. The PRA1 gene encodes a yeast vacuolar protease, YscA endoprotease A, which localizes to the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short-lived and abnormal proteins for ubiquitin-dependent degradation. Isolation of this ubc4 mutation was found to increase the copy number of the expression plasmid in the cell and lead to increased expression levels of the desired protein expressed from the plasmid (see eg International Publication No. WO99/00504, which is incorporated herein by reference in its entirety).

DXY1, a derivative of D88, has the following genotypes: [leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to the mutation isolated in D88, this strain also has a knockout of the YAP3 protease. This protease primarily causes cleavage at two basic residues (RR, RK, KR, KK) in proteins, but can also facilitate cleavage at single basic residues. Isolation of this yap3 mutation resulted in high levels of full-length HAS product (see, e.g., U.S. Pat. No. 5,965,386 and Kerry-Williams et al., Yeast 14:161-169, 1998, which are incorporated herein by reference in their entirety).

BXP10 has the following genotypes: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition to the isolated mutation in DXY1, this strain also has a knockout of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of the evolutionarily conserved family of dolichol-phosphate-D-mannose protein O-mannosyltransferases (Pmt). The transmembrane topology of Pmt1p suggests that it is a membrane-intrinsic protein of the endoplasmic reticulum with a role in O-linked glycosylation. This mutation serves to reduce/eliminate O-linked glycosylation of HAS fusions (see eg International Publication No. WO 00/44772, which is hereby incorporated by reference in its entirety). Studies have revealed that Hsp150 protein cannot be efficiently separated from rHA by ion exchange chromatography. Mutations in the HSP150 gene eliminate potential contaminants that have proven difficult to remove by standard purification techniques. See, eg, US Patent No. 5,783,423, which is incorporated herein by reference in its entirety.

The desired protein is produced in a conventional manner, for example from the coding sequence inserted into the host chromosome or on an episomal plasmid. Yeast is transformed with the coding sequence for the desired protein by any common method such as electroporation. Methods for transforming yeast by electroporation are disclosed in Becker and Guarente, Methods Enzymol. 194:182,1990.

Successfully transformed cells, ie, cells containing a DNA construct of the invention, can be identified by well known techniques. For example, cells resulting from the introduction of the expression construct can be cultured to produce the desired polypeptide. Cells can be harvested, lysed, and the DNA content assayed for the presence of DNA using methods such as those described by Southern, J. Mol. Biol. 98:503, 1975 or Berent et al., Biotech. Alternatively, antibodies can be used to detect the presence of protein in the supernatant.

Useful yeast plasmid vectors include pRS403-406 and pRS413-416, and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrating plasmids (YIp) and incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3. Plasmid pRS413-416 is a yeast centromere plasmid (YCp).

Preferred vectors for expression in yeast to produce albumin fusion proteins include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA, which are detailed in Example 1. Figure 2 shows a map of the pPPC0005 plasmid that can be used as a basic vector into which polynucleotides encoding therapeutic proteins can be cloned to form HA-fusions. It contains the PRB1 S. cerevisiae promoter (PRB1p), fusion leader (FL), DNA encoding HA (rHA) and the ADH1 S. cerevisiae termination sequence. The sequence of the fusion leader sequence consists of the first 19 amino acids of the human serum albumin (SEQ ID NO: 3) signal peptide and the last 5 amino acids of the mating factor α1 promoter (SLDKR, see EP-A-387319, which is incorporated in its entirety This article is used as a reference).

Plasmids pPPC0005, pScCHSA, pScNHSA and pC4:Has were deposited in the American Type Culture Collection on April 11, 2001, 10801 University Boulevard, Manassas, Virginia 20110-2209, and were given the deposit numbers ATCC PTA-3278, PTA- 3276, PTA-3279 and PTA-3277. Another vector that can be used to express albumin fusion proteins in yeast, the pSAC35 vector, is described in Sleep et al., BioTechnology 8:42, 1990, which is incorporated herein by reference in its entirety.

A yeast promoter that can be used to express albumin fusion proteins is the MET25 promoter. See, eg, Dominik Mumburg, Rolf Muller and Martin Funk, Nucleic Acids Research 22(25):5767-5768, 1994. The length of the Met25 promoter is 383 bases (base -382 to -1), and the genes expressed through this promoter are also called Met15, Met17 and YLR303W. A preferred embodiment uses a sequence in which the Not I site for cloning at the 5' end is underlined and the ATG start codon at the 3' end is underlined:

GCGGCCGC CGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTT

GCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTG

AAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAA

GTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAA

GTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAA

AATAAGATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTT

TTCCTTTTTCTTGCTCTCTTGTCTTTTTCATCTACTATTTTCCTTCGTGTAAT

ACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACA A

TG (SEQ ID NO: 5)

Other promoters that can be used to express albumin fusion proteins in yeast include the following:

a) cbh1 promoter:

TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTC

GCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGAT

GTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCT

ATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCT

TCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCG

AAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATAT

AATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGT

ACTGAGCTTGGACATAACTGTTCCGTACCCCACCTTTCTCAACCT

TTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTA

TTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAAT

AATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTG

TTCGAAGCCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAG

GCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCA

CGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAA

AGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAA

GTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAA

TTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTG

TGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGT

TTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCG

CTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTC

TGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGAC

AGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGA

GTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTA

TAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCAC

TGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCC

CTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAA

TGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCA

GCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT

TTCGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAG

ATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAAT

GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCC

ATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTT

TTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC

(SEQ ID NO: 113)

b) cysD promoter of Aspergillus nidulans:

AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTC

TTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCC

TATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATT

CACGCAATTACGATAGTATTTGGCCTTTTTTCGTTTGGCCCAGGTCA

ATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGTTGTG

CTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTG

GGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAAGAGAGGA

TTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC

(SEQ ID NO: 114)

c) a modified cbh1 promoter having the sequence:

TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTC

GCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGAT

GTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCT

ATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCT

TCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCG

AAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATAT

AATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGT

ACTGAGCTTGGACATAACTGTTCCGTACCCCACCTTTCTCAACCT

TTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTA

TTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAAT

AATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTG

TTCGAAGCCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAG

GCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCA

CGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAA

AGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAA

GTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAA

TTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTG

TGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGT

TTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCG

CTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTC

TGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGAC

AGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGA

GTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTA

TAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCAC

TGAACAGGCAAAAG ATTGAGTTGAAACTGCCTAAGATCTCGGGCC

CTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAA

TGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCA

GCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT

TTCGAATAGAAAGAGAAGCTTAGCCTGCAGCCCTCTTATCGAGAAA

GAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTG

AAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCA

GCTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAGAAC

AATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGC

AAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCC

CTCATGCTTCTCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC

TTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCG

GACTGGCATC (SEQ ID NO: 115)

d) the cysD promoter of Aspergillus nidulans, having the sequence:

AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTC

TTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCC

TATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATT

CACGCAATTACGATAGTATTTGGCCTTTTTTCGTTTGGCCCAGGTCA

ATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGCTGCA

GCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCA

AAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGT

CTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCT

ACGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCT

CCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTATTTCAAGTT

GTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATA

TCAAAGCCATC (SEQ ID NO: 116)

Various methods have been developed for operably linking DNA to vectors via complementary cohesive ends. For example, complementary homopolymer bundles can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segments are then joined by hydrogen bonding between complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide another means of joining DNA segments and vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or Escherichia coli DNA polymerase I, which, with their 3′-5′ exonucleolytic activity, eliminate the protruding 5′ singlet. chain end, and fill the recessed 3' end with its polymerization activity.

Thus, the combination of these activities produces a segment of DNA that is filled in at the ends. The blunt-ended segments are then incubated with a large molar excess of adapter molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a DNA segment bearing a polylinker sequence at its terminus. These DNA segments are then cleaved with an appropriate restriction enzyme and ligated with an expression vector that has been cleaved with an enzyme that produces ends compatible with the ends of the DNA segment.

Synthetic adapters containing sites for various restriction endonucleases are commercially available from a number of sources, including International Biotechnologies Inc, New Haven, CT, USA.

An ideal method for modifying DNA according to the invention, if for example HA variants are to be prepared, is to use the polymerase chain reaction disclosed in Saiki et al., Science 239:487-491, 1988. In this method, the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers, which themselves incorporate into the amplified DNA. Specific primers may contain restriction endonuclease recognition sites and may be used for cloning into expression vectors by methods known in the art.

Exemplary genera of yeast envisioned for use in the practice of the invention as hosts for expressing albumin fusion proteins are Pichia (Hansenula), Saccharomyces, Kluyveromyces, ), Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen ), Debaryomyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus ), Endomycopsis and the like. A preferred genus is a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, and Torulaspora. Examples of Saccharomyces are S. cerevisiae, S. italicus and S. rouxii.

Examples of Kluyveromyces species are K. fragilis, K. lactis and K. marxianus. A suitable Torula yeast species is T. delbrueckii. Examples of Pichia pastoris (Hansenula) are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and Pichia pastoris (P. pastoris). Methods for S. cerevisiae transformation are generally taught in EP251744, EP258067 and WO90/01063, all incorporated herein by reference.

Preferred exemplary species of Saccharomyces cerevisiae include S. cerevisiae, S. italicus, S. diastaticus and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. fragilis and K. lactis. Preferred exemplary species of Hansenula include Hpolymorpha (now P. angusta), H. anomala (now P. anomala), and Pichia crushed Yeast (Pichia capsulata). Additional preferred exemplary species of Pichia include P. pastoris. Preferred exemplary species of Aspergillus include A. niger and A. nidulans. Preferred exemplary species of Yarrowia include Y. lipolytica. A number of preferred yeast species are available from the ATCC. For example, the following preferred yeast species are available from ATCC and can be used to express albumin fusion proteins: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743yap3 mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743hsp150 mutant (ATCC Accession No. 4021266 )；Saccharomyces cerevisiae Hansen，teleomorph株BY4743pmt1突变体(ATCC登录号4023792)；Saccharomycescerevisiae Hansen，teleomorph(ATCC登录号20626；44773；44774和62995)；Saccharomyces diastaticus Andrews et Gilliland ex van der Walt，teleomorph(ATCC登录号62987); Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph (ATCC Accession No. 76492); Pichia angusta (Teunisson et al.) Kurtzman, teleomorph deposited as Hansenula polymorpha de Morais et Maia, teleomorph (ATCC Accession No. 26012); Aspergillus van Tieghem, anamorph (ATCC Accession No. 9029); Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 16404); Aspergillus nidulans (Eidam) Winter, anamorph (ATCC Accession No. 48756); and Yarrowia lipolytica (Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC Accession No. 201847).

Promoters suitable for Saccharomyces cerevisiae include genes associated with PGK1, GAL1 or GAL10, CYC1, PHO5, TRP1, ADH1, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, Triose phosphate isomerase, phosphoglucose isomerase, glucokinase genes, α-mating factor pheromone, promoter related to (a mating factor pheromone), PRB1 promoter, GUT2 promoter, GPD1 promoter , and hybrid promoters involving a hybrid of part of the 5' regulatory region with part of the 5' regulatory region of another promoter or with an upstream activation site (for example the promoter of EP-A-258067).

A conveniently regulated promoter for Schizosaccharomyces pombe is the thiamine-repressible promoter from the nmt gene as described by Maundrell, J. Biol. Chem. 265:10857-10864, 1990, and Glucose represses the jbp1 gene promoter as described by Hoffman and Winston, Genetics 124:807-816, 1990.

Methods for transforming Pichia pastoris to express foreign genes are taught, for example, by Cregg et al., 1993, and in various Philips patents (e.g., US4857467, incorporated herein by reference), while Pichia expression kits are commercially available from Invitrogen BV, Leek, Netherlands and Invitrogen Corp., San Diego, California. Suitable promoters include AOX1 and AOX2. Gleeson et al., J.Gen.Microbiol.132:3459-3465, 1986 include information about Hansenula vectors and transformation, suitable promoters are MOX1 and FMD1; and EP 361991, Fleer et al., 1991 and from Rhone- Other publications by Poulenc Rorer teach how to express foreign proteins in Kluyveromycess pp. A suitable promoter is PGK1.

The transcription termination signal is preferably the 3' flanking sequence of a eukaryotic gene, which contains the correct signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may be, for example, those in the gene which are naturally linked to the expression control sequence used, ie correspond to the promoter. Alternatively, they may be different, in which case the termination signal of the S. cerevisiae ADH1 gene is preferred.

Desired albumin fusion proteins can be initially expressed using a secretion leader sequence, which can be any leader sequence effective in the yeast of choice. Useful leader sequences in yeast include any of the following:

a) MPIF-1 signal sequence (eg, amino acids 1-21 of GenBank accession number AAB51134), MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 6)

b) Calponin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 7)

c) the pre-pro region of the HAS signal sequence (eg MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO: 8)

d) the pre region of the HAS signal sequence (e.g. MKWVTFISLLFLFSSAYS, SEQ ID NO: 9) or a variant thereof, such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO: 10)

e) invertase signal sequence (e.g. MLLQAFFLFLLAGFAAKISA, SEQ ID NO: 11)

f) Yeast mating factor alpha signal sequence (eg MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO: 12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR) ID: 1

g) Kluyveromyces lactis (K.Lactis) killing toxin leader sequence

h) a hybrid signal sequence (eg MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO: 13)

i) HSA/MFa-1 hybrid signal sequence (also known as HSA/kex2) (eg MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO: 14)

j) K. lactis killer/MFα-1 fusion leader sequence (eg MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 15)

k) Immunoglobulin Ig signal sequence (eg MGWSCIILFLVATATGVHS, SEQ ID NO: 16)

l) Fibrin B precursor signal sequence (e.g. MERAAPSRRVPPLLLLLGGLALLAAGVDA, SEQ ID NO: 17)

m) Clusterin precursor signal sequence (e.g. MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 18)

n) IGF-binding protein 4 signal sequence (e.g. MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 19)

o) variants of the pre-pro region of the HAS signal sequence, e.g.

MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO: 20),

MKWVTFISLLFLFAGVLG (SEQ ID NO: 21),

MKWVTFISLLFLFSGVLG (SEQ ID NO: 22),

MKWVTFISLLFLFGGVLG (SEQ ID NO: 23),

Modified HAS leader HSA#64-MKWVTFISLLFLFAGVSG (SEQ ID NO: 24);

Modified HAS leader HSA#66-MKWVTFISLLFLFGGVSG (SEQ ID NO: 25);

Modified HAS(A14) leader-MKWVTFISLLFLFAGVSG (SEQ ID NO: 26);

Modified HAS(S14) leader (also known as modified HAS#65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 27),

Modified HAS (G14) leader - MKWVTFISLLFLFGGVSG (SEQ ID NO: 28), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO: 29)

p) consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO: 30)

q) acid phosphatase (PH05) leader (eg MFKSVVYSILAASLANA, SEQ ID NO: 31)

r) The pre sequence of MFoz-1

s) pre sequence of beta glucanase (BGL2)

t) Kill toxin leader

u) The pre sequence of killing toxin

v) Kluyveromyces lactis killing toxin prepro (29 amino acids; 16 pre amino acids and 13 pro amino acids) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO: 32)

w) Saccharification yeast (S.diastaticus) glucoamylase II secretion leader sequence

x) S. carlsbergensis alpha-galactosidase (MEL1) secretion leader sequence

y) Candida glucoamylase leader sequence

z) The hybrid leader disclosed in EP-A-387319 (incorporated herein by reference)

aa) the gp67 signal sequence (in conjunction with the baculovirus expression system) (eg amino acids 1-19 of GenBank accession number AAA72759) or

bb) the natural leader of therapeutic protein X;

cc) Saccharomyces cerevisiae invertase (SUC2) leader, disclosed in JP62-096086 (authorization number 911036516, incorporated herein by reference); or

dd) Anvilase-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO: 33)

ee) Modified TA57 propeptide leader variant #1-

MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR (SEQ ID NO: 34)

ff) Modified TA57 propeptide leader variant #2-

MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESA FATQTNSGGLDVVGLISMAEEGEPKR (SEQ ID NO: 35)

gg) consensus signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 111)

jj) Modified HSA/kex2 signal sequence - MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID NO: 112)

kk) consensus signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 05)

Other Methods for Recombinant and Synthetic Production of Albumin Fusion Proteins

The present invention also relates to vectors containing polynucleotides encoding albumin fusion proteins of the present invention, host cells, and the production of albumin fusion proteins by synthetic and recombinant techniques. A vector can be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral multiplication usually occurs only in complementary host cells.

A polynucleotide encoding an albumin fusion protein of the present invention can be ligated to a vector containing a selectable marker for replication in a host. Typically, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or complexed with a charged lipid. If the vector is a virus, it can be packaged in vitro with an appropriate packaging cell line and transduced into host cells.

The polynucleotide insert should be operably linked to an appropriate promoter, such as bacteriophage lambda PL promoter, E. coli lac, trp, phoA and tac promoters, SV40 early and late promoters, retroviral LTR, and the like. Other suitable promoters are known to those skilled in the art. Sites for transcription initiation and termination, as well as ribosome binding sites for translation in the transcribed region, will also be included in the expression construct. The coding region of the transcript expressed by the construct preferably includes a translation initiation codon at the beginning of the polypeptide to be translated and an appropriate stop codon (UAA, UGA or UAG) at the terminus.

As mentioned above, the expression vector preferably comprises at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin for culture in E. coli or other bacteria or ampicillin resistance. Representative examples of suitable hosts include, but are not limited to: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris pastoris, ATCC Accession No. 201178)); insect cells, such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells, such as CHO, COS, NSO, 293, and Bowes melanoma cells; and plant cells. Suitable media and culture conditions for the above-mentioned host cells are known in the art.

Preferred vectors for bacteria include pQE70, pQE60, and pQE-9, available from QIAGEN; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46, available from Stratagene Cloning Systems; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, available from Pharmacia Biotech. Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, commercially available from Stratagene; and pSVK3, pBPV, pMSG and pSVL, commercially available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (both available from Invitrogen, Carlbad, CA). Other suitable vectors will be apparent to those skilled in the art.

In one embodiment, the polynucleotide encoding the albumin fusion protein of the present invention can be fused with a signal sequence that will direct the protein of the present invention to a specific compartment of a prokaryotic or eukaryotic cell and/or direct the protein of the present invention. Proteins are secreted from prokaryotic or eukaryotic cells. For example, in E. coli it may be desirable to direct the expression of proteins to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) fused to albumin fusion proteins of the invention in order to direct expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, the MBP , ompA signal sequence, the signal sequence of the periplasmic E. coli heat labile enterotoxin B subunit, and the signal sequence of alkaline phosphatase. Some vectors for constructing fusion proteins that will guide protein localization are commercially available, such as pMAL series vectors (especially pMAL-p series) can be purchased from New England Biolabs. In a specific embodiment, the polynucleotide encoding the albumin fusion protein of the present invention can be fused to the pelB pectin lyase signal sequence to improve the efficiency of expression and purification of the polypeptide in Gram-negative bacteria. See US Patent Nos. 5,576,195 and 5,846,818, the contents of which are incorporated herein by reference in their entirety.

In order to guide the secretion of the albumin fusion protein of the present invention in mammalian cells, examples of signal peptides that can be fused thereto include, but are not limited to:

a) MPIF-1 signal sequence (eg, amino acids 1-21 of GenBank accession number AAB51134), MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 6)

b) Calponin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 7)

c) the pre-pro region of the HAS signal sequence (eg MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO: 8)

d) the pre region of the HAS signal sequence (e.g. MKWVTFISLLFLFSSAYS, SEQ ID NO: 9) or a variant thereof, such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO: 10)

e) invertase signal sequence (eg MLLQAFFLFLLAGFAAKISA, SEQ ID NO: 11)

f) Yeast mating factor alpha signal sequence (eg MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO: 12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR) ID: 1

g) Kluyveromyces lactis (K.Lactis) killing toxin leader sequence

h) a hybrid signal sequence (eg MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO: 13)

i) HSA/MFa-1 hybrid signal sequence (also known as HSA/kex2) (eg MKWVSFISLLFLFS SAYRSLDKR, SEQ ID NO: 14)

j) K. lactis killer/MFα-1 fusion leader sequence (eg MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 15)

k) Immunoglobulin Ig signal sequence (eg MGWSCIILFLVATATGVHS, SEQ ID NO: 16)

l) Fibrin B precursor signal sequence (e.g. MERAAPSRRVPPLLLLLGGLALLAAGVDA, SEQ ID NO: 17)

m) Clusterin precursor signal sequence (e.g. MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 18)

n) IGF-binding protein 4 signal sequence (e.g. MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 19)

o) variants of the pre-pro region of the HAS signal sequence, e.g.

MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO: 20),

MKWVTFISLLFLFAGVLG (SEQ ID NO: 21),

MKWVTFISLLFLFSGVLG (SEQ ID NO: 22),

MKWVTFISLLFLFGGVLG (SEQ ID NO: 23),

Modified HAS leader HSA#64-MKWVTFISLLFLFAGVSG (SEQ ID NO: 24);

Modified HAS leader HSA#66-MKWVTFISLLFLFGGVSG (SEQ ID NO: 25);

Modified HAS(A14) leader-MKWVTFISLLFLFAGVSG (SEQ ID NO: 26);

Modified HAS(S14) leader (also known as modified HAS#65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 27),

Modified HAS (G14) leader - MKWVTFISLLFLFGGVSG (SEQ ID NO: 28), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO: 29)

p) consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO: 30)

q) acid phosphatase (PH05) leader (eg MFKSVVYSILAASLANA, SEQ ID NO: 31)

r) pre sequence of MFoz-1

s) pre sequence of beta glucanase (BGL2)

t) Kill toxin leader

u) The pre sequence of killing toxin

v) Kluyveromyces lactis killing toxin prepro (29 amino acids; 16 pre amino acids and 13 pro amino acids) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO: 32)

w) Saccharification yeast (S.diastaticus) glucoamylase II secretion leader sequence

x) S. carlsbergensis alpha-galactosidase (MEL1) secretion leader sequence

y) Candida glucoamylase leader sequence

z) The hybrid leader disclosed in EP-A-387319 (incorporated herein by reference)

aa) the gp67 signal sequence (in conjunction with the baculovirus expression system) (eg amino acids 1-19 of GenBank accession number AAA72759) or

bb) the natural leader of therapeutic protein X;

cc) Saccharomyces cerevisiae invertase (SUC2) leader, disclosed in JP62-096086 (authorization number 911036516, incorporated herein by reference); or

dd) Anvilase-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO: 33)

ee) Modified TA57 propeptide leader variant #1-

MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESA FATQTNSGGLDVVGLISMAKR (SEQ ID NO: 34)

ff) Modified TA57 propeptide leader variant #2-

MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAEEGEPKR (SEQ ID NO: 35)

gg) consensus signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 111)

jj) Modified HSA/kex2 signal sequence - MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID NO: 112)

kk) consensus signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 105)

In a preferred embodiment, a modified HAS/kex2 signal sequence (SEQ ID NO: 112) is fused to the amino terminus of an albumin fusion protein, including fusion proteins comprising albumin and a Therapeutic protein described herein, and WO93 /15199, WO97/24445, WO03/60071, WO03/59934, and PCT/US04/01369, each of which is incorporated herein by reference in its entirety. The modified HSA/kex2 signal sequence is based on the HSA/kex2 signal sequence (SEQ ID NO: 14) disclosed, for example, in Sleep et al., Biotechnology 8:42-46, 1990; and U.S. Pat. No. 5,302,697, combining the two This article is incorporated by reference in its entirety. The modified HSA/kex2 leader sequence disclosed herein contains a non-conservative amino acid substitution (Arg to Gly) at residue 19 of the parental signal peptide. It was found that the modified HSA/kex2 signal sequence resulted in unexpectedly better expression yields and/or better cleavage efficiencies of albumin fusion proteins compared to unmodified HSA/kex2 signal sequences when expressed in yeast. The invention also encompasses variants of the modified HSA/kex2 signal peptide. Specifically, the 19th Gly residue of SEQ ID NO: 112 can be replaced by a Pro residue. Other conservative substitution variants of the modified HSA/kex2 signal sequence are also contemplated. The invention also encompasses nucleic acids encoding the modified HSA/kex2 signal sequence of SEQ ID NO: 112 and conservative substitution variants thereof.

Vectors utilizing glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase-based vectors is that glutamine synthase-negative cell lines (eg, murine myeloma cell lines, NSO) are readily available. Glutamine synthase expression systems can also be functional in glutamine synthase expressing cells, such as Chinese hamster ovary (CHO) cells, by providing additional inhibitors that prevent endogenous genes from functioning. The glutamine synthase expression system and its components are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are incorporated herein by reference in their entirety. Alternatively, glutamine synthase expression vectors can be purchased from Lonza Biologics (Portsmouth, NH). Expression and production of monoclonal antibodies in murine myeloma cells using the GS expression system See Bebbington et al., Bio/technology 10:169, 1992; and Biblia and Robinson, Biotechnol.Prog.11:1, 1995, incorporated herein as refer to.

The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses cells containing the vector constructs operably linked to one or more heterologous control regions (such as promoters and/or enhancers) by techniques known in the art. The host cell of the nucleotide sequence of the present invention. The host cell may be a higher eukaryotic cell, such as a mammalian cell (eg, a human-derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. A host strain can be chosen that is capable of regulating the expression of the inserted gene sequence, or modifying and processing the gene product in the specific pattern desired. The presence of certain inducers can increase expression from certain promoters; thus the expression of genetically engineered polypeptides can be controlled. In addition, different host cells have characteristic and specific mechanisms for protein translation, post-translational processing and modification (eg, phosphorylation, cleavage). Selection of an appropriate cell line ensures that the expressed foreign protein is subject to desired modification and processing.

The nucleic acid and nucleic acid constructs of the present invention can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, transfection, or other method to achieve. These methods are described in many standard laboratory manuals, such as Davis et al., "Basic Methods In Molecular Biology", 1986. It is specifically contemplated that the polypeptides of the invention may in fact be expressed by host cells lacking recombinant vectors. In addition to encompassing host cells containing the vector constructs described herein, the invention also encompasses host cells that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Therapeutic protein can be replaced with an albumin fusion protein corresponding to the Therapeutic protein). ) and/or primary cells, secondary cells, and immortalized cells. Genetic material operably linked to an endogenous polynucleotide activates, alters, and/or amplifies the endogenous polynucleotide.

Alternatively, heterologous polynucleotides (e.g., polynucleotides encoding albumin proteins or fragments or variants thereof) and/or heterologous control regions (e.g., promoters and/or enhancer) is operably linked to an endogenous polynucleotide encoding a Therapeutic protein (see, e.g., U.S. Patent No. 5,641,670 issued June 24, 1997; International Publication No. WO96/29411; International Publication No. WO94/12650; Koller et al Sci USA 86:8932-8935, 1989; and Zijlstra et al., Nature, 342:435-438, 1989, the disclosure of each of which is incorporated herein by reference in its entirety).

Albumin fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, Affinity chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, and lectin chromatography. Most preferably, purification is performed using high performance liquid chromatography ("HPLC").

In a preferred embodiment, the albumin fusion protein of the present invention is purified by anion exchange chromatography, including but not limited to Q-Sepharose, DEAE Sepharose, poros HQ, porosDEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/ Chromatography was performed on Source Q and DEAE, Fractogel Q and DEAE columns.

In specific embodiments, the albumin fusion protein of the present invention is purified by cation exchange chromatography, including but not limited to SP-Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns and their equivalents and comparables.

In specific embodiments, albumin fusion proteins of the present invention are purified using hydrophobic interaction chromatography, including but not limited to phenyl, butyl, methyl, octyl, hexyl-Sepharose, poros-phenyl, butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl, Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables.

In a specific embodiment, the albumin fusion protein of the present invention is purified by size exclusion chromatography, including but not limited to Sepharose S100, S200, S300, superdex resin columns and their equivalents and comparables.

In specific embodiments, albumin fusion proteins of the invention are purified using affinity chromatography, including but not limited to dye-like affinity columns, peptide affinity columns, and antibodies that are selective for HAS or "fusion target" molecules affinity column.

In a preferred embodiment, the albumin fusion protein of the present invention is purified by one or more of the chromatographic methods described above. In other preferred embodiments, the albumin fusion protein of the present invention is purified with one or more of the following chromatographic columns: Q Sepharose FF column, SP Sepharose FF column, QSepharose high-efficiency column, Blue Sepharose FF column, Blue column, Phenyl Sepharose FF, DEAE Sepharose FF, or Methyl.

In addition, albumin fusion proteins of the present invention can be purified using the methods described in PCT International Publication WO 00/44772, which is incorporated herein by reference in its entirety. Those skilled in the art can readily adapt the methods described therein for the purification of albumin fusion proteins of the invention.

Albumin fusion proteins of the invention can be recovered from the products of chemical synthesis procedures and from products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending on the host employed in the recombinant production scheme, the polypeptides of the invention may be glycosylated or unglycosylated. Additionally, albumin fusion proteins of the invention may also include initially modified methionine residues in some cases as a result of host-mediated processing. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon is generally efficiently eliminated post-translationally from any protein in all eukaryotic cells. Although the N-terminal methionine of most proteins is also efficiently cleared in most prokaryotic cells, for some proteins this prokaryotic clearance process is ineffective, depending on the properties of amino acids.

In one embodiment, Pichia pastoris is used to express albumin fusion proteins of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast that can metabolize methanol as a sole carbon source. The main step in the methanol metabolic pathway is the oxidation of methanol to formaldehyde by O ₂ . This reaction is catalyzed by alcohol oxidase. To metabolize methanol as the sole carbon source, Pichia pastoris must produce high levels of alcohol oxidase, partly due to the relatively low affinity of alcohol oxidase for _O. Consequently, the promoter region of one of the two alcohol oxidase genes (AOX1) was highly active in a growth medium that relied on methanol as the primary carbon source. In the presence of methanol, the alcohol oxidase produced from the AOX1 gene accounts for about 30% of the total soluble protein of Pichia pastoris. See Ellis, SB et al., Mol. Cell. Biol. 5:1111-21, 1985; Koutz, PJ et al., Yeast 5:167-77, 1989; Tschopp, JF et al., Nucl. Acids Res. 15:3859 -76, 1987. Thus, heterologous coding sequences, such as eg polynucleotides of the invention, are expressed at particularly high levels in Pichia pastoris cultured in the presence of methanol under the transcriptional regulation of all or part of the AOX1 regulatory sequence.

In one embodiment, the plasmid vector pPIC9K was used in the Pichia system essentially as described in "Pichia Protocols: Methods in Molecular Biology", eds. D.R. Higgins and J. Cregg, The Humana Press, Totowa, NJ, 1998. Expression of DNA encoding albumin fusion proteins of the invention listed herein. This expression plasmid allows the expression and secretion of the polypeptides of the invention through the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (ie leader) located upstream of the multiple cloning site.

Those skilled in the art will readily appreciate that many other yeast vectors can be used in place of pPIC9K, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL- S1, pPIC3.5K, and PAO815, as long as the proposed expression constructs provide appropriately positioned transcription, translation, secretion (if desired), etc. signals, including the desired AUG in the same reading frame.

In another embodiment, high level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the invention can be achieved by cloning a heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha and achieved by growing yeast cultures in the absence of methanol.

In addition, the albumin fusion protein of the present invention can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.; and Hunkapiller et al., Nature, 310: 105-111 , 1984). For example, polypeptides corresponding to fragments of polypeptides can be synthesized using a polypeptide synthesizer. In addition, non-canonical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions, if desired. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline acid, homocitrulline, sulfoalanine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluorinated amino acids, tagged amino acids such as b -Methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and general amino acid analogs. In addition, amino acids may be in the D-form (dextrorotatory) or L-form (left-handed).

The invention encompasses albumin fusion proteins of the invention that undergo various modifications during or after translation, such as glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and Linkage of antibody molecules or other cellular ligands, etc. Any of numerous chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, _NaBH4 ; acetylation, formylation, oxidation, reduction ; metabolic synthesis in the presence of tunicamycin; and the like.

Other post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing, attachment of chemical moieties to amino acid backbones, N-linked or O-linked Chemical modification of carbohydrate chains, and addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Albumin fusion proteins can also be modified with detectable labels, such as enzymes, fluoresceins, isotopes, or affinity tags, allowing detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and avidin /biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent substances include Luminol; examples of bioluminescent substances include luciferase, luciferin, and aequorin; and examples of suitable radioactive substances include iodine ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C ), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹¹ In, ¹¹² In, ¹¹³ In, ^115m In), technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y , ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, and ⁹⁷ Ru.

In specific embodiments, albumin fusion proteins of the invention, or fragments thereof, or variants thereof, are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to ¹⁷⁷ Lu, ⁹⁰ Y , ¹⁶⁶ Ho, and ¹⁵³ Sm. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelator ^is111In . In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator ^is90Y . In a specific embodiment, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody or fragment thereof of the invention via a linker molecule. Examples of linker molecules that can be used to conjugate DOTA to polypeptides are generally known in the art, see, e.g., DeNardo et al., Clin. Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug.Chem 10(4):553-7, 1999; and Zimmerman et al., Nucl. Med. Biol. 26(8):943-50, 1999; incorporated herein by reference in their entirety.

As noted above, albumin fusion proteins of the invention may be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. It will be appreciated that in a given polypeptide, the same type of modification may be present to the same or varying degrees at several positions. Polypeptides of the invention may be branched, for example as a result of ubiquitination, and they may also be circular, with or without branches. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes or may result from synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, lipids or lipid Covalent attachment of phosphatidylinositol derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, pyroglutamine Acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, PEGylation, proteolytic processing, phosphorylation, iso Pennylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see e.g. PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd edition, T.E. Creighton, W.H. Freeman and Company, New York, 1993; POST-TRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, edited by B.C. Johnson, Academic Press, New York, pg.1-12, 1983; Seifter et al., Meth.Enzymol.182:626-646 , 1990; Rattan et al., Ann.N.Y.Acad.Sci.663:48-62, 1992)

Albumin fusion proteins and antibodies of the invention that bind a Therapeutic protein or fragment or variant thereof can be fused to a marker sequence, such as a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN Corporation, 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. For example, hexahistidine facilitates the purification of fusion proteins as described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Other peptide tags that can be used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984), and the "flag" tag.

In addition, albumin fusion proteins of the invention may be conjugated to therapeutic moieties, such as cytotoxins, eg cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, eg alpha-emitters, such as eg ²¹³ Bi. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, Doxorubicin, daunorubicin, dihydroxyanthraxedione, mitoxantrone, mithromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine , lidocaine, propranolol, and puromycin and its analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil diazepam), alkylating agents (e.g., dichloromethyldiethyl Ammonium (nitrogen mustard), thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, chain Ureacin, mitomycin C, and cis-dichlorodiamminoplatinum(II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunorubicin) and doxorubicin), Antibiotics (such as dactinomycin (formerly known as actinomycin), bleomycin, mithromycin, and anthranimycin (AMC)) and antimitotic agents (such as vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response and the therapeutic or drug moiety is not considered limited to classical chemotherapeutics. For example, a drug moiety can be a protein or polypeptide with a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet Derived growth factor, tissue plasminogen activator, apoptosis agent such as TNF-α, TNF-β, AIM I (see International Publication No. WO97/33899), AIM II (see International Publication No. WO97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6:1567-1574, 1994), VEGI (see International Publication No. WO99/23105), thrombotic or antiangiogenic agents, such as angiostatin or endostatin; or biological Chemical response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other growth factors. Techniques for conjugating these therapeutic moieties to proteins such as albumin fusion proteins are well known in the art.

Albumin fusion proteins can also be attached to a solid support, which is particularly useful for immunoassays or purification of polypeptides to which, with which, or with which albumin fusion proteins of the invention bind, bind, or associate. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Albumin fusion proteins, with or without conjugation to a therapeutic moiety, can be administered alone or in combination with cytotoxic factors and/or cytokines that are useful as therapeutic agents.

In embodiments where the albumin fusion proteins of the invention comprise only the VH domain of an antibody that binds a Therapeutic protein, it may be necessary and/or desirable to co-express a fusion protein of the VL domain of the same antibody that binds the Therapeutic protein such that all The VH-albumin fusion protein and VL protein are associated post-translationally (covalently or non-covalently).

In embodiments where the albumin fusion protein of the invention comprises only the VL domain of an antibody that binds a Therapeutic protein, it may be necessary and/or desirable to co-express a fusion protein of the VH domain of the same antibody that binds the Therapeutic protein such that all The VL-albumin fusion protein and VH protein are associated post-translationally (covalently or non-covalently).

Some therapeutic antibodies are bispecific, meaning that the antibody that binds the Therapeutic protein is an artificial hybrid antibody that has two different heavy/light chain pairs and two different binding sites. To generate an albumin fusion protein corresponding to the Therapeutic protein, it is possible to create an albumin fusion protein that has scFv fragments fused to both the N- and C-termini of the albumin protein moiety. More specifically, a scFv fused to the N-terminus of albumin would correspond to the heavy chain/light chain (VH/VL) pair of the original antibody that binds the Therapeutic protein, while an scFv fused to the C-terminus of albumin would correspond to the binding Another pair of heavy chain/light chain (VH/VL) of the original antibody to the Therapeutic protein.

The invention also provides chemically modified derivatives of the albumin fusion proteins of the invention that provide additional advantages, such as increased solubility, stability, and circulation time or reduced immunogenicity of the polypeptide (see US Pat. No. 4,179,337). The chemical moiety for the derivative can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. Albumin fusion proteins can be modified at random positions within the molecule or at predetermined positions within the molecule and can contain one, two, three or more attached chemical modules.

The polymers can be of any molecular weight and can be branched or unbranched. For polyethylene glycols, for ease of handling and manufacture, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" means that in polyethylene glycol preparations some molecules may be larger or smaller than the stated molecular weight). Other molecular weights may also be used, depending on the desired therapeutic properties (e.g., desired sustained release time, effect, if any, on biological activity, ease of handling, degree or lack of antigenicity, and the effect of polyethylene glycol on other known effects of therapeutic proteins or analogs). For example, polyethylene glycol can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500、10,000、10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500、20,000、25,000、30,000、35,000、 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

As mentioned above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72, 1996; Vorobjev et al., Nucleosides Nucleotides 18:2745-2750, 1999; and Caliceti et al. Al, Bioconjug. Chem. 10:638-646, 1999, the disclosure of each of which is incorporated herein by reference.

The effect on functional or antigenic regions of the protein should be considered when attaching polyethylene glycol molecules (or other chemical moieties) to proteins. A variety of attachment methods are available to those skilled in the art such as, for example, the method disclosed in EP0401384 (coupling of PEG to G-CSF), incorporated herein by reference; see also Malik et al., Exp.Hematol. 20:1028- 1035, 1992, which reported PEGylation of GM-GSF with tresyl chloride. For example, polyethylene glycol can be covalently attached via amino acid residues through reactive groups such as free amino or carboxyl groups. A reactive group is a group that can bind to an activated polyethylene glycol molecule. Amino acid residues with free amino groups may include lysine residues and N-terminal amino acid residues; amino acids with free carboxyl groups may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Thiol groups can also be used as reactive groups for attachment of polyethylene glycol molecules. Attachment to an amino group, such as to the N-terminus or a lysine group, is preferred for therapeutic purposes.

As noted above, polyethylene glycol can be attached to proteins through linkage to any of a variety of amino acid residues. For example, polyethylene glycol can be attached to the protein through covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries can be used to attach polyethylene glycol to specific amino acid residues (such as lysine, histidine, aspartic acid, glutamic acid, or cysteine) of a protein or to more than one amino acid residue of a protein. A type of amino acid residue (eg, lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof).

It may be particularly desirable to chemically modify the protein at the N-terminus. Taking polyethylene glycol as an example of a composition, various polyethylene glycol molecules (according to molecular weight, branching conditions, etc.), the ratio between polyethylene glycol molecules and protein (polypeptide) molecules in the reaction mixture, The choice is made between the type of PEGylation reaction to be performed, and the method to obtain the selected N-terminal PEGylated protein. A method of obtaining selected N-terminally PEGylated preparations (ie, separating this module from other mono-PEGylated modules if necessary) may be by purifying N-terminally PEGylated species from a population of PEGylated protein molecules. Selective chemical modification of proteins at the N-terminus can be achieved by reductive alkylation, which takes advantage of the differential reactivity of the different types of primary amino groups (lysine versus N-terminus) available for derivatization in a particular protein. Substantially selective derivatization of proteins at the N-terminus is achieved with carboxyl-containing polymers under appropriate reaction conditions.

As noted above, PEGylation of albumin fusion proteins of the invention can be accomplished by a variety of methods. For example, polyethylene glycol can be attached to the albumin fusion protein either directly or through an intermediate linker. A linkerless system for attaching polyethylene glycol to proteins is described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304, 1992; Francis et al., Intern. J. of Hematol.68 : 1-18, 1998; US Patent No. 4,002,531; US Patent No. 5,349,052; WO95/06058; and WO98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without intermediate linkers uses trifluoroethanesulfonated MPEG, which is trifluoroethanesulfonic acid chloride (ClSO ₂ CH ₂ CF ₃ ) It is produced by modifying monomethoxypolyethylene glycol (MPEG). After the protein was reacted with trifluoroethanesulfonated MPEG, polyethylene glycol was attached directly to the amino group of the protein. Thus, the present invention includes protein-polyethylene glycol conjugates produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonic acid group.

Polyethylene glycol can also be attached to proteins using a variety of different intermediate linkers. For example, U.S. Patent No. 5,612,460 discloses urethane linkers for attaching polyethylene glycol to proteins, the entire disclosure of which is incorporated herein by reference. Protein-polyethylene glycol conjugates in which polyethylene glycol is attached to the protein via a linker can also be produced by reacting the protein with a compound such as MPEG-succinimidyl succinate, with 1, 1'-carbonyldiimidazole-activated MPEG, MPEG-2,4,5-trichloropentyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinates derivative. Many other polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO98/32466, the entire disclosure of which is incorporated herein by reference. PEGylated protein products generated using the reaction chemistry described herein are included within the scope of the present invention.

The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention (ie, the degree of substitution) can also vary. For example, a PEGylated protein of the invention can have an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules attached thereto. Similarly, the average degree of substitution ranges from 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11 per protein molecule , 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 polyethylene glycol modules. Methods for determining the degree of substitution are discussed in the literature, see, eg, Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304, 1992.

Polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell culture by standard methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction layers chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Most preferably, purification is performed using high performance liquid chromatography ("HPLC"). When the polypeptide is denatured during isolation and/or purification, well known techniques for protein refolding can be employed to restore the active conformation.

The presence and amount of albumin fusion proteins of the invention can be determined using an immunoassay well known in the art, ie, ELISA. In one ELISA protocol that can be used to detect/quantify the albumin fusion protein of the present invention, the following steps are included: coating the ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, (with One or more different concentrations) are added to a solution containing an albumin fusion protein of the invention, and a secondary antibody specific for the Therapeutic protein conjugated to a detectable label (as described herein or otherwise known in the art) is added , and detect the presence of secondary antibodies. In an alternative to this protocol, an ELISA plate can be coated with an antibody specific to the Therapeutic protein and the labeled secondary antibody can be an antibody specific to human albumin.

Uses of polynucleotides

Each of the polynucleotides identified in the present invention can be used as reagents in a variety of ways. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the invention can be used to generate albumin fusion proteins of the invention. As described in more detail below, polynucleotides of the invention (encoding albumin fusion proteins) can be used in recombinant DNA methods to generate, by genetic engineering, albumin proteins encoded by polynucleotides encoding albumin fusion proteins of the invention. Cells, cell lines or tissues of protein fusion proteins.

The polynucleotides of the invention can also be used in gene therapy. One goal of gene therapy is to correct a genetic defect by inserting a normal gene into an organism with a defective gene. The polynucleotides disclosed in the present invention provide a means of targeting these genetic defects in a highly precise manner. Another purpose is to insert new genes that are not present in the host genome, thus giving the host cell new characteristics. Other non-limiting examples of gene therapy contemplated by the invention are described in more detail elsewhere herein (see, eg, the section entitled "Gene Therapy", and Examples 61 and 62).

Uses of peptides

Each of the polypeptides identified in the present invention can be used in a variety of ways. The following description should be considered exemplary and utilizes known techniques.

Albumin fusion proteins of the invention can be used to provide immunological probes for differential identification of tissues (e.g., immunohistochemical assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29 : 577-580, 1981) or cell type (eg immunocytochemical assay).

Albumin fusion proteins can be used to determine polypeptide levels in biological samples using classical immunohistological methods known to those skilled in the art (see, e.g., Jalkanen et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen et al. , J. Cell. Biol. 105:3087-3096, 1987). Other methods that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable assay labels are known in the art and include enzyme labels such as glucose oxidase; radioactive isotopes such as iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^115m In, ¹¹³ In, ¹¹² In, ¹¹¹ In), technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), Molybdenum ( ⁹⁹ Mo), Xenon ( ¹³³ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, and ⁹⁷ Ru; luminescent labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin.

Albumin fusion proteins of the invention can also be detected in vivo by imaging. Labels or markers for in vivo imaging of proteins include substances detectable by X-ray radiography, nuclear magnetic resonance (NMR) or electron spin relaxation (ESR). For radiography, suitable labels include radioactive isotopes, such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include substances with a detectable characteristic spin, such as deuterium, which can be incorporated into albumin fusion proteins by labeling the nutrients provided to cell lines expressing the albumin fusion proteins of the invention.

Detectable imaging moieties such as radioactive isotopes (e.g. ¹³¹ I, ¹¹² In, ^99m Tc, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^115m In, ^113m In, ¹¹² In, ¹¹¹ In), technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), Molybdenum ( ⁹⁹ Mo), Xenon ( ¹³³ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ^{186 Re, 188 Re} , ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru), radiopaque substances, or substances detectable by nuclear magnetic resonance labeled albumin fusion protein introduced (such as parenteral, subcutaneous, or intraperitoneal) to be checked for immune system disorders animal. As will be understood in the art, the size of the subject and the imaging system used will determine the amount of imaging modules required to produce a diagnostic image. In the example of a radioisotope module, for human subjects, the amount of injected radioactivity is typically in the range of about 5 to 20 ^{millicuries99mTc} . The labeled albumin fusion protein will then preferentially be present in vivo with one or more reporter molecules, ligands or substrates (corresponding to the reporter molecule, ligand or substrate used to generate the Therapeutic protein of the albumin fusion protein of the invention ) in sites such as organs, cells, extracellular space, or matrix. Alternatively, where the albumin fusion protein comprises at least a fragment or variant of a therapeutic antibody, the labeled albumin fusion protein will preferentially exist in the body in the same range as the therapeutic antibody (used to generate the albumin fusion protein of the invention). The bound polypeptide/epitope accumulates at a site (eg organ, cell, extracellular space or matrix) of the corresponding polypeptide/epitope. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments", Chapter 13, "Tumor Imaging: The Radiochemical Detection of Cancer", eds. SW Burchiel and BAThodes, Masson Publishing Inc., 1982. Those skilled in the art can readily adapt the protocols described therein for use with albumin fusion proteins of the invention.

In one embodiment, the present invention provides that by administering an albumin fusion protein of the invention (e.g., a polypeptide encoded by a polynucleotide encoding an albumin fusion protein and/or antibody of the invention) associated with a heterologous polypeptide or nucleic acid molecule ) to specifically deliver the albumin fusion protein of the present invention to cells. In one embodiment, the invention provides a method of delivering a therapeutic protein into a target cell. In another embodiment, the invention provides delivery of single-stranded nucleic acid (such as antisense or ribozyme) or double-stranded nucleic acid (such as DNA that can integrate into the genome of the cell or replicate episomally and be transcribed) to a target cell method in .

In another embodiment, the invention provides a method of specifically destroying cells (eg, destroying tumor cells) by administering an albumin fusion protein of the invention associated with a toxin or a cytotoxic prodrug.

"Toxin" refers to one or more compounds that bind to and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or compounds that are not normally present in or on the cell's surface, under specified conditions any molecule or enzyme that causes cell death. Toxins that may be used in accordance with the methods of the invention include, but are not limited to, radioisotopes, compounds known in the art such as, for example, antibodies (or complement-fixing moieties thereof) that bind intrinsic or induced endogenous cytotoxic effector systems, thymidine kinase , endonuclease, α-toxin, ricin, abrin, pseudomonas exotoxin A, diphtheria toxin, saponin, bitter melon, gelonin, pokeweed Viral proteins, alpha-paurine and cholera toxin. "Toxin" also includes cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, such as alpha-emitters, such as, for example, ²¹³ Bi, or other radioisotopes, such as, for example, ¹⁰³ Pd, ¹³³ Xe, ¹³¹ I, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ^{169 Yb, 51 Cr} , ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ Yttrium, ¹¹⁷ Tin, ¹⁸⁶ Rhenium, ¹⁶⁶ Holmium , and ^188rhenium ; luminescent labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin. In a specific embodiment, the invention provides a method of specifically destroying cells (eg, destroying tumor cells) by administering a polypeptide or antibody of the invention associated with the radioactive ^isotope90Y . In another specific embodiment, the invention provides a method of specifically destroying cells (eg, destroying tumor cells) by administering a polypeptide or antibody of the invention associated with the ^{radioisotope111In} . In another specific embodiment, the invention provides methods of specifically destroying cells (eg, destroying tumor cells) by administering a polypeptide or antibody of the invention associated with the radioactive ^isotope131I .

Polypeptides of the invention can be labeled using techniques known in the art. These techniques include, but are not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; incorporated herein by reference).

The albumin fusion protein of the present invention can be used for diagnosis, treatment, prevention and/or prediction of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein below in the section entitled "Biological Activity".

Thus, the present invention provides a method for diagnosing a disorder, comprising (a) measuring the expression level of a certain polypeptide in cells or body fluids of an individual using the albumin fusion protein of the present invention; and (b) comparing the measured expression level of the polypeptide with a standard Polypeptide expression levels are compared, and an increase or decrease in measured polypeptide expression levels relative to a standard expression level is used as an indicator of a disorder. For cancer, the presence of relatively high numbers of transcripts in individual biopsies may indicate a predisposition to disease development or provide a means of detecting disease before actual clinical symptoms appear. A more definitive diagnosis of this type could allow medical staff to take preventive measures earlier or attack treatment to stop the cancer from growing or getting worse.

In addition, the albumin fusion proteins of the invention are useful in the treatment or prevention of diseases or conditions such as, for example, nervous disorders, immune system disorders, muscle disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative Disorders, and/or cancerous diseases and conditions. For example, a polypeptide of the invention can be administered to a patient to replace a missing or reduced polypeptide (eg insulin), to supplement a missing or lowered different polypeptide (eg hemoglobin S to supplement hemoglobin B, SOD, catalase, DNA repair proteins), inhibit the activity of polypeptides (such as oncogenes or tumor suppressor genes), activate the activity of polypeptides (such as binding to receptors), reduce the activity of membrane-bound receptors by competing with membrane-bound receptors for free ligands ( For example, use of soluble TNF receptors in reducing inflammation), or eliciting a desired response (eg, inhibiting blood vessel growth, enhancing immune responses to proliferating cells or tissues).

In particular, albumin fusion proteins comprising at least a fragment or variant of a therapeutic antibody are useful in the treatment of disease (as described above and elsewhere herein). For example, administration of an albumin fusion protein comprising at least a fragment or variant of a therapeutic antibody can bind and/or neutralize a polypeptide to which the therapeutic antibody used to generate the albumin fusion protein specifically binds, and/or reduce the amount of protein used to generate the albumin fusion protein. Overproduction of the polypeptide to which the therapeutic antibody of the protein fusion protein specifically binds. Similarly, administration of an albumin fusion protein comprising at least a fragment or variant of a therapeutic antibody activates a polypeptide to which the therapeutic antibody used to generate the albumin fusion protein specifically binds by binding to a membrane (receptor) bound polypeptide And realize.

At a minimum, albumin fusion proteins of the invention can be used as molecular weight markers on SDS-PAGE gels or in molecular sieve gel filtration columns, using methods well known to those skilled in the art. The albumin fusion proteins of the invention can also be used to generate antibodies, which in turn can be used to measure protein expression of therapeutic proteins, albumin proteins, and/or albumin fusion proteins of the invention from recombinant cells as a tool for assessing host cell transformation or biological method of learning samples. In addition, the albumin fusion proteins of the invention can be used to test the biological activities described herein.

diagnostic assay

The compounds of the present invention are useful in the diagnosis, treatment, prevention and/or prediction of various disorders in mammals, especially humans. Such disorders include, but are not limited to, those in the corresponding rows of Table 1 and herein under the headings "Immune Competence", "Blood-Related Disorders", "Hyperproliferative Disorders", "Kidney Disorders", "Cardiovascular Disorders", "Respiratory Disorders" , "anti-angiogenic activity", "diseases at the cellular level", "wound healing and epithelial cell proliferation", "nervous activity and neurological diseases", "endocrine disorders", "reproductive system disorders", "infectious diseases", "regenerative ", and/or "Gastrointestinal Disorders" for the disorder documented for each Therapeutic protein.

For many disorders, gene expression levels relative to " A "standard" gene expression level is a substantial change (increase or decrease) in expression levels in tissues or body fluids taken from individuals without the disorder. Accordingly, the present invention provides a diagnostic method useful in the diagnosis of a disorder, comprising measuring the expression level of a gene encoding a polypeptide in tissues, cells or body fluids obtained from an individual, and comparing the measured gene expression level with a standard gene expression level In comparison, an increase or decrease in gene expression levels relative to a standard is indicative of a disorder. These diagnostic assays can be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.

The present invention can also be used as a predictive indicator whereby patients showing increased or decreased gene expression will suffer poor clinical outcomes.

"Determining the expression level of a gene encoding a polypeptide" is intended to qualitatively or quantitatively measure or estimate the level of a particular polypeptide of the invention (e.g., a polypeptide corresponding to a therapeutic protein disclosed in Table 1) or mRNA encoding a polypeptide in a first biological sample The level of , either directly (eg, by measuring or estimating absolute levels of protein or mRNA) or relative (eg, by comparing to polypeptide or mRNA levels in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to the polypeptide level or mRNA level of a standard obtained from a second biological sample from an individual without the disorder, or from a second biological sample without the disorder. The average level of the population of individuals with the disorder is determined. Those skilled in the art will appreciate that once a standard polypeptide level or mRNA level is known, it can be repeatedly used as a standard for comparison.

"Biological sample" means any biological sample taken from an individual, cell line, tissue culture, or other source that contains a polypeptide (including portions thereof) or mRNA of the invention. As noted above, biological samples include bodily fluids (such as serum, plasma, urine, semen, synovial fluid, and spinal fluid) and tissue sources found to express polypeptides or mRNAs in full length or fragments thereof. Methods for obtaining biopsies and body fluids from mammals are well known in the art. When the biological sample includes mRNA, biopsy tissue is the preferred source.

Total cellular RNA can be isolated from biological samples by any suitable technique, such as the one-step guanidinium thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159, 1987 . The level of mRNA encoding a polypeptide of the invention can then be determined using any suitable method. These include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription coupled polymerase chain reaction (RT-PCR), and reverse transcription coupled ligase chain reaction (RT-LCR).

The invention also relates to diagnostic methods, such as quantitative methods, for detecting the level of a polypeptide bound to, bound by, or associated with an albumin fusion protein of the invention in a biological sample (e.g., cells and tissues). and diagnostic assays, including the determination of normal and abnormal levels of polypeptides. Thus, for example, a diagnostic assay according to the invention for detecting abnormal levels of a polypeptide bound to, bound by, or associated with an albumin fusion protein relative to a normal control tissue sample can be used to detect the presence of tumors. exist. Assay techniques that can be used to determine the level of a polypeptide bound to, bound by, or associated with an albumin fusion protein of the invention in a sample derived from a host are well known to those skilled in the art. Such assays include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. The level of polypeptide in a biological sample can be determined by any method known in the art.

A variety of techniques can be used to determine polypeptide levels in biological samples. For example, classical immunohistological methods can be used (Jalkanen et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987) to study peptide expression in tissues. Other methods that can be used to detect polypeptide gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase, and radioactive isotopes, such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In) and technetium ( ^99m Tc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

Tissues or cell types to be analyzed generally include those known or suspected to express the gene of interest (such as, for example, carcinoma). The protein isolation method adopted in the present invention can be, for example, such as in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Where noted, it is incorporated herein by reference in its entirety. Isolated cells can be derived from cell culture or from a patient. Analysis of cells from culture may be an essential step in evaluating cells that can be used as part of cell-based gene therapy techniques or testing the effect of compounds on gene expression.

For example, albumin fusion proteins can be used to quantitatively or qualitatively detect the presence of a polypeptide bound to, bound by, or associated with an albumin fusion protein of the invention. This can be achieved, for example, by immunofluorescence techniques using fluorescently labeled albumin fusion proteins coupled to detection by light microscopy, flow cytometry, or fluorometric assays.

In a preferred embodiment, at least a serum protein comprising a fragment or variant of an antibody that specifically binds to at least one therapeutic protein disclosed in the present invention or otherwise known in the art (such as a therapeutic protein disclosed in Table 1) Protein fusion proteins can be used to quantitatively or qualitatively detect the presence of gene products or conservative variants or peptide fragments thereof. This can be achieved, for example, by immunofluorescence techniques, which employ fluorescently labeled antibodies coupled to detection by light microscopy, flow cytometry, or fluorometric assays.

The albumin fusion proteins of the invention can also be used histologically as in immunofluorescence, immunoelectron microscopy or non-immunological assays for in situ detection of proteins bound to or by albumin fusion proteins of the invention. , or a polypeptide associated therewith. In situ detection can be achieved by removing a histological specimen from the patient and applying thereto a labeled antibody or polypeptide of the invention. The albumin fusion protein is preferably applied by overlaying the labeled albumin fusion protein on the biological sample. By such a procedure it is possible not only to determine the presence of the polypeptide bound to, by or associated with the albumin fusion protein, but also its distribution in the examined tissue. Using the present invention, those skilled in the art will readily appreciate that any of a wide variety of histological methods, such as staining procedures, can be modified to achieve in situ detection.

Immunoassays and non-immunoassays for the detection of polypeptides bound to, bound by, or associated with albumin fusion proteins typically involve taking samples, such as biological fluids, tissue extracts, freshly harvested cells, or Lysates of cells that have been incubated in cell culture, incubated in the presence of detectably labeled antibodies capable of binding the gene product or conservative variants or peptide fragments thereof, and treated by any of a variety of techniques well known in the art to detect bound antibodies.

The biological sample can be contacted with and immobilized on a solid support or carrier, such as nitrocellulose or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support can then be washed with a suitable buffer and subsequently treated with a detectably labeled albumin fusion protein of the invention. The solid support can then be washed again with buffer to remove unbound antibody or polypeptide. Antibodies are optionally subsequently labeled. The amount of bound label on the solid support can then be detected by conventional methods.

"Solid support or carrier" means any support capable of binding a polypeptide (e.g., an albumin fusion protein, or a polypeptide bound to, bound by, or associated with an albumin fusion protein of the invention). Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. For the purposes of the present invention, the nature of the carrier can be either soluble or insoluble to some extent. The support can have virtually any possible structural configuration as long as the conjugated molecule is capable of binding the polypeptide. Thus, the support may be spherical in shape, such as a bead, or cylindrical, such as the inner wall of a test tube, or the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet, test strip, or the like. Preferred supports include polystyrene beads. Those skilled in the art will also know of many other carriers suitable for binding the antibody or antigen, or will be able to determine a suitable carrier by routine experimentation.

The binding activity of a given batch of various albumin fusion proteins can be determined according to well known methods. Those skilled in the art will be able to determine the operation and optimal assay conditions for each assay by routine experimentation.

In addition to determining the level of a polypeptide in a biological sample taken from an individual, the polypeptide can be detected in vivo by imaging. For example, in one embodiment of the invention, an albumin fusion protein of the invention is used to image diseased or neoplastic cells.

Markers or markers for in vivo imaging of albumin fusion proteins of the present invention include substances detectable by X-ray radiography, NMR, MRI, CAT-scan or ESR. For radiography, suitable labels include radioactive isotopes, such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include species with a detectable characteristic spin, such as deuterium, which can be incorporated into albumin fusion proteins by labeling nutrients of engineered cell lines (or bacterial or yeast strains).

Additionally, an albumin fusion protein of the invention whose presence is detectable can be administered. For example, an albumin fusion protein of the invention labeled with a radiopaque compound or other suitable compound can be administered and imaged in vivo, as discussed above for labeled antibodies. Additionally, such polypeptides may find use in in vitro diagnostic procedures.

Introducing ^{( e.g.} , ^parenteral , subcutaneous or intraperitoneal) in mammals whose disorder is to be examined. As will be understood in the art, the size of the subject and the imaging system used will determine the amount of imaging modules required to produce a diagnostic image. In the example of a radioisotope module, for human subjects, the amount of injected radioisotope is typically in the range of about 5 to 20 ^mCi99mTc . The labeled albumin fusion protein will then preferentially accumulate in vivo at sites containing polypeptides or other substances bound to, bound by, or associated with the albumin fusion protein of the invention. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments", Chapter 13, "Tumor Imaging: The Radiochemical Detection of Cancer", eds. SW Burchiel and BARhodes, Masson Publishing Inc., 1982.

One method of detectably labeling an albumin fusion protein of the invention is to link it to a reporter enzyme and use the ligated product in an enzyme immunoassay (EIA) (Voller, A., "The enzymeLinked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J.Clin.Pathol.31:507-520, 1978; Butler, J.E., Meth.Enzymol.73: 482-523, 1981; Maggio, E. ed., 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E. et al., eds., 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo. The reporter enzyme bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a way as to produce a detectable chemical moiety, for example by spectrophotometric, fluorometric or visual means. Reporter enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate Isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase enzymes, glucoamylase and acetylcholinesterase. Alternatively, detection can be performed by colorimetric methods using a chromogenic substrate for the reporter enzyme. It can also be detected by visual comparison of the extent of enzymatic reaction of the substrate with similarly prepared standards.

Albumin fusion proteins can also be radiolabeled and used in a variety of other immunoassays. For example, it is possible to use albumin fusion proteins in radioimmunoassays (RIAs) by radiolabeling them (see e.g. Weintraub, B., Principles of radioimmunoassay, Seventh training Course on Radioligand Assay Techniques, The Endocrine Society, 1986 March 2008, incorporated herein by reference). Radioisotopes can be detected by means including, but not limited to, gamma counters, scintillation counters, or autoradiography.

Additionally, chelating molecules are known in the art and can be used to label albumin fusion proteins. Chelating molecules can be attached to albumin fusion proteins of the invention in order to label the protein with metal ions, including radionuclides or fluorescent labels. See, eg, Subramanian, R. and Meares, C.F., "Bifunctional Chelating Agents for Radiometal-labeled monoclonal Antibodies," in Cancer Imaging with Radiolabeled Antibodies, ed. D.M. Goldenberg, Kluwer Academic Publications, Boston; Saji, H.ive, " of radiolabeled imaging and therapeutic agents: bifunctional radiopharmaceuticals", Crit. Rev. Ther. Drug Carrier Syst. 16: 209-244, 1999; Srivastava, S.C. and Mease, R.C., "Progress in research on ligands, nuclides odib and techniques for monoclonal labeling" , Int.J.Rad.Appl.Instrum.B 18:589-603, 1991; and Liu.S. and Edwards, D.S., "Bifunctional chelators for therapeuticlanthanide radiopharmaceuticals", Bioconjug.Chem.12:7-34, 2001. Any chelating agent that can covalently bind to the albumin fusion can be used in the present invention. The chelating agent may also include a linker module linking the chelating module to the albumin fusion protein.

In one embodiment, albumin fusion proteins of the invention are attached to an acyclic chelating agent, such as diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DPTA), DPTA's analogue substances, and derivatives of DPTA. As a non-limiting example, the chelating agent may be 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (IB4M-DPTA, also known as MX-DTPA), 2-Methyl-6-(ρ-nitrobenzyl)-1,4,7-triazaheptane-N,N,N',N",N"-pentaacetic acid (nitro-IB4M-DTPA or nitro-MX-DTPA); 2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), or N-[2-amino-3- (ρ-nitrophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N',N"-pentaacetic acid (nitro-CHX-A-DTPA).

In another embodiment, albumin fusion proteins of the invention are attached to an acyclic terpyridine chelator, such as 6,6"-bis[[N,N,N",N"-tetrakis(carboxymethyl)amino ]methyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine (TMT-amine).

In a specific embodiment, the macrocyclic chelator attached to the albumin fusion protein of the invention is 1,4,7,10-tetraazacyclododecane-N,N,N,N"'-tetra Acetic acid (DOTA). In other specific embodiments, DOTA is attached to the albumin fusion protein of the present invention via a linker molecule. Examples of linker molecules that can be used to conjugate DOTA to polypeptides are generally known in the art, see For example, DeNardo et al., Clin. Cancer Res. 4(10): 2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4): 553-7, 1999; and Zimmerman et al., Nucl. Med. Biol .26(8):943-50, 1999, which is incorporated herein by reference in its entirety. In addition, U.S. Patent Nos. 5,652,361 and 5,756,065, which disclose chelating agents that can be conjugated to antibodies and methods for their production and use, are incorporated herein in their entirety Incorporated herein by reference. Although US Patents 5,652,361 and 5,756,065 focus on conjugating chelators to antibodies, those skilled in the art can readily adapt the methods disclosed therein to conjugate chelators to other polypeptides.

According to M.Moi et al., J.Amer.Chem.Soc.49:2639,1989 (2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N ', N", N"'-tetraacetic acid); S.V.Deshpande et al., J.Nucl.Med.31:473, 1990; G.Ruser et al., Bioconj.Chem.1:345, 1990; C.J.Broan et al. , J.C.S.Chem.Comm.23:1739,1990; and C.J.Anderson et al., J.Nucl.Med.36:850,1995 described the use of bifunctional chelating agents based on macrocyclic ligands, which are activated by arm or functional The conjugation is achieved by attaching a group to the carbon backbone of the ligand.

In one embodiment, a macrocyclic chelating agent, such as a polyazamacrocyclic chelating agent, optionally containing one or more carboxyl, amino, hydroxamic acid, phosphonic acid, or phosphoric acid groups, is attached to the scavenger of the present invention. protein fusion protein. In another embodiment, the chelating agent is a chelating agent selected from DOTA, DOTA analogs, and DOTA derivatives.

In one embodiment, suitable chelator molecules that can be attached to albumin fusion proteins of the invention include DOXA (1-oxo-4,7,10-triazacyclododecanetriacetic acid), NOTA (1,4, 7-triazacyclononanetriacetic acid), TETA (1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT (4′-(3-amino-4-methoxy-benzene base)-6,6"-bis(N',N'-dicarboxymethyl-N-methylhydrazine)-2,2':6',2"-terpyridine) and its analogues and derivatives. See eg Ohmono et al., J. Med. Chem. 35: 157-162, 1992; Kung et al., J. Nucl. Med. 25: 326-332, 1984; Jurisson et al., Chem. Rev. 93: 1137- 1156, 1993; and US Patent No. 5,367,080. Other suitable chelating agents include those disclosed in US Patent Nos. 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO89/00557;

In another embodiment, suitable macrocyclic carboxylic acid chelating agents useful in the present invention include 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA); 1,4,8,12-tetraazacyclopentadecane-N,N',N",N"'-tetraacetic acid (15N4); 1,4,7-triazacyclononane -N, N', N"-triacetic acid (9N3); 1,5,9-triazacyclododecane-N, N', N"-triacetic acid (12N3); and 6-bromoacetamido- Benzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (BAT).

A preferred chelator that can be attached to an albumin fusion protein of the invention is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1 , 4,7,10-tetraacetic acid, also known as MeO-DOTA-NCS. Salts of α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid can also be used or ester.

Albumin fusion proteins of the invention to which the above-mentioned chelating agents are covalently attached can be labeled (via the coordination site of the chelating agent) with a radionuclide suitable for therapeutic, diagnostic, or both therapeutic and diagnostic purposes. Examples of suitable metals include Ag, At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y, and Yb. Examples of radionuclides for diagnostic purposes are Fe, Gd, ¹¹¹ In, ⁶⁷ Ga or ⁶⁸ Ga. In another embodiment, the radionuclide used for diagnostic purposes ^{is111In or67Ga} . Examples of radionuclides used for therapeutic purposes are ¹⁶⁶ Ho, ¹⁶⁵ Dy, ⁹⁰ Y, ^115m In, ⁵² Fe or ⁷² Ga. In one embodiment, the radionuclide used for diagnostic purposes ^{is166Ho or90Y} . Examples of radionuclides ^for therapeutic and diagnostic purposes include153Sm, ^177Lu , ^159Gd , ^{175Yb or47Sc} . In one embodiment, the radionuclide ^is153Sm , ^177Lu , ^{175Yb or159Gd} .

Preferred metal radionuclides include ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ⁴⁷ Sc, 67 Ga, ⁵¹ Cr, ¹⁷⁷ mSn, ⁶⁷ Cu ^{, 167} Tm, ⁹⁷ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ¹⁷⁷ mSn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁵ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ²⁰³ Pb and ¹⁴¹ Ce.

In a specific embodiment, an albumin fusion protein of the invention to which a chelating agent is covalently attached can be labeled with a metal ion selected ^from90Y , ^111In , ^177Lu , ^166Ho , ^{215Bi and225Ac} .

Additionally, gamma emitting radionuclides such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, and ¹⁶⁹ Yb have been approved for diagnostic imaging or are under investigation, while beta emitters such as ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, and ⁹⁰ Y are available application in tumor therapy. Other useful radionuclides include gamma emitters such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, and ¹⁶⁹ Yb, beta emitters such as ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, ¹⁸⁸ Re, and ⁹⁰ Y, and others of interest. Radionuclides such as ²¹¹ At, ²¹² Bi, ¹⁷⁷ Lu, ⁸⁶ Rb, ¹⁰⁵ Rh, ¹⁵³ Sm, ¹⁹⁸ Au, ¹⁴⁹ Pm, ⁸⁵ Sr, ¹⁴² Pr, ²¹⁴ Pb, ¹⁰⁹ Pd, ¹⁶⁶ Ho, ²⁰³ Tl and ⁴⁴ Sc. Albumin fusion proteins of the invention to which a chelating agent is covalently attached can be labeled with radionuclides as described above.

In another embodiment, albumin fusion proteins of the invention to which a chelating agent is covalently attached can be labeled with paramagnetic metal ions, including ions of transition metals and lanthanide metals, such as atomic numbers 21-29, 42, 43, 44, or 57-71 metals, especially Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm , Yb and Lu ions. Paramagnetic metals useful in magnetic resonance imaging compositions include elements with atomic numbers 22 to 29, 42, 44, and 58-70.

In another embodiment, albumin fusion proteins of the invention to which a chelator is covalently attached can be labeled with fluorescent metal ions, including lanthanide metals, particularly La, Ce, Pr, Nd, Pm, Sm, Eu (e.g. ¹⁵² Eu), Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

In another embodiment, an albumin fusion protein of the invention to which a chelating agent is covalently attached may be labeled with a heavy metal-containing reporter, which may include Mo, Bi, Si, and W atoms.

It is possible to label albumin fusion proteins with fluorescent compounds. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence is detectable due to fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde (ophthaldehyde) and fluorescamine.

Albumin fusion proteins may also be detectably labeled with fluorescent emitting metals, such ^as152Eu or other lanthanide metals. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Albumin fusion proteins can also be detectably labeled by conjugation with chemiluminescent compounds. The presence of the chemiluminescence-labeled albumin fusion protein can then be confirmed by detecting the luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts and oxalates.

Likewise, bioluminescent compounds can also be used to label the albumin fusion proteins of the invention. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

genetically modified organism

The invention also includes transgenic organisms expressing albumin fusion proteins of the invention. Genetically modified organisms are genetically modified organisms into which recombinant, foreign or cloned genetic material has been transferred. Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene may contain one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, which may be necessary for optimal expression and secretion of the encoded protein. A transgene can be designed to direct the expression of the encoded protein in a manner that facilitates recovery of the protein by the organism or by products produced by the organism, such as the organism's milk, blood, urine, eggs, hair, or seeds. The transgene may consist of a nucleic acid sequence derived from the genome of the same species as the target animal species or a different species. The transgene can integrate into the genome at a locus where the particular nucleic acid sequence is not normally found otherwise or at a normal locus for the transgene.

The term "germline cell line transgenic organism" refers to a transgenic organism in which genetic alterations or genetic information have been introduced into germline cells, thereby conferring on the transgenic organism the ability to pass the genetic information on to offspring. If such offspring actually have some or all of this alteration or genetic information, then they are also genetically modified organisms. The alteration or genetic information may be foreign to the biological species to which the recipient belongs, foreign only to a particular individual recipient, or may be genetic information already possessed by the recipient. In the latter case, the expression of the altered or introduced gene may differ from the native gene.

A transgenic organism can be a transgenic animal or a transgenic plant. Transgenic animals can be produced by a number of different methods, including transfection, electroporation, microinjection, gene targeting in embryonic stem cells, and infection with recombinant viruses and retroviruses (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. People, Hypertension 22(4):630-633, 1993; Brenin et al., Surg.Oncol.6(2):99-110, 1997; Tuan ed., Recombinant Gene Expression Protocols, "Methods in Molecular Biology", No.62 , Humana Press, 1997). The method of introducing nucleic acid fragments into recombinant competent mammalian cells can be any method that facilitates co-transformation of multiple nucleic acid molecules. Detailed procedures for generating transgenic animals, including the methods disclosed in US Patent No. 5,489,743 and US Patent No. 5,602,307, are readily available to those skilled in the art.

A number of recombinant or transgenic mice have been produced, including those expressing an activated oncogene (US Patent No. 4,736,866); those expressing the simian SV40T-antigen (U.S. Patent No. 5,728,915); those that do not express Interferon Regulatory Factor 1 (IRF-1) ( U.S. Patent No. 5,731,490); exhibiting dopaminergic dysfunction (U.S. Patent No. 5,723,719); expressing at least one human gene involved in blood pressure control (U.S. Patent No. 5,731,489); exhibiting association with naturally occurring Alzheimer's disease (US Patent No. 5,720,936); mediates decreased cell adhesion (US Patent No. 5,602,307); possesses the bovine growth hormone gene (Clutter et al., Genetics 143(4): 1753-1760, 1996 ); or, (McCarthy, The Lancet 349(9049):405, 1997) mice capable of generating a fully human antibody response.

While mice and rats are still the choice for most transgenic experiments, in some cases it is preferable or even necessary to use other animal species. The transgenic process has been successfully applied to a variety of non-mouse animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cattle, and guinea pigs (see, e.g., Kim et al., Mol. Reprod. Dev. 46(4 ):515-526,1997; Houdebine, Reprod.Nutr.Dev.35(6):609-617,1995; Petters, Reprod.Fertil.Dev.6(5):643-645,1994; Schnieke et al., Science 278(5346):2130-2133, 1997; and Amoah, J. Animal Science 75(2):578-585, 1997).

To direct secretion of the protein of the invention encoded by the transgene into the milk of the transgenic animal, it can be placed under the control of a promoter which is preferentially activated in mammary epithelial cells. Promoters that preferably control genes encoding milk proteins, such as casein, β-lactoglobulin, orotate, or whey protein (see, e.g., DiTullio, BioTechnology 10:74-77, 1992; Clark et al. , BioTechnology 7:487-492, 1989; Gorton et al., BioTechnology 5:1183-1187, 1987; and Soulier et al., FEBS Letts.297:13, 1992). The transgenic mammals selected will produce large amounts of milk and have a long lactation period, such as goats, cows, camels or sheep.

Albumin fusion proteins of the invention may also be expressed in transgenic plants, eg, plants in which the DNA transgene has been inserted into the nucleus or plastid genome. Plant transformation procedures for introducing foreign nucleic acids into plant cells or protoplasts are known in the art. See for example Methods in Enzymology Vol.153, "Recombinant DNA Part D", 1987, edited by Wu and Grossman, Academic Press and European Patent Application EP693554. Methods for producing genetically engineered plants are also described in US Patent No. 5,283,184, US Patent No. 5,482,852 and European Patent Application EP693554, all of which are incorporated herein by reference.

drug or therapeutic composition

The albumin fusion proteins of the present invention or formulations thereof can be administered by any conventional method, including parenteral (eg, subcutaneous or intramuscular) injection or intravenous infusion. Treatment may consist of a single dose or of multiple doses over a period of time.

Although it is possible to administer the albumin fusion proteins of the invention alone, it is preferably provided in the form of a pharmaceutical formulation together with one or more acceptable carriers. The carrier must be "acceptable" in the sense of being compatible with the albumin fusion protein and not deleterious to its recipient. Typically, the carrier will be sterile and pyrogen-free water or saline. Albumin fusion proteins of the invention are particularly suitable for formulation in aqueous vehicles such as sterile pyrogen-free water, saline or other isotonic solutions due to their extended shelf-life in solution. For example, the pharmaceutical compositions of the invention may be pre-formulated in aqueous form, eg, weeks or months or longer prior to distribution.

For example, formulations containing albumin fusion proteins can be prepared taking into account the increased shelf life of albumin fusion proteins in aqueous formulations. As discussed above, the shelf life of many of these therapeutic proteins was significantly increased or extended after fusion with HA.

Where aerosol administration is suitable, albumin fusion proteins of the invention may be formulated as aerosols using characterization procedures. The term "aerosol" includes any aerosol suspension phase of an albumin fusion protein of the invention capable of inhalation into the bronchioles or nasal passages. In particular, aerosol formulations include an aerosol suspension of droplets of an albumin fusion protein of the invention, which may be generated in a metered dose inhaler or nebulizer or in a nebuliser. Aerosols also include dry powder compositions of a compound of the invention suspended in air or other carrier gas which can be delivered by insufflation, for example, with an inhalation device. See Ganderton and Jones, "Drug Delivery to the Respiratory Tract", Ellis Horwood, 1987; Gonda, Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313, 1990; and Raebum et al., Pharmacol.Toxicol.Methods 27:143- 159, 1992.

Formulations of the invention are also generally non-immunogenic, in part because the components of the albumin fusion protein used are derived from suitable species. For example, when used in humans, both the Therapeutic protein and the albumin portion of the albumin fusion protein are typically human. In some cases where neither of the two components is derived from humans, the component can be humanized by substituting key amino acids such that a particular epitope appears to the human immune system to be human in nature rather than foreign .

The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the albumin fusion protein with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then shaping the product if necessary.

Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation suitable for its intended recipient; and aqueous or non-aqueous sterile suspensions. Liquids may contain suspending and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules, vials, or syringes, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier such as Water is ready for injection. Extemporaneous injections and suspensions can be prepared from sterile powders. Since many albumin fusion proteins of the invention exhibit prolonged serum half-lives, dosage formulations may contain lower molar concentrations or lower doses of the Therapeutic protein moiety than non-fused standard formulations of a given Therapeutic protein.

As an example, when the albumin fusion protein of the present invention comprises one of the proteins listed in the "Therapeutic Protein: X" column of Table I as one or more Therapeutic protein domains, the efficacy of the albumin fusion protein can be compared to the therapeutic protein alone. The dosage form was calculated based on the potency of the therapeutic protein, taking into account the increased serum half-life and shelf life of albumin fusion proteins compared to natural therapeutic proteins. For example, if the therapeutic protein is typically administered at 0.3 to 30.0 IU/kg/week or 0.9 to 12.0 IU/kg/week, then in three or seven doses over a year or more. In an albumin fusion protein consisting of full-length HA fused to a Therapeutic protein, an equivalent dose on a unit would represent a greater weight of agent, but dosing frequency could be reduced to, for example, twice a week, once a week, or more. few.

The formulations or compositions of the invention can be packaged or included in a kit with instructions or a package insert for extending the shelf life of the albumin fusion protein components. For example, such instructions or package inserts may give recommended storage conditions, such as time, temperature and light, taking into account the extended or extended shelf life of the albumin fusion proteins of the invention. Such instructions or package inserts may also give specific advantages to the albumin fusion proteins of the invention, such as ease of storage for formulations that may be required for use outside of field, controlled hospital, clinical or clinic conditions. As noted above, the formulations of the invention can be in aqueous form and can be stored under less than ideal conditions without appreciable loss of therapeutic activity.

Albumin fusion proteins of the invention may also be included in nutraceuticals. For example, certain albumin fusion proteins of the invention can be administered in natural products, including milk or milk products obtained from transgenic mammals expressing albumin fusion proteins. Such compositions may also include plants or plant products obtained from transgenic plants expressing albumin fusion proteins. Albumin fusion proteins may also be provided in powder or tablet form with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, December 1993.

The present invention also provides an effective amount of an albumin fusion protein of the present invention or a polynucleotide encoding an albumin fusion protein of the present invention ("albumin fusion polynucleotide") administered to a subject in a pharmaceutically acceptable carrier. Methods of treating and/or preventing a disease or disorder, such as, for example, any one or more of the diseases or disorders disclosed herein.

Taking into account the individual patient's clinical condition (especially the side effects of treatment with albumin fusion protein and/or polynucleotide alone), delivery site, method of administration, administration regimen, and other factors known to practitioners, according to the best Albumin fusion proteins and/or polynucleotides are formulated and administered in a manner consistent with medical practice. Accordingly, an "effective amount" for use in the present invention will be determined by such considerations.

As a general recommendation, the total pharmaceutically effective amount of parenterally administered albumin fusion protein will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight per dose, although as noted above this will be therapeutically judge. More preferably, this dosage is at least 0.01 mg/kg/day, and most preferably for humans the hormone is between about 0.01 and 1 mg/kg/day. If administered continuously, the albumin fusion protein is typically administered at a dosage rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 daily injections or by continuous subcutaneous infusion, eg, using a minipump. Intravenous bag solutions are also available. The length of treatment required to observe a change and the interval after treatment for response to occur will vary depending on the desired effect.

As noted above, the albumin fusion proteins of the invention have greater plasma stability than the Therapeutic protein moiety (or fragment or variant thereof) alone. This increase in plasma stability should be taken into account when determining the effective amount of albumin fusion protein administered per dose and the dosing regimen. In particular, greater plasma stability may allow administration of albumin fusion proteins at lower doses at the same frequency of administration, or may allow administration of albumin fusion proteins less frequently. Preferably, the higher stability allows for less frequent administration of the albumin fusion proteins of the invention at fewer times. More preferably, the albumin fusion protein can be administered every two weeks. Still more preferably, the albumin fusion protein may be administered every three, four, five or more weeks, depending on the pharmacokinetics of the albumin fusion protein. For example, as discussed above, the pharmacokinetics of IFN-α-HSS fusion proteins support a dosing regimen every 2-4 weeks or longer, and even at intervals of 4 weeks or more.

Albumin fusion proteins and/or polynucleotides can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powder, ointment, gel, drops, or transdermal patch), buccal , or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to any non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or preparation auxiliary material. The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Albumin fusion proteins and/or polynucleotides of the invention are also suitable for administration by sustained release systems. Examples of sustained-release albumin fusion proteins and/or polynucleotides are oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (via powder, ointment, gel, drops, or transdermal patch) , buccal, or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or preparation auxiliary material. The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Other examples of slow-release albumin fusion proteins and/or polynucleotides include suitable polymeric materials (such as, for example, semipermeable polymeric matrices in the form of shaped products, such as films or microcapsules), suitable hydrophobic materials (such as, for example, in acceptable oils). emulsions) or ion exchange resins, and small amounts of soluble derivatives (such as, for example, small amounts of soluble salts).

Sustained-release matrices include polylactide (US Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and L-glutamic acid-γ-ethyl ester (Sidman et al., Biopolymers 22:547-556, 1983 ), poly(2-hydroxyethylmethacrylate) (Langer et al., J.Biomed.Mater.Res.15:167-277, 1981; and Langer, Chem.Tech.12:98-105, 1982) , ethylene vinyl acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyrate (EP133,988).

Slow-release albumin fusion proteins and/or polynucleotides also include liposome-entrapped albumin fusion proteins and/or polynucleotides of the invention (see generally Langer, Science 249:1527-1533, 1990; Treat et al., at In Liposomes in the Therapy of Infectious Disease and Cancer, eds. Lopez-Berestein and Fidler, Liss, New York, pp. 317-327 and 353-365, 1989). Liposomes containing albumin fusion proteins and/or polynucleotides are prepared by methods known per se: DE3,218,121; Epstein et al., Proc.Natl.Acad.Sci.(USA) 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034, 1980; EP52,322; EP36,676; 118008; US Patent Nos. 4,485,045 and 4,544,545; and EP102,324). Typically, liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid component is greater than about 30 mol% cholesterol, and the ratio selected is adjusted for optimal efficacy.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are delivered by a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989).

Other controlled release systems are discussed in the review by Langer, Science 249:1527-1533, 1990.

With regard to parenteral administration, in one embodiment, albumin fusion proteins and/or polynucleotides are generally formulated in unit dosage injectable forms (solutions, suspensions, or emulsions) at the desired degree of purity with pharmaceutically acceptable The receptive carrier is formulated by admixture with a carrier which is nontoxic to recipients at the dosages and concentrations employed and which is compatible with the other ingredients of the formulation. For example, the formulation is preferably free of oxidizing agents and other compounds known to be detrimental to therapeutic efficacy.

Typically, the formulation is prepared by uniformly and intimately contacting the albumin fusion protein and/or polynucleotide with a liquid carrier or a finely divided solid carrier or both. Then, as needed, the product is shaped into the desired dosage form. Preferably, the carrier is a parenteral vehicle, more preferably a solution isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles, such as fixed oils and ethyl oleate, and liposomes can also be employed in the present invention.

The carrier suitably contains minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Such materials are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, succinates, acetic acid, and other organic acids or salts thereof; antioxidants, such as ascorbic acid; low molecular weight ( less than about 10 residues) polypeptides, such as polyarginine or tripeptide; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine acids, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates, including cellulose or its derivatives, glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannose alcohol or sorbitol; a counterion, such as sodium; and/or a nonionic surfactant, such as polysorbate (including, for example, Tween-20), poloxamer, or PEG.

Albumin fusion proteins are typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It is understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any drug used for therapeutic administration can be sterile. Sterility is readily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). The albumin fusion protein and/or polynucleotide is typically placed in a container having a sterile access port, such as an intravenous solution bag or vial with a hypodermic needle-punctureable stopper.

Albumin fusion proteins and/or polynucleotides are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 5 ml of a sterile-filtered 1% (w/v) aqueous solution of albumin fusion protein and/or polynucleotide is filled into a vial, and the resulting mixture is lyophilized. The lyophilized albumin fusion protein and/or polynucleotide was rehydrated with bacteriostatic water for injection to prepare a perfusate.

In a specific and preferred embodiment, the albumin fusion protein formulation comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromolar sodium caprylate/mg fusion protein, 15 micrograms/ml polysorbate 80, pH 7.2. In another specific and preferred embodiment, the albumin fusion protein formulation consists of 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromolar sodium caprylate/mg fusion protein, 15 micrograms/ml polysorbate 80, pH 7.2 . The pH and buffer are chosen to match physiological conditions and salt is added as a tonicifier. Sodium caprylate was chosen for its reported ability to increase the thermal stability of proteins in solution. Finally, polysorbate was added as a general surfactant, which lowers the surface tension of the solution and reduces non-specific adsorption of the albumin fusion protein to the container closure system.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components comprising albumin fusion proteins and/or polynucleotides of the invention. A notice issued by a governmental executive agency regulating the manufacture, use or sale of a drug or biological product, which notice reflects the agency's approval for the manufacture, use or sale of a drug for human administration, may be provided with such a container. In addition, albumin fusion proteins and/or polynucleotides can be used in combination with other therapeutic compounds.

)、MPL和小棒杆菌(Corynebacterium parvum)的非活体制品。在一个具体的实施方案中，本发明的清蛋白融合蛋白和/或多核苷酸与明矾联合施用。在另一个具体的实施方案中，本发明的清蛋白融合蛋白和/或多核苷酸与QS-21联合施用。可与 本发明的清蛋白融合蛋白和/或多核苷酸联合施用的其它佐剂包括但不限于单磷酰脂质免疫调节剂、Adju Vax 100a、QS-21、QS-18、CRL1005、铝盐、MF-59、和病毒体(Virosomal)佐剂技术。可与本发明的清蛋白融合蛋白和/或多核苷酸联合施用的疫苗包括但不限于致力于针对MMR(麻疹、腮腺炎、风疹)、小儿麻痹症(polio)、水痘、破伤风/白喉、甲肝、乙肝、流感嗜血杆菌(Haemophilus influenzae)B、百日咳(whooping cough)、肺炎、流感、莱姆氏病、轮状病毒、霍乱、黄热病、日本脑炎、脊髓灰质炎(poliomyelitis)、狂犬病、伤寒、和百日咳(pertussis)提供保护的疫苗。联合施用既可以是伴随施用，例如作为混合物、分开但同时或并行施用；也可以是顺序施用。这包括其中联合药剂作为治疗性混合物一起施用的呈现，也包括其中联合药剂分开但同时施用的程序，例如经由分开的静脉内线路进入同一个体。“联合”施用还包括这样的分开施用，即先给予化合物或药剂中的一种，然后给予第二种。 The albumin fusion protein and/or polynucleotide of the present invention can be administered alone or in combination with an adjuvant. Adjuvants that can be administered with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc. .), BCG (eg

), MPL and non-living preparations of Corynebacterium parvum. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with alum. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with QS-21. Other adjuvants that can be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, monophosphoryl lipid immunomodulators, Adju Vax 100a, QS-21, QS-18, CRL1005, aluminum salts , MF-59, and Virosomal adjuvant technology. Vaccines that may be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, vaccines directed against MMR (measles, mumps, rubella), polio, varicella, tetanus/diphtheria, Hepatitis A, Hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, Vaccines that provide protection against rabies, typhoid, and pertussis. Joint administration can be either concomitant, for example as a mixture, separate but simultaneously or concurrently; also sequential. This includes presentations where the combined agents are administered together as a therapeutic mixture, as well as procedures where the combined agents are administered separately but simultaneously, for example via separate intravenous lines into the same individual. "Combined" administration also includes separate administration in which one of the compounds or agents is administered first and then the second.

Albumin fusion proteins and/or polynucleotides of the invention can be administered alone or in combination with other therapeutic agents. Other therapeutic protein and/or polynucleotide agents that can be administered in conjunction with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory drugs, conventional immunization Therapeutic agents, and/or therapeutic treatments described below. Joint administration can be either concomitant, for example as a mixture, separate but simultaneously or concurrently; also sequential. This includes presentations where the combined agents are administered together as a therapeutic mixture, as well as procedures where the combined agents are administered separately but simultaneously, for example via separate intravenous lines into the same individual. "Combined" administration also includes separate administration in which one of the compounds or agents is administered first and then the second.

)、双香豆素、4-羟基香豆素、茴茚二酮(例如MIRADON^TM)、醋硝香豆素(acenocoumarol)(例如醋硝香豆素(nicoumalone)，SINTHROME^TM)、1，3-茚二酮、苯丙香豆素(例如MARCUMAR^TM)、双香豆素乙酸乙酯(例如TROMEXA^TM)、及阿斯匹林。在一个具体的实施方案中，本发明的组合物与肝素和/或华法林联合施用。在另一个具体的实施方案中，本发明的组合物与华法林联合施用。在另一个具体的实施方案中，本发明的组合物与华法林和阿斯匹林联合施用。在另一个具体的实施方案中，本发明的组合物与肝素联合施用。在另一个具体的实施 方案中，本发明的组合物与肝素和阿斯匹林联合施用。 In one embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an anticoagulant. Anticoagulants that can be administered in conjunction with the compositions of the present invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g.

), dicoumarol, 4-hydroxycoumarin, anindone (eg MIRADON ^TM ), acenocoumarol (eg acenocoumarol (nicoumalone), SINTHROME ^TM ), 1,3 - indandione, phenprocoumon (eg MARCUMAR ^(TM ), dicoumarin ethyl acetate (eg TROMEXA ^(TM )), and aspirin. In a specific embodiment, the composition of the invention is administered in combination with heparin and/or warfarin. In another specific embodiment, the composition of the invention is administered in combination with warfarin. In another specific embodiment, the composition of the invention is administered in combination with warfarin and aspirin. In another specific embodiment, the composition of the invention is administered in combination with heparin. In another specific embodiment, the composition of the invention is administered in combination with heparin and aspirin.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with thrombolytic agents. Thrombolytic drugs that can be administered with the compositions of the present invention include, but are not limited to, plasminogen, lys-plasminogen, α2-antiplasmin, streptokinase (eg KABIKINASE ^™ ), antiresplace (eg EMINASE ^™ ) , tissue plasminogen activator (t-PA, altevase, ACTIVASE ^TM ), urokinase (eg ABBOKINASE ^TM ), sauruplase, (prourokinase, single-chain urokinase), and aminocaproic acid (eg AMICAR ^TM ) . In a specific embodiment, the composition of the invention is administered in combination with tissue plasminogen activator and aspirin.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiplatelet agents. Antiplatelet drugs that can be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (eg, PERSANTINE ^™ ), and ticlopidine (eg, TICLID ^™ ).

In a specific embodiment, it is contemplated that the prevention, diagnosis and/or treatment of thrombosis, thrombosis, thrombosis, Arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In a specific embodiment, the prevention of occlusion of saphenous vein grafts is envisaged by the combination of anticoagulants, thrombolytics and/or antiplatelet drugs with albumin fusion proteins and/or polynucleotides of the invention. grafts), reduce the risk of perioperative thrombosis that may be associated with angioplasty, reduce the risk of stroke in patients with atrial fibrillation, including nonrheumatic atrial fibrillation, and reduce the risk of embolism associated with prosthetic heart valves and/or mitral valve disease . Other uses of the therapeutic agents of the invention alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic agents include, but are not limited to, the prevention of Obstructions in dialysis units, and cardiopulmonary bypass units).

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are combined with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors ( NNRTI), and/or protease inhibitor (PI) in combination. NRTIs that can be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, RETROVIR ^™ (zidovudine/AZT), VIDEX ^™ (didanosine/ddI), HIVID ^TM (zalcitabine, zalcitabine/ddC), ZERIT ^TM (stavudine, stavudine/d4T), EPIVIR ^TM (lamivudine, lamivudine/3TC), and COMBIVIR ^TM (zidovudine/lamivudine Certainly). NNRTIs that can be administered in combination with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, VIRAMUNE ^™ (nevirapine, nevirapine), RESCRIPTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirenz, efavirenz). Protease inhibitors that can be administered in combination with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, CRIXIVAN ^™ (indinavir, indinavir), NORVIR ^™ (ritonavir, ritonavir), INVIRASE ^™ ( saquinavir), and VIRACEPT ^™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors can be combined with albumin fusion proteins and/or multinuclear Any combination of nucleotides is useful for treating AIDS and/or preventing or treating HIV infection.

(阿德福韦二匹伏酯，阿德福韦的活性原药；其活性形式是PMEA-pp)；TENOFOVIR^TM(bis-POCPMPA，PMPA原药；Gilead)；DAPD/DXG(DAPD的活性代谢物；Triangle/Abbott)；D-D4FC(与3TC相关，具有针对AZT/3TC抗性病毒的活性)；GW420867X(Glaxo Wellcome)；ZIAGEN^TM(阿巴卡韦，abacavir/159U89；Glaxo Wellcome Inc.)；CS-87(3’-叠氮-2’，3’-二脱氧尿苷；WO99/66936)；和β-L-FD4C和β-L-FddC的携带S-酰基-2-乙硫酯(SATE)的原药形式(WO98/17281)。 Other NRTIs include LODENOSINE ^™ (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott); COVIRACIL ^™ (emtricitabine/FTC; structurally related to lamivudine (3TC) but highly active in vitro 3 to 10-fold; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retaining activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adelford Adefovir (FDA declined to approve anti-HIV treatment; Gilead Sciences);

(Adefovir dipivoxil, the active drug of adefovir; its active form is PMEA-pp); TENOFOVIR ^TM (bis-POCPMPA, the original drug of PMPA; Gilead); DAPD/DXG (the active metabolite of DAPD Triangle/Abbott); D-D4FC (related to 3TC with activity against AZT/3TC-resistant viruses); GW420867X (Glaxo Wellcome); ZIAGEN ^TM (abacavir, abacavir/159U89; Glaxo Wellcome Inc.) ; CS-87 (3'-azido-2',3'-dideoxyuridine;WO99/66936); and S-acyl-2-ethylthioesters of β-L-FD4C and β-L-FddC (SATE) in technical form (WO98/17281).

Other NNRTIs include COACTINON ^TM (Emivirine, Emivirine/MKC-442, a potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE ^TM (AG-1549/S-1153, a next-generation NNRTI targeting viruses with the K130N mutation Agouron); PNU-142721 (20 to 50 times more active than its predecessor delavirdine and active against the K130N mutant; Pharmacia &Upjohn); DPC-961 and DPC-963 (second generation of efavirenz Derivatives, designed to be active against viruses with the K103N mutation; DuPont); GW-420867X (25-fold more active than HBY097 and active against the K103N mutant; Glaxo Wellcome); CALANOLIDEA (naturally occurring drug from the rubber tree; Viruses containing one or both of the Y181C and K103N mutations are active); and Propolis (WO99/49830).

Other protease inhibitors include LOPINAVIR ^™ (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR ^™ (PNU-140690, a non-peptide dihydroxypyrone; Pharmacia & Upjohn ); PD-178390 (a non-peptide dihydroxypyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myres Squibb); L-756,423 (an indinavir analog; Merck); DMP -450 (cyclized urea compound; Avid &DuPont); AG-1776 (peptidomimetic active against protease inhibitor-resistant viruses in vitro; Agouron); VX-175/GW-433908 (amprenavir, amprenavir Phosphate Technical; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASE ^™ (Aprenavir; Glaxo Welcome Inc.).

Other antiretrovirals include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (peptide derived from residues 643-678 of the HIV gp41 transmembrane protein ectodomain that binds gp41 in the resting state and prevents transition to the fusion state; Trimeris) and T-1249 (Second Generation Fusion Inhibitor; Trimeris).

Other antiretrovirals include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (bicyclam), SDF-1 and its analogs, and ALX40-4C (cationic peptide), T22 (18 amino acid peptide; Trimeris) and T22 Analogs T134 and T140; CCR5 antagonists such as RANTES(9-68), AOP-RANTES, NNY--RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (distamycin analog) . Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc. can also inhibit fusion.

Other antiretroviral drugs include integrase inhibitors. Integrase inhibitors include dicaffeoylquinine (DFQA) acid; L-cichoric acid (dicaffeoyltartaric (DFQA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR ^TM (AR 177, possibly Oligonucleotides on the cell surface rather than true integrase inhibitors; Arondex); and naphthols, such as disclosed in WO98/50347.

Other antiretrovirals include hydroxyurea-like compounds such as BCX-34 (purine nucleoside phosphatase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX ^™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors, such as VX-497 (Vertex); and mycopholic acids, such as CellCept (mycophenolate mofetil; Roche).

Other antiretroviral drugs include inhibitors of viral integrase, inhibitors of nuclear translocation of viral genomes such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY- RANTES, RANTES-IgG fusion proteins, soluble complexes of RANTES and glycosaminoglycans (GAGs), and AMD-3100; nucleocapsid zinc finger inhibitors, such as dithiane compounds; targets of HIV Tat and Rev; and drug Pharmacoenhancers such as ABT-378.

Other antiretroviral and adjuvant therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN ^™ (aldesleukin, aldesleukin/L2-7001; Chiron), IL - 4. Antagonism of IL-10, IL-12 and IL-13; interferon, such as IFN-α2a, IFN-α2b, or IFN-β; TNF, NFκB, GM-CSF, M-CSF, and IL-10 agents; agents that modulate immune activation, such as cyclosporine and prednisone; vaccines, such as Remune ^™ (HIV Immunogen), APL400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope Glycoproteins, rgp120CM235, MN rgp120, SF-2rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptides derived from discontinuous gp120C3/C4 domains, fusion competent immunogens, and Gag, Pol, Nef and Tat vaccines; gene-based therapies such as the Gene Suppressor Element (GSE; WO98/54366), and intrakine (a genetically modified CC chemokine that targets the ER to block the surface of newly synthesized CCR5 Expression (Yang et al., PNAS 94:11567-72, 1997; Chen et al., Nat.Med.3:1110-16, 1997)); antibodies, such as anti-CXCR4 antibody 12G5, anti-CCR5 antibody 2D7, 5C7, PA8, PA9, PA10, PA11, PA12 and PA14, anti-CD4 antibody Q4120 and RPA-T4, anti-CCR3 antibody 7B11, anti-gp120 antibody 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibody, anti- TNF-α antibody, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists, such as TCDD, 3,3', 4,4', 5-pentachlorobiphenyl, 3,3', 4, 4'-tetrachlorobiphenyl, and α-naphthoflavone (WO98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO99/56764).

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiviral drugs. Antiviral drugs that can be administered in combination with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, remantidine, maxamine, or thymalfasin (thymalfasin). In particular, interferon albumin fusion proteins can be administered in combination with any of these agents. In addition, interferon alpha albumin fusion protein can also be administered in combination with any of these agents, preferably, interferon alpha-2a or 2b albumin fusion protein can be administered in combination with any of these agents. In addition, interferon beta albumin fusion proteins can also be administered in combination with any of these agents. Additionally, any IFN hybrid albumin fusion protein can be administered in combination with any of these agents.

In a most preferred embodiment, the interferon albumin fusion protein is administered in combination with ribavirin. In another preferred embodiment, the interferon alpha albumin fusion protein is administered in combination with ribavirin. In another preferred embodiment, the interferon alpha 2a albumin fusion protein is administered in combination with ribavirin. In another preferred embodiment, the interferon alpha 2b albumin fusion protein is administered in combination with ribavirin. In another preferred embodiment, the interferon beta albumin fusion protein is administered in combination with ribavirin. In another preferred embodiment, the hybrid interferon albumin fusion protein is administered in combination with ribavirin.

In other embodiments, albumin fusion proteins and/or polynucleotides of the present invention may be administered in combination with anti-opportunistic infection drugs. Anti-opportunistic agents that can be administered in combination with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , ATOVAQUONE ^™ , ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ 、RIFABUTIN ^TM 、CLARITHROMYCIN ^TM 、AZITHROMYCIN ^TM 、GANCICLOVIR ^TM 、FOSCARNET ^TM 、CIDOFOVIR ^TM 、FLUCONAZOLE ^TM 、ITRACONAZOLE ^TM 、KETOCONAZOLE ^TM 、ACYCLOVIR ^TM 、FAMCICOLVIR ^TM 、PYRIMETHAMINE ^TM 、LEUCOVORIN ^TM 、NEUPOGEN ^TM (非格司亭，filgrastim/ G-CSF), and LEUKINE ^™ (sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used prophylactically in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , and/or ATOVAQUONE ^™ for the treatment or prevention of opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used prophylactically in any combination with ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , and/or ETHAMBUTOL ^™ for the treatment or prevention of opportunistic birds Mycobacterium avium coinfection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the present invention are used prophylactically in any combination with RIFABUTIN ^™ , CLARITHROMYCIN ^™ , and/or AZITHROMYCIN ^™ for the treatment or prevention of opportunistic Mycobacterium tuberculosis (Mycobacterium tuberculosis) infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the present invention are used prophylactically in any combination with GANCICLOVIR ^™ , FOSCARNET ^™ , and/or CIDOFOVIR ^™ for the treatment or prevention of opportunistic cytomegalovirus Infect. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the present invention are used prophylactically in any combination with FLUCONAZOLE ^™ , ITRACONAZOLE ^™ , and/or KETOCONAZOLE ^™ for the treatment or prevention of opportunistic fungal infections. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the present invention are used prophylactically in any combination with ACYCLOVIR ^™ and/or FAMCICOLVIR ^™ for the treatment or prevention of opportunistic type I and/or type II simple Herpes virus infection. In another specific embodiment, the albumin fusion protein and/or polynucleotide of the present invention and PYRIMETHAMINE ^™ and/or LEUCOVORIN ^™ are used prophylactically in any combination for the treatment or prevention of opportunistic murine Toxoplasma gondii (Toxoplasma gondii) infection . In another specific embodiment, albumin fusion proteins and/or polynucleotides of the present invention are used prophylactically in any combination with LEUCOVORIN ^™ and/or NEUPOGEN ^™ for the treatment or prevention of opportunistic bacterial infections.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antibiotics. Antibiotics that may be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, amoxicillin, β-lactamase, aminoglycosides, β-lactams (glycopeptides), β-lactams Enzymes, clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillin, quinolones, rapamycin, rifampicin, chain Trimethoprim, sulfonamides, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an immune stimulant. Immune stimulants that may be administered in conjunction with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, levamisole (eg ERGAMISOL ^™ ), isoprenin (eg INOSIPLEX ^™ ), interferon (eg interferon alpha) , and interleukins (such as IL-2).

3(muromonab-CD3)、SANDIMMUNE^TM、NEORAL^TM、SANGDYA^TM(环孢菌素)、 (FK506，他克莫司)、 

(霉酚酸吗乙酯， 其中的活性代谢物是霉酚酸)、IMURAN^TM(咪唑硫嘌呤)、糖皮质类固醇、肾上腺皮质类固醇诸如DELTASONE^TM(强的松)和HYDELTRASOL^TM(强的松龙)、FOLEX^TM和MEXATE^TM(氨甲喋呤)、OXSORALEN-ULTRA^TM (甲氧沙林)和RAPAMUNE^TM(西罗莫司)。 In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunosuppressants. Immunosuppressants that can be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone Pine, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressants that act by inhibiting the function of responsive T cells. Other immunosuppressants that can be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen ), rapamycin, leflunomide, mizoribine (BREDININ ^TM ), brequinar, deoxyspergualin, and azaspirane (SKF105685) 、ORTHOCLONE

3 (muromonab-CD3), SANDIMMUNE ^TM , NEORAL ^TM , SANGDYA ^TM (cyclosporine), (FK506, tacrolimus),

(mycophenolate mofetil, the active metabolite of which is mycophenolic acid), IMURAN ^TM (azathioprine), glucocorticoids, adrenal corticosteroids such as DELTASONE ^TM (prednisone) and HYDELTRASOL ^TM (prednisolone ), FOLEX ^™ and MEXATE ^™ (methotrexate), OXSORALEN-ULTRA ^™ (methoxsalen) and RAPAMUNE ^™ (sirolimus).

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that can be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, GAMMAR ^™ , IVEEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S/D ^™ , ATGAM ^™ (antithymocyte spheres protein), and GAMIMUNE ^™ . In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immunoglobulin preparations in transplantation therapy (eg, bone marrow transplantation).

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered alone or as part of a combination therapy, either in vivo to a patient or in vitro to a cell, to treat cancer. In a specific embodiment, an albumin fusion protein, particularly an IL-2-albumin fusion protein, is administered repeatedly during passive immunotherapy of cancer, such as adoptive cell transfer therapy for metastatic melanoma, as described by Dudley et al. (Science Express, September 19, 2002, www.scienceexpress.org, incorporated herein by reference in its entirety).

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with anti-inflammatory drugs. Anti-inflammatory drugs that may be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, corticosteroids (e.g., betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), NSAIDs (eg, diclofenac, diflunisal, etodolac, fenoprofen, frofex Ning, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamic acid ester, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin , phenylbutazone (benzobutazone), piroxicam, sulindac, tenoxicam, tiaprofen, and tolmetin), and antihistamines, aminoaryl carboxylic acid derivatives, aryl Acetic acid derivatives, aryl butyric acid derivatives, aryl carboxylic acids, aryl propionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamide, e-acetylaminocaproic acid Acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixitine, bentacic acid, benzydamine, bucolon, bixenpyramide, deetazol , Imofazone, Guaiazulene, Nabumetone, Nimesulide, Augustine, Oxaceiro, Renitoline, Perisozole, Perafoxime, Proquinone, Proxazole, and Tenidap.

In another embodiment, the composition of the invention is administered alone or in combination with an anti-angiogenic drug. Anti-angiogenic agents that can be administered in combination with the compositions of the present invention include, but are not limited to, angiostatin (Entremed, Rockville, MD), troponin-1 (Boston Life Sciences, Boston, MA), anti-invasion factors, Yellow acid and its derivatives, paclitaxel (Taxol), suramin, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, VEGI, plasminogen activator inhibitor-1, plasminogen activation Inhibitor-2, and various forms of the lighter "d-group" transition metals.

Lighter "group d" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. These transition metal species can form transition metal complexes. Suitable complexes of the above transition metals include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo-vanadium complexes, such as vanadates and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl-based complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate, including vanadyl sulfate hydrates, such as vanadyl sulfate mono- and trihydrate.

Representative examples of tungsten and molybdenum complexes also include oxygen complexes. Suitable tungsten oxide complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten(IV) oxide and tungsten(VI) oxide. Suitable oxymolybdenum complexes include molybdates, molybdenum oxide and oxymolybdenum based complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum(IV) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable oxymolybdenum-based complexes include, for example, oxymolybdenum acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxy derivatives derived from, for example, glycerol, tartaric acid and sugars.

A wide variety of other anti-angiogenic factors can also be used in the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulfate; sulfated chitin derivatives (made from snow crab shells) (Murata et al., Cancer Res. 51:22-26, 1991); Glycan-peptidoglycan complex (SP-PG) (the function of this compound is enhanced by the presence of steroids such as estrogen and tamoxifen citrate); staurosporine; substrate metabolism modulators including e.g. proline Analogues, cis-hydroxyproline, d,L-3,4-dehydroproline, Thiaproline, α,α-bipyridine, fumaric acid aminopropionitrile; 4-propyl-5-(4-pyridyl )-2(3H)-oxazolone; methotrexate; mitoxantrone; heparin; interferon; 2-macroglobulin-serum; -17326,1992); Chymotrypsin inhibitor (Tomkinson et al., Biochem J.286:475-480,1992); Tetradecyl sulfate cyclodextrin; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); gold sodium thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest. 79: 1440-1446, 1987); anticollagenase-serum; α2 - Antiplasmin (Holmes et al., J.Biol.Chem.262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Clobenzalide disodium (N-(2)-carboxylate Phenyl-4-chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316, 1992); and metalloproteinase inhibitors such as BB94.

Other anti-angiogenic factors that can be used in the context of the present invention include thalidomide (Celgene, Warren, NJ); angiostatic steroids; AGM-1470 (H. Brem and J. Folkman, J. Pediatr. Surg. 28:445 -51, 1993); Integrin αvβ3 antagonist (C.Storgard et al., J.Clin.Invest.103:47-54, 1999); Carboxyaminoimidazole; Carboxyaminotriazole (CAI) (National Cancer Institute, Bethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470 (Tap Pharmaceuticals, Deerfield, IL); ZD-0101 AstraZeneca (London, UK ); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; ); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinostat (AG-3340); Purlytin; Suradista (FCE26644); tetrathiomolybdate; Xeloda (capecitabine); and 5-fluorouracil.

Anti-angiogenic agents that may be administered in conjunction with the compositions of the present invention may act through a variety of mechanisms including, but not limited to, inhibition of proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, antagonizing vascular Function of inducers such as growth factors, and inhibition of integrin receptors expressed on proliferating endothelial cells occurs. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and that can be administered in combination with the compositions of the invention include, but are not limited to, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven , CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK) and Metastat (Aetema, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and that can be administered in combination with the compositions of the present invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, Gaithersburg, MD). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting inducers of angiogenesis and that can be administered in combination with the compositions of the invention include, but are not limited to, Angiozyme (Ribozyme, Boulder, CO), anti-VEGF antibodies (Genentech, S. San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, CA), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, NJ), and SU-6668 (Sugen). Other anti-angiogenic agents work by indirectly inhibiting angiogenesis. Examples of indirect inhibitors of angiogenesis that can be administered in combination with the compositions of the present invention include, but are not limited to, IM-862 (Cytran, Kirkland, WA), interferon alpha, IL-12 (Roche, Nutley, NJ), and polysulfate Pentosan (Georgetown University, Washington, DC).

In a particular embodiment, it is envisaged to treat, prevent and/or ameliorate autoimmune diseases, such as for example the autoimmune diseases described herein, by using the compositions of the invention in combination with anti-angiogenic agents.

In a specific embodiment, it is envisaged to treat, prevent and/or ameliorate arthritis by the combination of the composition of the invention and an anti-angiogenic agent. In a more specific embodiment, it is envisaged to treat, prevent and/or ameliorate rheumatoid arthritis by using the composition of the invention in combination with an anti-angiogenic agent.

In another embodiment, a polynucleotide encoding a polypeptide of the invention is administered in combination with an angiogenic protein or a polynucleotide encoding an angiogenic protein. Examples of angiogenic proteins that may be administered in conjunction with the compositions of the present invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factors alpha and beta, platelets Derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and oxidative nitrogen synthase.

In other embodiments, compositions of the invention are administered in combination with chemotherapeutic agents. Chemotherapeutic agents that may be administered in conjunction with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., bischloroethylmethylamine, cyclophosphamide, cyclophosphamide, ifosf amide, melphalan (L-sarcolysin), and chlorambucil), ethyleneimine and methylmelamine (such as hexamethylmelamine and thiotepa), alkylsulfonates (such as An), nitrosoureas (such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin), triazenes (such as Dacarbazine (DTIC; dimethyltriazolamide), folic acid analogs (such as methotrexate (methotrexate)), pyrimidine analogs (such as fluorouracil (5-fluorouracil; 5-FU), fluorouracil glycosides (fludeoxyuridine; FudR), and cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6- - Thioguanine; TG), and pentostatin (2′-deoxycomorphycin)), vinca alkaloids (such as vinblastine (VLB, vinblastine sulfate)) and vincristine (vincristine sulfate alkali)), epipodophyllotoxins (such as etoposide and teniposide), antibiotics (such as dactinomycin (actinomycin D), daunorubicin (daunomycin; daunorubicin) , doxorubicin, bleomycin, plicamycin (mitomycin), and mitomycin (mitomycin C), enzymes (such as L-asparaginase), biological response modifiers (such as interferon-α and interferon-α-2b), platinum complexes (such as cisplatin (cis-DDP) and carboplatin), anthracenedione (mitoxantrone), substituted ureas ( e.g. hydroxyurea), methylhydrazine derivatives (e.g. procarbazine (N-methylhydrazine; MIH)), corticosteroids (e.g. prednisone), progestins (e.g. hydroxyprogesterone caproate, medroxyprogesterone, acetate medroxyprogesterone, and megestrol acetate), estrogens (such as diethylstilbestrol (DES), diethylstilbestrol diphosphate, estradiol, and ethinyl estradiol), antiestrogens (such as tamoxifen), androgens (testosterone propionate and fluoxymesterone), antiandrogens (such as flutamide), gonadotropin-releasing hormone analogs (such as leuprolide), other hormones and hormone analogs (such as methyltestosterone, estramustine , estramustine phosphate sodium, chlorestradiol, and testolactone), and other agents (eg, dacarbazine, glutamate, and mitotane).

In one embodiment, the composition of the present invention is administered in combination with one or more of the following drugs: infliximab (also known as Remicade ^™ Centocor, Inc.), trocate (Roche, RO-32-3555), leflunomide (also known as Arava ^™ from Hoechst Marion Roussel), Kineret ^™ (IL-1 receptor antagonist, also known as Anakinra from Amgen, Inc.).

In a specific embodiment, a composition of the invention is administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or with a combination of one or more components of CHOP. In one embodiment, the composition of the invention is administered in combination with an anti-CD20 antibody, a human monoclonal anti-CD20 antibody. In another embodiment, the composition of the invention is administered in combination with any combination of an anti-CD20 antibody and CHOP, or an anti-CD20 antibody and one or more components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, the composition of the invention is administered in combination with Rituximab. In another embodiment, the composition of the invention is administered with Rituximab and CHOP, or any combination of Rituximab and one or more components of CHOP, particularly cyclophosphamide and/or prednisone. In another embodiment, the composition of the invention is administered with tositumomab and CHOP, or any combination of tositumomab and one or more components of CHOP, especially cyclophosphamide and/or prednisone. Anti-CD20 antibodies can optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.

In another specific embodiment, the compositions of the invention are administered in combination with Zevalin ^™ . In another embodiment, the composition of the present invention is administered with Zevalin ^™ and CHOP, or any combination of Zevalin ^™ and one or more components of CHOP, especially cyclophosphamide and/or prednisone. Zevalin( ^TM) can be associated with one or more radioactive isotopes. Particularly preferred isotopes ^{are90Y and111In} .

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with cytokines. Cytokines that can be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, ILI5, anti-CD40, CD40L, IFN -γ and TNF-α. In another embodiment, albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including but not limited to IL-1α, IL-1β, IL-2, IL-3, IL4, IL -5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, L-17 , IL-18, IL-19, IL-20 and IL-21.

In one embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with TNF family members. TNF, TNF-related molecules, or TNF-like molecules that can be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, soluble forms of TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (International publication number WO96 /14328), AIM-I (International Publication No. WO97/33899), endokine-α (International Publication No. WO98/07880), OPG, and neutrokine-α (International Publication No. WO98/18921), OX40, and nerve growth factor ( NGF), and soluble forms of Fas, CD30, CD27, CD40, and 4-IBB, TR2 (International Publication No. WO96/34095), DR3 (International Publication No. WO97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO98/30693), TRANK, TR9 (International Publication No. WO98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO98/06842), and TR12, and soluble forms of CD154, CD70, and CD153.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered in conjunction with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, glial-derived growth factor (GDGF), disclosed in European Patent No. EP-399816; platelet-derived growth factor ( PDGF-A), disclosed in European Patent No. EP-682110; platelet-derived growth factor-B (PDGF-B), disclosed in European Patent No. EP-282317; placental growth factor (PIGF), disclosed in International Publication No. WO92/06194 ; Placental Growth Factor-2 (PIGF-2), disclosed in Hauser et al., Growth Factors 4:259-268,1993); Vascular Endothelial Growth Factor (VEGF), disclosed in International Publication No. WO90/13649; Vascular Endothelial Growth Factor -A (VEGF-A), disclosed in European Patent No. EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), disclosed in International Publication No. WO96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Endothelial growth factor B-186 (VEGF-B186), disclosed in International Publication No. WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), disclosed in International Publication No. WO98/02543; Vascular Endothelial Growth Factor-D (VEGF -D), disclosed in International Publication No. WO98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), disclosed in German Patent No. DE19639601. The above references are incorporated herein by reference in their entirety.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that may be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6 , FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that can be administered in combination with albumin fusion proteins and/or polynucleotides of the present invention include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) (sargragrastim, sargramostim, LEUKINE ^™ , PROKINE ^TM ), granulocyte colony stimulating factor (G-CSF) (filgrastim, filgrastim, NEUPOGEN ^TM ), macrophage colony stimulating factor (M-CSF, CSF-1), erythropoietin (epoetin alfa , epoetin alfa, EPOGEN ^TM , PROSCRIT ^TM ), stem cell factors (SCF, c-kit ligand, gray factor), megakaryocyte colony-stimulating factor, PIXY321 (GMCSF/IL-3 fusion protein), interleukins, especially IL-1 to Any one or more of IL-12, interferon gamma, or thrombopoietin.

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with adrenergic blocking agents such as, for example, acebutolol, atenolol, beta Betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxyprene oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.

In another embodiment, albumin fusion proteins and/or polynucleotides of the present invention are administered in combination with antiarrhythmic drugs (such as adenosine, amidoarone, bretylium, digitalis, Digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, Moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with diuretics, such as carbonic anhydrase inhibitors (e.g., acetazolamide, dichlorphenamide, and methazolamide ), osmotic diuretics (such as glycerol, isosorbide, mannitol, and urea), diuretics that inhibit Na ⁺ -K ⁺ -2Cl ^- symport (such as furosemide, bumetanide, azosemide thiazide, piretanide, tripamide, ethacrynic acid, mozolomide, and torsemide), thiazide and thiazide-like diuretics (such as bendrofluthiazide, benthiazide, chlorothiazide, hydrochlorothiazide, hydrofluorothiazide, methiazide, polythiazide, trichlorothiazide, chlorthalidone, indapamide, metolazone, and quinetazone), potassium-sparing diuretics (such as amiloride and ammonia phenterene), and mineralocorticoid receptor antagonists (such as spironolactone, canrenone, and canrenoate potassium).

In one embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with the treatment of endocrine and/or hormonal imbalance disorders. Treatment of endocrine and/or hormonal imbalance disorders includes, but is not limited ^to127I , radioactive isotopes of iodine such ^{as131I and123I} ; recombinant growth hormone, such as HUMATROPE ^™ (recombinant somatotropin); growth hormone analogs, such as PROTROPIN ^™ ( human ghrelin); dopamine agonists such as PARLODEL ^™ (bromocriptine); somatostatin analogs such as SANDOSTATIN ^™ (octreotide); gonadotropin preparations such as PREGNYL ^™ , APL ^™, and PROFASI ^™ (chilli gonadotropin (CG)), PERGONAL ^TM (menopausal gonadotropin), and METRODIN ^TM (urinary follicle-stimulating hormone (uFSH)); synthetic human gonadotropin-releasing hormone preparations such as FACTREL ^TM and LUTREPULSE ^TM (gonadorelin hydrochloride ); synthetic gonadotropin agonists such as LUPRON ^™ (leuprolide acetate), SUPPRELIN ^™ (histrelin acetate), SYNAREL ^™ (nafarelin acetate), and ZOLADEX ^™ (goserelin acetate) ; synthetic thyrotropin-releasing hormone preparations, such as RELEFACT TRH ^TM and THYPINONE ^TM (prorelin); recombinant human TSH, such as THYROGEN ^TM ; synthetic preparations of thyroid hormone natural isomer sodium salt, such as LT ₄ ^TM , SYNTHROID ^TM and LEVOTHROID ^TM (levothyroxine), LT ₃ ^TM , CYTOMEL ^TM, and TRIOSTAT ^TM (iodothyroine sodium), and THYROLAR ^TM (liotrix); antithyroid compounds such as 6-n - Propylthiouracil (Propylthiouracil), 1-Methyl-2-Mulcapylimidazole and TAPAZOLE ^TM (methimazole), NEO-MERCAZOLE ^TM (carbimazole); beta-adrenergic receptors Antibody antagonists, such as propranolol and esmolol; Ca2 ⁺ channel blockers; dexamethasone and iodinated radiological contrast agents, such as TELEPAQUE ^™ (iopanic acid) and ORAGRAFIN ^TM (Sodium Ipodate).

Other treatments for endocrine and/or hormonal imbalance disorders include but are not limited to estrogens or conjugated estrogens such as ESTRACE ^™ (estradiol), ESTINYL ^™ (ethinyl estradiol), PREMARIN ^™ , ESTRATAB ^™ , ORTHO-ES ^™ , OGEN ^TM and piperazine estrone thioester (estrone), ESTROVIS ^TM (ethinyl estradiol), ESTRADERM ^TM (estradiol), DELESTROGEN ^TM and VALERGEN ^TM (estradiol valerate), DEPO-ESTRADIOL CYPIONATE ^TM and ESTROJECT LA ^™ (estradiol cypionate); antiestrogens such as NOLVADEX ^™ (tamoxifen), SEROPHENE ^™ , and CLOMID ^™ (clomiphene); progestins such as DURALUTIN ^™ (hydroxyprogesterone caproate ketone), MPA ^TM and DEPO-PROVERA ^TM (megestrol acetate), PROVERA ^TM and CYCRIN ^TM (MPA), MEGACE ^TM (megestrol acetate), NORLUTIN ^TM (norethindrone), and NORLUTATE ^TM and AYGESTIN ^TM (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM ^™ (subcutaneous implant of norgestrel); antiprogestins such as RU 486 ^™ (mifepristone); hormonal contraceptives such as ENOVID ^TM (norethindrone plus mestranol), PROGESTASERT ^TM (progesterone-releasing intrauterine contraceptive device), LOESTRIN ^TM , BREVICON ^TM , MODICON ^TM , GENORA TM , NELONA ^TM , NORINYL ^{TM ,} OVACON-35 ^TM , and OVACON-50 ^TM (ethinyl estradiol/ norethindrone), LEVLEN ^TM , NORDETTE ^TM , TRI-LEVLEN ^TM and TRIPHASIL-21 ^TM (ethinyl estradiol/levonorgestrel), LO/OVRAL ^TM and OVRAL ^TM (ethinyl estradiol diol/norgestrel), DEMULEN ^TM (ethinyl estradiol/ norethindrol), NORINYL ^TM , ORTHO-NOVUM ^TM , NORETHIN ^TM , GENORA ^TM , and NELOVA ^TM (norethindrone/mestranol) , DESOGEN ^TM and ORTHO-CEPT ^TM (ethinyl estradiol/desogestrel), ORTHO-CYCLEN ^TM and ORTHO-TRICYCLEN ^TM (ethinyl estradiol/norgestimate), MICRONOR ^TM and NOR - QD ^™ (norethindrone), and OVRETTE ^™ (norgestrel).

Other treatments for endocrine and/or hormonal imbalance disorders include, but are not limited to, testosterone esters such as primobolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50 ^™ (testosterone), TESTEX ^™ (propionic acid testosterone), DELATESTRYL ^TM (testosterone enanthate), DEPO-TESTOSTERONE ^TM (testosterone cypionate), DAQNOCRINE ^TM (danazol), HALOTETIN ^TM (fluoxymesterone), ORETON METHYL ^TM , TESTRED ^TM and VIRILON ^TM (methyltestosterone ), and OXANDRIN ^TM (oxandrolone); testosterone transdermal systems, such as TESTODERM ^TM ; androgen receptor antagonists and 5-α-reductase inhibitors, such as ANDROCUR ^TM (cyproterone acetate), EULEXIN ^TM ( flutamide), and PROSCAR ^TM (finasteride); corticotropin preparations, such as CORTROSYN ^TM (cosyntropin); adrenocorticosteroids and their synthetic analogues, such as ACLOVATE ^TM (alclomethasone dipropionate), CYCLOCORT ^TM (amcinonide), BECLOVENT ^TM and VANCERIL ^TM (beclomethasone dipropionate), CELESTONE ^TM (betamethasone), BENISONE ^TM and UTICORT ^TM (betamethasone benzoate), DDIPROSONE ^TM (betamethasone dipropionate) CELESTONEPHOSPHATE ^TM (betamethasone sodium phosphate), CELESTONE SOLUSPAN ^TM (betamethasone phosphate and sodium acetate), BETA-VAL ^TM and VALISONE ^TM (betamethasone valerate), TEMOVATE ^TM (clobetasol propionate ), CLODERM ^TM (clocotorone trimethylacetate), CORTEF ^TM and HYDROCORTONE ^TM (cortisol (hydrocortisone)), HYDROCORTONE ACETATE ^TM (cortisol acetate (hydrocortisone)), LOCOID ^TM (butylene Acid Cortisol (Hydrocortisone)), HYDROCORTONE PHOSPHATE ^TM (Sodium Cortisol (Hydrocortisone) Phosphate), A-HYDROCORT ^TM and SOLU CORTEF ^TM (Sodium Cortisol (Hydrocortisone) Succinate), WESTCORT ^TM (cortisol valerate (hydrocortisone)), CORTISONE ACETATE ^TM (cortisone acetate), DESOWEN ^TM and TRIDESILON ^TM (desonide), TOPICORT ^TM ( Dexamethasone), DECADRON ^TM (dexamethasone), DECADRON LA ^TM (dexamethasone acetate), DECADRON PHOSPHATE ^TM and HEXADROL PHOSPHATE ^TM (dexamethasone sodium phosphate), FLORONE ^TM and MAXIFLOR ^TM (diflurasone diacetate ), FLORINEF ACETATE ^TM (fludrocortisone acetate), AEROBID ^TM and NASALIDE ^TM (flunisolide), FLUONID ^TM and SYNALAR ^TM (fluocinolone), LIDEX ^TM (fluocinonide acetate), FLUOR-OP ^TM and FML ^TM (fluorometholone), CORDRAN ^TM (fludrosolone), HALOG ^TM (hacinonide), HMS LIZUIFILM ^TM (methidone), MEDROL ^TM (methylprednisolone), DEPO-MEDROL ^TM and MEDROL ACETATE ^TM ( Methylprednisolone acetate), A-METHTMRED ^TM and SOLUMEDROL ^TM (methylprednisolone sodium succinate), ELOCON ^TM (mometasone furoate), HALDRONE ^TM (paramethasone acetate), DELTA-CORTEF ^TM (prednisolone) , ECONOPRED ^TM (prednisolone acetate), HYDELTRASOL ^TM (prednisolone sodium phosphate), HYDELTRA-TBA ^TM (prednisolone tert-butyl acetate), DEmSONE ^TM (prednisone), ARISTOCORT ^TM and KENACORT ^TM (triamcinolone), KENALOG ^TM (triamcinolone acetonide), ARISTOCORT ^TM and KENACORTDIACETATE ^TM (triamcinolone diacetate), and ARISTOSPAN ^TM (triamcinolone acetonide); inhibitors of corticosteroid biosynthesis and action, such as CYTADREN ^TM (aminoglutimide), NIZORAL ^TM (ketoconazole), MODRASTANE ^TM (trolosteine), and METOPIRONE ^TM (metyrapone); bovine, porcine, or human insulin or mixtures thereof; insulin analogues; Recombinant human insulin, such as HUMULIN ^TM and NOVOLIN ^TM ; oral hypoglycemic agents, such as ORAMIDE ^TM and ORINASE ^TM (tolbutamide), DIABINESE ^TM (chlorpropamide), TOLAMIDE ^TM and TOLINASE ^TM (tolasulfame), DYMELOR ^TM (hexylurea acetate), glibenclamide, MICRONASE ^TM , DIBETA ^TM and GLYNASE ^TM (grid GLUCOTROL ^TM (glipizide), and DIAMICRON ^TM (gliclazide), GLUCOPHAGE ^TM (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN ^™ (octreotide); and diazoxides such as PROGLYCEM ^™ (diazoxide).

和 

)、雌二醇(例如 

和 

)、哌嗪雌酮硫酯、和氯烯雌醚；孕酮药物(例如 

(甲羟孕酮)、 

(醋酸炔诺酮)、 

孕酮、和醋酸甲地孕酮)；以及雌激素/孕酮联合疗法，诸如例如缀合雌激素/甲羟孕酮(例如PREMPRO^TM和 

)和醋酸炔诺酮/乙炔雌二醇(例如FEMHRT^TM)。 In one embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with the treatment of uterine motility disorders. Treatment of uterine motility disorders includes, but is not limited to, estrogenic agents, such as conjugated estrogens (e.g.

and

), estradiol (eg

and

), piperazinestrone thioester, and chlorphenestradiol; progesterone drugs (such as

(Medroxyprogesterone),

(Norethindrone Acetate),

progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogen/medroxyprogesterone (e.g., PREMPRO ^™ and

) and norethindrone acetate/ethinyl estradiol (eg FEMHRT ^™ ).

In another embodiment, the albumin fusion protein and/or polynucleotide of the present invention is administered in combination with drugs effective in the treatment of iron deficiency and hypohemochromic anemia, including but not limited to ferrous sulfate (ferrous sulfate, FEOSOL ^™ ), ferrous fumarate (such as FEOSTAT ^TM ), ferrous gluconate (such as FERGON ^TM ), polysaccharide-iron complex (such as NIFEREX ^TM ), iron dextran injection (such as INFED ^TM ), ketone sulfate, pyridoxine pyroxidine, riboflavin, vitamin B ₁₂ , cyanocobalamin injection (such as REDISOL ^TM , RUBRAMIN PC ^TM ), hydroxocobalamin, folic acid (such as FOLVITE ^TM ), leucovorin (leucovorin, 5-CHOH4PteGlu, calcium folinate) or WELLCOVORIN (calcium salt of leucovorin), transferrin, or ferritin.

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents for the treatment of mental disorders. Psychiatric drugs that can be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antipsychotics (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, , loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanics (eg, carbamazepine, divalproex, lithium carbonate, and lithium citrate), antidepressants (eg, amitriptyline, amoxapine, bupropion , citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone , nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), anxiolytics (such as alprazolam , buspirone, chlordigepam, chlordiazepoxide, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (such as d-amphetamine, methylphenidate, and pemoline).

In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents useful in the treatment of neurological disorders. Neurological agents that can be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antiepileptic drugs (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, Primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam , lorazepam, and clonazepam), antiparkinsonian drugs (eg, levodopa/carbidopa, selegiline, amantadine, bromocriptine, pergolide, ropinirole , pramipexole, benztropine; biperiden; eprobazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (eg, riluzole).

In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with vasodilators and/or calcium channel blockers. Vasodilators that can be administered in conjunction with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, angiotensin converting enzyme (ACE) inhibitors (e.g. papaverine, isoxulin, benazepril, Captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril , spirapril, trandolapril, and bufenin), and nitrates (eg, isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blockers that may be administered in conjunction with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, amlodipine, bepridil, diltazol, felodipine, flunarizine Nifedipine, Isradipine, Nicardipine, Nifedipine, Nimodipine, and Verapamil.

In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with therapeutic agents for gastrointestinal disorders. Therapeutic agents for gastrointestinal disorders that may be administered in combination with albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, H2 histamine receptor antagonists (e.g., TAGAMET ^™ (cimetidine), ZANTAC ^™ (Ray Nitidine), PEPCTD ^TM (famotidine), and AXID ^TM (nizatidine)); H ⁺ , K ⁺ ATPase inhibitors (such as PREVACID ^TM (lansoprazole) and PRILOSEC ^TM (Ogilvy bismuth subsalicylate)); bismuth compounds (such as PEPTO-BISMOL ^™ (bismuth subsalicylate) and DE-NOL ^™ (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogues ( For example, CYTOTEC ^™ (misoprostol)); muscarinic cholinergic antagonists; laxatives (such as surfactant laxatives, stimulant laxatives, saline, and osmotic laxatives); antidiarrheals (such as LOMOTIL ^™ (diphenoxylate), MOTOFEN ^™ (diphenoxin), and IMODIUM ^™ (loperamide hydrochloride)), synthetic analogs of somatostatin, such as SANDOSTATIN ^™ (octreotide), antiemetics (such as ZOFRAN ^™ ( ondansetron), KYTRIL ^TM (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thioethyl D2 antagonists (eg, metoclopr amines, trimebenzine, and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations, such as pancreatin and pancreatic lipase.

In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in conjunction with other therapeutic or prophylactic regimens such as, for example, radiation therapy.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of a pharmaceutical composition comprising an albumin fusion protein of the invention. Optionally, a notice issued by an administrative agency of government regulating the manufacture, use, or sale of a drug or biological product, reflecting the agency's approval for manufacture, use, or sale of a drug for human administration, is optionally provided with such container .

gene therapy

Constructs encoding albumin fusion proteins of the invention can be used to deliver therapeutically effective doses of albumin fusion proteins as part of a gene therapy regimen. A preferred method for introducing a nucleic acid into a cell in vivo is through the use of a viral vector comprising a nucleic acid encoding an albumin fusion protein of the invention. The advantage of infecting cells with a viral vector is that most of the target cells are able to obtain the nucleic acid. In addition, molecules within the viral vector, eg, encoded by the cDNA contained in the viral vector, are efficiently expressed in cells that have taken up the viral vector nucleic acid.

Retroviral and adeno-associated viral vectors can be used as recombinant gene delivery systems for in vivo transfer of exogenous nucleic acid molecules encoding albumin fusion proteins. These vectors efficiently deliver nucleic acids into cells, and the transfected nucleic acids are stably integrated into the host chromosomal DNA. The development of specialized cell lines (termed "packaging cells") that produce only replication-defective retroviruses has increased the utility of retroviruses in gene therapy, and the identified defective retroviruses are used for gene therapy purposes. Metastasis (for review see Miller, A.D., Blood 76:271, 1990). By standard techniques, replication-defective retroviruses can be packaged into virions, which can be used to infect target cells through the use of a helper virus. Protocols for generating recombinant retroviruses and for infecting cells with such viruses in vitro or in vivo can be found in "Current Protocols in Molecular Biology", eds. Ausubel, F.M. et al., Greene Publishing Associates, 1989, Sections 9.10-9.14 and Other standard experimental guidelines.

Another viral gene delivery system that can be used in the present invention uses adenovirus-derived vectors. The genome of the adenovirus can be manipulated such that it encodes and expresses the gene product of interest, but inactivates its ability to replicate during the normal cytolytic viral life cycle. See, eg, Berkner et al., BioTechniques 6:616, 1988; Rosenfeld et al., Science 252:431-434, 1991; and Rosenfeld et al., Cell 68:143-155, 1992. Suitable adenovirus vectors derived from adenovirus strain Ad5 type d1324 or other adenovirus strains (eg Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses are advantageous in certain circumstances because they cannot infect non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., 1992, supra). In addition, virus particles are relatively stable, easy to purify and concentrate, and, as mentioned above, can be engineered to affect their spectrum of infection. In addition, the imported adenoviral DNA (and the foreign DNA contained therein) is not integrated into the host cell genome, but remains as an episome, thus avoiding the possibility of the introduction of DNA integrated into the host genome (such as retroviral DNA) Potential problems arising from insertional mutagenesis in this situation. In addition, the capacity of the adenoviral genome to carry foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., cited above; Haj-Ahmand et al., J. Virol. 57:267, 1986 ).

In another embodiment, the non-viral gene delivery systems of the invention rely on the endocytic pathway for uptake of the subject nucleotide molecules by target cells. Exemplary gene delivery systems of this type include liposome-derived systems, polylysine conjugates, and artificial viral envelopes. In one representative embodiment, a nucleic acid molecule encoding an albumin fusion protein of the invention can be entrapped into a liposome whose surface is positively charged and (optionally) tagged with an antibody directed against a target tissue cell surface antigen (e.g. lipofectin) (Mizuno et al., No Shinkei Geka 20:547-551, 1992; PCT Publication WO91/06309; Japanese Patent Application 1047381; and European Patent Publication EP-A-43075).

The gene delivery system of the gene encoding the albumin fusion protein of the present invention can be introduced into the patient by a variety of methods. For example, a pharmaceutical preparation of a gene delivery system can be introduced, for example, through an intravenous injection system, and the specific transduction of proteins in target cells is mainly provided by the gene delivery vehicle. Transfection-specific, transcriptional regulatory sequences attributable to the control of receptor gene expression Cell type or tissue type expression or a combination thereof. In other embodiments, the initial delivery of the recombinant gene is more limited to relatively localized animals. For example, gene delivery vehicles can be introduced by catheter (see US Patent No. 5,328,470) or by directed injection (eg, Chen et al., PNAS 91:3054-3057, 1994). Pharmaceutical preparations of gene therapy constructs may consist essentially of the gene delivery system in an acceptable diluent, or may comprise a slow release matrix in which the gene delivery vehicle is embedded. Where intact albumin fusion proteins can be produced by recombinant cells, such as retroviral vectors, pharmaceutical preparations may comprise one or more cells that produce albumin fusion proteins.

other gene therapy

The invention also encompasses gene therapy for the treatment or prevention of disorders, diseases and conditions. Gene therapy involves the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into animals to effect expression of albumin fusion proteins of the invention. This approach requires that a polynucleotide encoding an albumin fusion protein of the invention be operably linked to a promoter and any other genetic elements necessary for expression of the fusion protein in the target tissue. Such gene therapy and delivery techniques are known in the art, see eg WO90/11092, which is incorporated herein by reference.

Thus, for example, cells from a patient can be engineered ex vivo with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the invention, and the engineered cells can then be provided to A patient to be treated with an albumin fusion protein of the invention. Such methods are well known in the art. For example, see Belldegrun, A. et al., J. Natl. Cancer Inst. 85:207-216, 1993; Ferrantini, M. et al., Cancer Research 53:1107-1112, 1993; Ferrantini, M. et al., J. .Immunology 153: 4604-4615, 1994; Kaido, T. et al., Int. J. Cancer 60: 221-229, 1995; Ogura, H. et al., Cancer Research 50: 5102-5106, 1990; Santodonato, L. . et al., Human Gene Therapy 7:1-10, 1996; Santodonato, L. et al., Gene Therapy 4:1246-1255, 1997; and Zhang, J.-F. et al., Cancer GeneTherapy 3:31-38 , 1996, which is incorporated herein by reference. In one embodiment, the cells to be engineered are arterial cells. Arterial cells can be reintroduced into the patient by injection directly into the artery, into the tissue surrounding the artery, or by injection through a catheter.

As discussed in more detail below, polynucleotide constructs can be delivered to animal cells by any method of delivery of injectable materials, such as injection into the interstitial space of tissue (heart, muscle, skin, lung, liver, etc.). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, polynucleotides encoding albumin fusion proteins of the invention are delivered as naked polynucleotides. The term "naked" polynucleotide, DNA or RNA refers to sequences free from any delivery vehicle that assists, facilitates or accelerates entry into cells, including viral sequences, viral particles, liposome formulations, lipofection or precipitation reagents, and the like. However, polynucleotides encoding albumin fusion proteins of the present invention can also be delivered in liposome preparations, lipofection preparations, etc., which can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

Polynucleotide vector constructs for gene therapy are preferably constructs that neither integrate into the host genome nor contain sequences that allow replication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1 and pRc/CMV2 available from Invitrogen. Other suitable vectors will be apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used to drive expression of a polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenovirus major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, Such as MMT promoter, metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globulin promoter; viral thymidine kinase promoter, such as herpes simplex thymidine kinase promoter; retrovirus LTR; b-actin promoter; and human growth hormone promoter. The promoter may also be the native promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention.

A major advantage of introducing naked nucleic acid sequences into target cells, unlike other gene therapy techniques, is the transient nature of polynucleotide synthesis in cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for the production of desired polypeptides for a period of up to 6 months.

The polynucleotide constructs can be delivered to the interstitial tissue of animals, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, Intestines, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue. Tissue interstitial spaces include intercellular, fluid, mucopolysaccharide layers between reticular fibers of organ tissues, elastic fibers in vessel walls or chamber walls, collagen fibers in fibrous tissues, or the same in connective tissue or bone spaces incorporated into muscle cells matrix. The space occupied by circulating plasma and lymphatic fluid of the lymphatic ducts is also similar. Delivery to the interstitial space of muscle tissue is preferred for reasons discussed below. They can be conveniently delivered by injection to tissues containing these cells. They are preferably delivered to and expressed in persistent, non-dividing differentiated cells, although delivery and expression may be accomplished in undifferentiated or less fully differentiated cells such as, for example, blood stem cells or skin fibroblasts. Muscle cells are particularly efficient in their ability to uptake and express polynucleotides in vivo.

For naked nucleic acid sequence injections, effective doses of DNA or RNA will range from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably, the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, those of ordinary skill in the art will appreciate that this dosage will vary with the tissue site of injection. Appropriate and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art, and may depend on the condition being treated and the route of administration.

The preferred route of administration is by parenteral injection into the interstitial space of the tissue. However, other parenteral routes may also be used, such as aerosol inhalation, particularly suitable for delivery to the lung or bronchial tissue, larynx or nasal mucosa. In addition, naked DNA constructs can be delivered to arteries during angioplasty through the catheter used in the procedure.

Delivery of naked polynucleotides is by any method known in the art, including, without limitation, direct needle injection at the site of delivery, intravenous injection, topical application, catheter infusion, and the so-called "gene gun." These delivery methods are known in the art.

Constructs can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofection, precipitation reagents, and the like. These delivery methods are known in the art.

In certain embodiments, polynucleotide constructs are complexed into liposome preparations. Liposome preparations useful in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred since tight charge complexes can be formed between cationic liposomes and polyanionic nucleic acids. It has been shown that cationic liposomes mediate plasmid DNA in a functional form (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987, which is incorporated herein by reference); mRNA (Malone et al. , Proc.Natl.Acad.Sci.USA 86:6077-6081,1989, which is incorporated herein by reference); and purified transcription factors (Debs et al., J.Biol.Chem.265:10189-10192,1990, Intracellular delivery which is incorporated herein by reference).

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy]propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available from GIBCO BRL, Grand Island, N.Y., The trademark is Lipofectin (see also Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987, which is incorporated herein by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg, the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonium)propane) liposomes as described in PCT Publication No. WO90/11092, which is incorporated herein by reference. The preparation of DOTMA liposomes is described in the literature, see eg P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is incorporated herein by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are also readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be readily prepared from readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and the like. These materials can also be mixed with DOTMA and DOTAP starting materials in appropriate proportions. Methods for preparing liposomes from these materials are well known in the art.

For example, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylethanolamine (DOPE) are commercially available in various combinations for the preparation of conventional liposomes, with or without the addition of Add cholesterol. Thus, for example, DOPG/DOPC liposomes can be prepared by drying 50 mg each of DOPG and DOPC in a sonicated vial under nitrogen flow. The samples were placed under vacuum pump overnight and hydrated with deionized water the next day. The samples were then sonicated in capped vials with a Heat Systems Sonicator Model 350 equipped with an inverted cup (water bath) probe at maximum setting for 2 hours while the water bath was circulating at 15°C. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion of the nuclear pore membrane to produce unilamellar vesicles of various sizes. Other methods are also known and available to those skilled in the art.

Liposomes may comprise multilamellar vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV), preferably SUVs. Various liposome-nucleic acid complexes are prepared using methods well known in the art. See, eg, Straubinger et al., Methods of Immunology 101:512-527, 1983, which is incorporated herein by reference. For example, nucleic acid-containing MLVs can be prepared by depositing thin films of phospholipids on the walls of glass test tubes, followed by hydration with a solution of the material to be encapsulated. SUVs were prepared by prolonged sonication of MLVs to generate a homogeneous population of unilamellar liposomes. The material to be captured is added to the treated MLV suspension followed by sonication. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer such as 10 mM Tris/NaCl, sonicated, and the treated liposomes are directly mixed with DNA mixed. Due to the association of positively charged liposomes with cationic DNA, liposomes and DNA form very stable complexes. SUVs can be used for small nucleic acid fragments. LUVs are prepared by a number of methods known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta 394:483, 1975; Wilson et al., Cell 17:77, 1979); ether injection (Deamer, D. and Bangham, A. ., Biochim.Biophys.Acta 443:629,1976; Ostro et al., Biochem.Biophys.Res.Commun.76:836,1977; Fraley et al., Proc.Natl.Acad.Sci.USA76:3348,1979); Detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145, 1979); and reverse-phase distillation (REV) (Fraley et al., J. Biol. Chem. 255: 10431, 1980; Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75: 145, 1978; Schaefer-Ridder et al., Science 215: 166, 1982), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ratio is from about 5:1 to about 1:5. More preferably, the ratio is from about 3:1 to about 1:3. Still more preferably, the ratio is about 1:1.

US Patent No. 5,676,954, which is incorporated herein by reference, reports injecting mice with genetic material complexed with cationic liposome carriers. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055 and International Publication No. WO 94/9469 (which are incorporated herein by reference) provide cations for transfection of DNA into cells and mammals. Lipid. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055 and International Publication No. WO 94/9469 provide methods of delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered ex vivo or in vivo with retroviral particles containing RNA comprising a sequence encoding an albumin fusion protein of the invention. Retroviruses from which retroviral plasmid vectors can be derived include, but are not limited to, Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus, human immunodeficiency virus, Myeloproliferative sarcoma virus, and mammary tumor virus.

Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of transfectable packaging cells include, but are not limited to, PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17 described in Miller, Human Gene Therapy 1:5-14, 1990 - H2, RCRE, RCRIP, GP+E-86, GP+envAm12 and DAN cell lines, which are incorporated herein by reference in their entirety. Packaging cells can be transduced with the vector by any method known in the art. Such methods include, but are not limited to, electroporation, the use of liposomes, and _CaPO4 precipitation. In an alternative, the retroviral plasmid vector can be encapsulated in liposomes, or conjugated to lipids, and administered to the host.

The producer cell line produces infectious retroviral vector particles comprising a polynucleotide encoding an albumin fusion protein of the invention. Eukaryotic cells can then be transduced with such retroviral vector particles in vitro or in vivo. The transduced eukaryotic cells will express the fusion protein of the invention.

In certain other embodiments, cells are engineered ex vivo or in vivo with polynucleotides contained in adenoviral vectors. The adenovirus can be manipulated to encode and express the fusion protein of the invention while inactivating its ability to replicate during the normal cytolytic viral life cycle. Adenoviral expression is achieved without the need for integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years and have an excellent safety profile (Schwartz et al., Am. Rev. Respir. Dis. 109:233-238, 1974). Finally, many examples of adenovirus-mediated gene transfer have been demonstrated, including the transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al., Science 252:431-434, 1991; Rosenfeld et al., Cell 68:143-155, 1992). In addition, extensive studies attempting to identify adenoviruses as causative agents of human cancer have been uniformly negative (Green, M. et al., Proc. Natl. Acad. Sci. USA 76:6606, 1979).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503, 1993; Rosenfeld et al., Cell 68:143-155, 1992; Genet. Ther. 4:759-769, 1993; Yang et al., Nature Genet. 7:362-369, 1994; Wilson et al., Nature 365:691-692, 1993; and U.S. Patent No. 5,652,224, which is incorporated herein Reference. For example, the adenoviral vector Ad2 is useful and can be cultured in human 293 cells. These cells contain the El region of the adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene deleted in the vector. In addition to Ad2, other various adenoviruses (eg, Ad3, Ad5 and Ad7) can also be used in the present invention.

Preferably, the adenoviruses used in the present invention are replication defective. Replication-defective adenoviruses require the help of a helper virus and/or a packaging cell line to form infectious particles. The virus thus obtained is capable of infecting cells and expressing the polynucleotide of interest operably linked to the promoter, but cannot replicate in most cells. Replication-defective adenoviruses may have all or part of one or more of the following genes deleted: E1a, E1b, E3, E4, E2a, or L1 to L5.

In certain other embodiments, cells are engineered with adeno-associated virus (AAV) ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97, 1992). It is also one of the few viruses that can integrate its DNA into non-dividing cells. Vectors of as little as 300 base pairs containing AAV can be packaged and integrated, but the space for foreign DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, eg, US Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, a suitable AAV vector for use in the invention will include all sequences necessary for DNA replication, encapsidation, and host cell integration. The polynucleotide constructs are inserted into AAV vectors using standard cloning methods, such as found in Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, 1989. The recombinant AAV vector is then transfected into a packaging cell line infected with a helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once packaging cells are transfected or infected, they will produce infectious AAV viral particles comprising the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells ex vivo or in vivo. Transduced cells will contain the polynucleotide construct integrated into their genome and will express the fusion protein of the invention.

Another approach to gene therapy involves the operably linking of heterologous control regions and endogenous polynucleotide sequences (e.g., encoding polypeptides of the invention) by homologous recombination (see, e.g., U.S. Patent No. 5,641,670 issued June 24, 1997; International Publication No. WO96/29411, published September 26, 1996; International Publication No. WO94/12650, published August 4, 1994; Koller et al., Proc.Natl.Acad.Sci.USA 86:8932-8935, 1989 and Zijlstra et al., Nature 342:435-438, 1989, which are incorporated herein by reference). This method involves the activation of a gene that is present in the target cell but is not normally expressed in the cell or is expressed at a lower than desired level.

A polynucleotide construct comprising a promoter flanked by targeting sequences is prepared using standard techniques known in the art. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination of the promoter-targeting sequence and the endogenous sequence. The targeting sequence will be sufficiently proximal to the 5' end of the desired endogenous polynucleotide sequence that the promoter will be operably linked to the endogenous sequence by homologous recombination.

Promoter and targeting sequences can be PCR amplified. Preferably, the amplified promoter contains distinct restriction enzyme sites at the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter, while the 5' end of the second targeting sequence contains the same restriction enzyme site as the 3' end of the amplified promoter. the same restriction enzyme sites. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cell, either as a naked polynucleotide, or with a transfection-enhancing agent such as liposomes, viral sequences, viral particles, whole virus, lipofection, as described in more detail above. , precipitant and other combinations. The P promoter-targeting sequence can be delivered by any method, including direct needle injection, intravenous injection, topical application, catheter infusion, particle accelerator, and the like. These methods are described in more detail below.

The promoter-targeting sequence construct is taken up by the cell. Homologous recombination occurs between the construct and the endogenous sequence, bringing the endogenous sequence under the control of the promoter. The promoter then drives expression of the endogenous sequence.

A polynucleotide encoding an albumin fusion protein of the invention may contain a secretion signal sequence that promotes secretion of the protein. Typically, the signal sequence is towards or located 5' to the coding region of the polynucleotide to be expressed. The signal sequence may be homologous or heterologous to the polynucleotide of interest, and may be homologous or heterologous to the cell to be transfected. Alternatively, signal sequences can be chemically synthesized using methods known in the art.

Any mode of administration of any of the aforementioned polynucleotide constructs may be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injections, systemic injections, catheter infusions, biolistic injections, particle accelerators (ie, "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (eg, Alza micropumps) , oral or suppository solid (tablet or pill) pharmaceutical preparations, and pour or topical application during surgery. For example, direct injection of naked calcium phosphate-precipitating plasmids into rat liver and rat spleen or direct injection of protein-coated plasmids into the portal vein resulted in gene expression of foreign genes in rat liver (Kaneda et al., Science 243:375, 1989).

A preferred method of topical administration is by direct injection. Preferably, the albumin fusion protein of the invention complexed with a delivery vehicle is administered by direct injection into the artery or topically into the arterial region. Local administration of the composition into the arterial region means injecting the composition into the artery a few centimeters, preferably a few millimeters.

Another method of topical administration is to place a polynucleotide construct of the invention in or around a surgical wound. For example, a patient can be operated on and the polynucleotide construct can be coated on the tissue surface within the wound, or the construct can be injected into the tissue area within the wound.

Therapeutic compositions useful for systemic administration include fusion proteins of the invention complexed to targeted delivery vehicles of the invention. Delivery vehicles suitable for systemic administration include liposomes containing ligands to target the vehicle to specific sites. In specific embodiments, delivery vehicles suitable for systemic administration include liposomes comprising an albumin fusion protein of the invention to target the vehicle to a specific site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injection can be performed by standard methods in the art. Aerosol delivery can also be performed using standard methods in the art (see, eg, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be accomplished by complexing the polynucleotide constructs of the invention with a carrier that resists degradation by digestive enzymes in the gut of the animal. Examples of such carriers include plastic capsules or tablets, such as are known in the art. Topical delivery can be performed by admixing a polynucleotide construct of the invention with a lipophilic agent (eg, DMSO) capable of penetrating the skin.

Determining the effective amount of a substance to be delivered may depend on a variety of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition requiring treatment and its severity, and the route of administration. The frequency of treatment will depend on factors such as the number of polynucleotide constructs administered per dose, and the subject's health status and medical history. The attending physician or veterinarian will determine the exact amount, how often, and when to take it.

The albumin fusion protein of the present invention can be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, with humans being particularly preferred.

biological activity

Albumin fusion proteins and/or polynucleotides encoding albumin fusion proteins of the invention can be used in assays that test for one or more biological activities. If albumin fusion proteins and/or polynucleotides exhibit activity in a particular assay, those therapeutic proteins corresponding to the fusion proteins are likely to be involved in the disease associated with that biological activity. Therefore, the fusion protein can be used to treat related diseases.

In a preferred embodiment, the present invention encompasses a method of treating a disease or disorder listed in the "Preferred Indication Y" column of Table 1, comprising treating, preventing or ameliorating said disease or disorder in an amount effective for treating, preventing or ameliorating said disease or disorder in need of such Treatment, prevention or improvement of patients administered albumin fusion protein of the present invention, the albumin fusion protein comprising the same as Table 1 "Therapeutic Protein X" column disclosed therapeutic protein (and Table 1 "preferred indication Y" column) Listed diseases or disorders are on the same row) corresponding to the Therapeutic Protein section.

In another preferred embodiment, the present invention encompasses methods of treating the diseases or disorders listed in the "Preferred Indications Y" column of Table 1 for specific therapeutic proteins, including effective treatment, prevention or amelioration of the diseases or disorders Amount of Disorder An albumin fusion protein of the invention comprising a Therapeutic protein moiety corresponding to a Therapeutic protein associated with an indication in the Examples is administered to a patient in need of such treatment, prevention or amelioration.

The present invention specifically contemplates albumin fusion proteins produced by cells when encoded by a polynucleotide encoding SEQ ID NO:Y. When these polynucleotides are used to express the encoded protein from a cell, the cell's natural secretion and processing steps produce a protein lacking the signal sequence specifically listed in Table 2, columns 4 and/or 11. The specific amino acid sequence of the listed signal sequence is shown in the specification or is well known in the art. Thus, most preferred embodiments of the invention include albumin fusion proteins produced by cells (which lack the leader sequences listed in columns 4 and/or 11 of Table 2). Most preferred are also polypeptides comprising SEQ ID NO: Y but not having the leader sequence listed in Table 2, columns 4 and/or 11. Compositions, including pharmaceutical compositions, comprising these two preferred embodiments are also preferred. The use of these albumin fusion proteins to treat, prevent, or ameliorate the diseases or disorders listed as specific therapeutic proteins in Table 1 "Preferred Indications: Y" is expressly contemplated.

In a preferred embodiment, the fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of the endocrine system (see for example the section "Endocrine Disorders" below), the nervous system (see for example the section "Neurological Disorders" below) , the immune system (see, e.g., the section "Immune activity" below), the respiratory system (see, e.g., the section "Respiratory Disorders" below), the cardiovascular system (see, e.g., the section "Cardiovascular Disorders" below), the reproductive system (see, e.g., the section "Reproductive Disorders" below), the Systemic disorders" section), diseases and/or disorders related to diseases and disorders of the digestive system (see, eg, "Gastrointestinal disorders" section below), diseases and/or disorders associated with cell proliferation (see, eg, "Hyperproliferative disorders" section below) " section), and/or blood-related diseases or disorders (see, eg, the section "Blood-related disorders" below).

In certain embodiments, the albumin fusion proteins of the invention are useful in the diagnosis and/or prediction of diseases and/or disorders associated with tissues in which the gene corresponding to the Therapeutic protein portion of the fusion protein of the invention is expressed.

Therefore, the fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, detection and/or treatment, including but not limited to prohormone activation, neurotransmitter activity, cell signaling, cell proliferation, cell differentiation , Diseases and/or disorders associated with the activity of cell migration.

More generally, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, prediction, prevention and/or treatment of diseases and/or disorders associated with the following systems.

immune activity

The albumin fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention can be used for treatment, prevention, diagnosis and/or by, for example, activating or inhibiting the proliferation, differentiation, or activity (chemotaxis) of immune cells Or to predict diseases, disorders and/or conditions of the immune system. Immune cells develop in a process called hematopoiesis, giving rise to myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders and/or conditions may be genetic, somatic (such as cancer and some autoimmune diseases), acquired (eg, acquired from chemotherapy or toxins), or infectious. In addition, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful as markers or detectors for specific immune system diseases or disorders.

In another embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat diseases and disorders of the immune system and/or inhibit or enhance the expression of the polypeptide of the present invention and its expression. An immune response produced by cells associated with a tissue.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treating, preventing, diagnosing and/or predicting immunodeficiency, including congenital and acquired immunodeficiency. Examples of B-cell immunodeficiencies in which immunoglobulin levels of B-cell function and/or B-cell numbers are reduced include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia Proteinemia, X-linked hyper-IgM immunodeficiency, non-X-linked hyper-IgM immunodeficiency, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia Gammaglobulinemia, adult-onset agammaglobulinemia, delayed agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, Recessive agammaglobulinemia (Swiss type), selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiency, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Ig, Ig heavy chain deficiency, κ chain deficiency, B-cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI ) (acquired), and transient hypogammaglobulinemia in infants and young children.

In specific embodiments, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat, prevent, diagnose and/or predict ataxia-telangiectasia or ataxia-telangiectasia Conditions related to expansion.

Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or numbers are reduced include, but are not limited to: DiGeorge's anomaly, severe combined immunodeficiency (SCID) (including but not limited to X-linked SCID, Autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, MHC class II deficiency (bare lymphocyte syndrome), Viller-Odd syndrome, ataxia telangiectasia) , thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphopenia, dominant T Immunodeficiency of cellular deficiency (uncharacterized), and immunodeficiency of uncharacterized cell-mediated immunity.

In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, prevent, diagnose and/or predict or be associated with DiGeorge's abnormality status.

Other immune deficiencies that can be treated, prevented, diagnosed and/or predicted with the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, chronic granulomatous disease, Chel-Health syndrome, Myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiency (including C1, C2, C3, C4, C5 , C6, C7, C8, and/or C9 deficiencies), reticulocyte hypoplasia, thymolymphoid hypoplasia-agenesis, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal Children with neutropenia, short limb dwarfism, and Nezrov's syndrome combined with Ig immunodeficiency.

In a preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat, prevent, diagnose and/or predict the immunodeficiency and/or conditions related to the above-mentioned immunodeficiency .

In a preferred embodiment, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful as agents for enhancing immune responsiveness in immunodeficient individuals. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful as agents for enhancing immune responsiveness in B-cell and/or T-cell immunodeficient individuals.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention, diagnosis and/or prediction of autoimmune disorders. Many autoimmune disorders are caused by immune cells misidentifying themselves as foreign substances. This misrecognition triggers an immune response that leads to the destruction of host tissues. Therefore, administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention capable of inhibiting the proliferation, differentiation, or chemotaxis of immune responses, particularly T cells, may be an effective treatment for preventing autoimmune disorders .

The autoimmune diseases and disorders that can be treated, prevented, diagnosed and/or predicted with the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention include but are not limited to one or more of the following : Systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmunity thrombocytopenic purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, purpura (e.g., Henry-Schörner's purpura), autoimmune cytopenias, Goodpasque syndrome, vulgaris Herpes, myasthenia gravis, Graves' disease (hyperthyroidism), and insulin-resistant diabetes.

Other disorders that may have autoimmune factors that can be treated, prevented, and/or diagnosed with albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, type II collagen-induced arthritis, Antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrine disease, Reiter's disease, stiff man syndrome, autologous Immune lung inflammation, autism, Guillain-Barr syndrome, insulin-dependent diabetes, and autoimmune inflammatory eye disease.

Other disorders that may have autoimmune factors that can be treated, prevented, diagnosed, and/or predicted with albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, sclerosis with anti-collagen antibodies. Dermatosis (usually characterized by, for example, nucleolar and other nuclear antibodies), mixed connective tissue disease (usually characterized by, for example, antibodies against extractable nuclear antigens (such as nucleoproteins), polymyositis (usually characterized by, for example, non-histone ANA ), pernicious anemia (usually characterized by, for example, anti-parietal cell, microsomal, and intrinsic factor antibodies), idiopathic Addison's disease (usually characterized by, for example, humoral and cell-mediated adrenal cytotoxicity), infertility (usually characterized by e.g. antisperm antibodies), glomerulonephritis (usually characterized by e.g. glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (usually characterized by e.g. IgG and complement in the basement membrane), Sjogren's syndrome (usually characterized by, for example, multitissue antibodies, and/or specific non-histone ANA (SS-B)), diabetes (often characterized by, for example, cell-mediated and humoral islet cell antibodies), and Adrenergic drug resistance (including that associated with asthma or cystic fibrosis) (often characterized by eg beta-adrenergic receptor antibodies).

Other disorders that may have an autoimmune component that can be treated, prevented, diagnosed and/or predicted with the albumin fusion proteins of the invention and/or polynucleotides encoding the albumin fusion proteins of the invention include, but are not limited to, chronic active hepatitis (usually characterized by e.g. smooth muscle antibodies), primary gallbladder sclerosis (usually characterized by e.g. mitochondrial antibodies), failure of other endocrine glands (in some cases usually characterized by e.g. tissue-specific antibodies), vitiligo (usually characterized by e.g. melanocyte antibodies), Vasculitis (usually characterized by, for example, Ig and complement in the vessel wall and/or low serum complement), post-MI (usually characterized by, for example, myocardial antibodies), cardiotomy syndrome (usually characterized by, for example, myocardial antibodies), Urticaria (usually characterized by, for example, IgG and IgM antibodies to IgE), atopic dermatitis (usually characterized by, for example, IgG and IgM antibodies to IgE), asthma (usually characterized by, for example, IgG and IgM antibodies to IgE), and Many other inflammatory, granulomatous, degenerative and atrophic disorders.

In a preferred embodiment, for example, the fusion protein of the invention and/or the polynucleotide encoding the albumin fusion protein of the invention are used to treat, prevent, diagnose and/or predict autoimmune diseases associated with the above-mentioned diseases and disorders and disorders and/or conditions. In a specific preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat, prevent and/diagnose rheumatoid arthritis.

In another specific preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention is used to treat, prevent and/diagnose systemic lupus erythematosus. In another specific preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention is used to treat, prevent and/diagnose idiopathic thrombocytopenic purpura.

In another specific preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention is used to treat, prevent and/diagnose IgA nephropathy.

In a preferred embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat, prevent, diagnose and/or predict autoimmune diseases and Disorders and/or Conditions.

In a preferred embodiment, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as immunosuppressants.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used for the treatment, prevention, prediction and/or diagnosis of diseases, disorders and/or conditions of hematopoietic cells. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the treatment or prevention of those diseases, disorders and/or conditions associated with a decrease in certain (or many) types of hematopoietic cells, including In efforts to, but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia, differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, are enhanced. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the treatment or prevention of those diseases, disorders and/or conditions associated with certain (or many) types of hematopoietic cell hyperplasia, including In an effort not limited to histiocytosis, the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, are enhanced.

Fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to treat, prevent, diagnose and/or predict allergies and conditions, such as asthma (especially allergic asthma) or other respiratory problems . In addition, these molecules are useful in the treatment, prevention, prediction and/or diagnosis of allergy, hypersensitivity to antigenic molecules, or blood group incompatibility.

In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treatment, prevention, diagnosis and/or prediction of IgE-mediated allergy. Such allergies include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to modulate IgE concentrations in vitro or in vivo.

In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to diagnose, predict, prevent and/or treat inflammatory conditions. For example, since the fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can inhibit the activation, proliferation and/or differentiation of cells involved in inflammatory responses, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. These inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection , nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, cytokine (eg, TNF or IL-1) overproduction, respiratory disorders (eg, asthma and allergy); gastric Bowel disorders (eg, inflammatory bowel disease); cancers (eg, stomach, ovary, lung, bladder, liver, and breast); CNS disorders (eg, multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury , neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion diseases); cardiovascular disorders (such as atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); and many other diseases, conditions, and disorders characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Greve Stuart's disease, systemic lupus erythematosus, diabetes, and allograft rejection).

Because inflammation is a fundamental defense mechanism, inflammatory disorders can affect virtually any tissue in the body. Therefore, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat tissue-specific inflammatory disorders, including but not limited to adrenalitis, alveolitis (alveolitis), cholangiocholecystitis, appendicitis, Balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, vocal cord inflammation, cochitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, cardiac Endometritis, esophagitis, eustachian tube inflammation, fibrous tissue inflammation, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, otitis interna, laryngitis, lymphangitis, mastitis , otitis media, meningitis, metritis, mucositis, myocarditis, myositis, tympanitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, tenosynovitis, peritonitis, pharyngitis, phlebitis, poliomyelitis , prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerachoroiditis, scrotumitis, sinusitis, spondylitis, adipose tissue inflammation, stomatitis, arthritis, eustachian tube inflammation, tendonitis , tonsillitis, urethritis, and vaginitis.

In a specific embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosing, predicting, preventing and/or treating organ transplant rejection and graft-versus-host disease. Organ rejection occurs by host immune cells destroying the transplanted tissue through an immune response. Similarly, GVHD also involves immune responses, but in this case, foreign transplanted immune cells destroy host tissues. Polypeptides, antibodies or polynucleotides of the invention and/or agonists or antagonists thereof that inhibit immune responses, particularly activation, proliferation, differentiation or chemotaxis of T cells, may be an effective treatment for preventing organ rejection or GVHD. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that inhibit immune responses, particularly activation, proliferation, differentiation or chemotaxis of T cells, may be prophylactic assays. Effective treatment for allergic and hyperacute xenograft rejection.

In other embodiments, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to diagnose, predict, prevent and/or treat immune complex diseases, including but not limited to serum sickness, streptococcal Postinfectious glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treating, detecting and/or preventing infectious agents. For example, by enhancing the immune response, particularly the proliferation, activation and/or differentiation of B and/or T cells, infectious diseases can be treated, detected and/or prevented. The immune response can be enhanced by enhancing an existing immune response or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also directly inhibit infectious agents (refer to the section of the application listing infectious agents, etc.) without eliciting an immune response.

In another embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as a vaccine adjuvant to enhance immune responsiveness to an antigen. In a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as an adjuvant to enhance tumor-specific immune response.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as an adjuvant to enhance the antiviral immune response. Antiviral immune responses that can be enhanced using the compositions of the invention as adjuvants include viruses and virus-associated diseases or symptoms described herein or otherwise known in the art. In a specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response against a virus, disease or condition selected from the group consisting of AIDS, meningitis, dengue, EBV, and hepatitis (eg hepatitis B). In another specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response against a virus, disease or condition selected from the group consisting of: HIV/AIDS, Respiratory Syncytial Virus, Dengue Virus, Rotavirus , Japanese encephalitis B, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin virus, chikungunya, Rift Valley fever, herpes simplex, and yellow fever.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention is used as an adjuvant to enhance antibacterial or antifungal immune response. Antibacterial or antifungal immune responses that may be enhanced using the compositions of the present invention as adjuvants include bacteria or fungi or bacteria and fungi bacteria or fungi associated diseases or symptoms described herein or otherwise known in the art. In a specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response against bacteria or fungi, diseases or conditions selected from the group consisting of tetanus, diphtheria, botulism, and meningitis B.

In another specific embodiment, the composition of the present invention is used as an adjuvant to enhance the immune response against bacteria or fungi, diseases or symptoms selected from the group consisting of Vibrio cholerae, Mycobacterium leprae ( Mycobacterium leprae), Salmonella typhi, Salmonella paratyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B Streptococcus, Shigella spp. ), enterotoxigenic Escherichia coli, enterohaemorrhagic Escherichia coli, and Borrelia burgdorferi.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as an adjuvant to enhance anti-parasitic immune response. Anti-parasitic immune responses that may be enhanced using the compositions of the present invention as adjuvants include parasites and parasite-associated diseases or conditions described herein or otherwise known in the art. In a specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response against parasites. In another specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response against Plasmodium (malaria) or Leishmania.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used to treat infectious diseases, including silicosis, sarcoidosis, and idiopathic pulmonary Fibrosis, for example by preventing the recruitment and activation of mononuclear phagocytes.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as antigens to generate antibodies to inhibit or enhance immune mediation against the polypeptide of the present invention reaction.

In one embodiment, an albumin fusion protein of the invention and/or a polynucleotide encoding an albumin fusion protein of the invention is administered to an animal (e.g. mouse, rat, rabbit, hamster, guinea pig, pig, minipig, Chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to enhance the immune system to produce greater amounts of one or more antibodies (e.g., IgG, IgA, IgM and IgE), induce the production of higher affinity antibodies and immunoglobulin class switching (such as IgG, IgA, IgM and IgE), and/or enhance the immune response.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as stimulators of B cell responsiveness to pathogens.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as activators of T cells.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as agents to elevate the immune status of an individual before receiving immunosuppressive therapy.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as agents for inducing higher affinity antibodies.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as agents for increasing serum immunoglobulin concentrations.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as agents to accelerate recovery in immunocompromised individuals.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention is used as a medicament for enhancing the immune response of the elderly population and/or neonates.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used prior to bone marrow transplantation and/or other transplantation (such as allogeneic or xenogeneic organ transplantation) , during, or after an immune system booster. For transplantation, the compositions of the invention may be administered before, concurrently with, and/or after transplantation. In a specific embodiment, the composition of the invention is administered after transplantation but before the recovery of the T cell population begins. In another specific embodiment, the composition of the invention is first administered after the T cell population has begun to recover after transplantation, but before the B cell population has fully recovered.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as agents to enhance immune responsiveness in individuals with acquired loss of B cell function. Conditions leading to acquired loss of B cell function that can be ameliorated or treated by administration of albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, HIV infection, AIDS, bone marrow transplantation, and B-cell chronic lymphocytic leukemia (CLL).

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as agents to enhance immune responsiveness in individuals with transient immunodeficiency. Conditions leading to transient immunodeficiency that may be improved or treated by administration of albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, recovery from viral infection (e.g., influenza), and Malnutrition-related conditions, recovery from infectious mononucleosis, stress-related conditions, recovery from measles, recovery from blood transfusions, and recovery from surgery.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as modulators of monocyte, dendritic cell, and/or B cell antigen presentation . In one embodiment, an albumin fusion protein of the invention and/or a polynucleotide encoding an albumin fusion protein of the invention enhances antigen presentation or antagonizes antigen presentation in vitro or in vivo. Additionally, in related embodiments, this enhancement or antagonism of antigen presentation can be used as an anti-tumor therapy or to modulate the immune system.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as a means to guide the development of an individual's immune system towards a humoral response (i.e. TH2) rather than a TH1 cellular response. potion.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as a means to induce tumor proliferation, thereby making it more susceptible to the effects of anti-neoplastic drugs. For example, multiple myeloma is a slowly dividing disease and therefore refractory to virtually all antineoplastic regimens. If these cells are forced to proliferate more quickly, their susceptibility profile is likely to change.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used in pathologies such as AIDS, chronic lymphocyte disorder and/or common variant immunodeficiency Stimulator of B cell production.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as a therapeutic method for the generation and/or regeneration of lymphoid tissue after surgery, trauma or genetic defect. In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used for pretreatment of bone marrow samples before transplantation.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as congenital genetic disorders leading to immune anergy/immunodeficiency such as observed in SCID patients. Gene-based therapies for disorders.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to activate monocytes/macrophages against parasitic diseases affecting monocytes such as Means of Leishmania.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of modulating secreted cytokines elicited by polypeptides of the invention.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in one or more of the applications described herein, just as they may be used in veterinary applications.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of blocking various aspects of the immune response against foreign agents or self. Examples of diseases or conditions in which it may be desirable to block certain aspects of the immune response include autoimmune disorders such as lupus, and arthritis, as well as immune responsiveness to skin allergies, inflammation, enteropathy, injury, and pathogen-associated Disease/disorder.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to prevent and treat autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus Treatment of B cell proliferation and Ig secretion associated with multiple sclerosis.

In another specific embodiment, the fusion protein of the present invention and/or the polypeptide, antibody, polynucleotide and/or agonist or antagonist of the polynucleotide encoding the albumin fusion protein of the present invention are used as B in endothelial cells and/or inhibitors of T cell migration. This activity disrupts tissue architecture or similar responses, and can be used, for example, to disrupt immune responses, and prevent sepsis.

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used as monoclonal gammopathy uncharacterized (MGUS), Waldenstrom Treatment of chronic hypergammaglobulinemia evident in diseases such as Cerlin's disease, related idiopathic monoclonal gammopathy, and plasmacytoma.

In another specific embodiment, the albumin fusion protein of the invention and/or the polynucleotide encoding the albumin fusion protein of the invention can be used, for example, to inhibit polypeptides in certain autoimmune and chronic inflammatory and infectious diseases Chemotaxis and activation of macrophages and their precursors, neutrophils, basophils, B lymphocytes and some T cell subsets such as activated and CD8 cytotoxic T cells and natural killer cells. Examples of autoimmune diseases are described herein, including multiple sclerosis and insulin-dependent diabetes.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also treat idiopathic hypereosinophilic syndrome by, for example, preventing eosinophil production and migration.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit complement-mediated cell lysis.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit antibody-dependent cellular cytotoxicity.

In another specific embodiment, the albumin fusion protein of the invention and/or the polynucleotide encoding the albumin fusion protein of the invention can also be used in the treatment of atherosclerosis, for example by preventing the infiltration of monocytes in the arterial wall .

In another specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat adult respiratory distress syndrome (ARDS).

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to stimulate wound and tissue repair, stimulate angiogenesis, and/or stimulate blood or lymphatic vessels Repair of disease or disorder. In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to stimulate regeneration of mucosal surfaces.

In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in the diagnosis, prediction, treatment and/or prevention of primary or acquired immunodeficiencies characterized by , insufficient production of serum immunoglobulins, recurrent infections, and/or disorders of immune system dysfunction. In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat or prevent joint, bone, skin, and/or parotid gland infection, blood-borne infection (such as sepsis, meningitis, pus toxic arthritis, and/or osteomyelitis), autoimmune diseases (such as disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disease associated with these infections, diseases, disorders, and/or malignancies Disorders or conditions, including but not limited to CVID, other primary immunodeficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g. severe shingles herpes), and/or Pneumocystiscarnii. Other diseases and disorders that can be prevented, diagnosed, predicted and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, Phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in the treatment and/or diagnosis of common variable immunodeficiency disease ("CVID"; also known as Individuals with "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or subclasses of this disorder.

In a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of cancer or neoplasm, including immune cells or immune Tissue-associated cancer or neoplasm. Examples of cancers or neoplasms that can be prevented, diagnosed or treated with the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma, EBV-transformed disease, and/or Diseases and disorders described elsewhere in the section entitled "Hyperproliferative Disorders".

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a method of treatment for reducing proliferation of large B-cell lymphoma cells.

In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means to reduce the involvement of B cells and Ig associated with chronic myelogenous leukemia.

In a specific embodiment, the compositions of the invention are used as agents to enhance immune responsiveness in B-cell immunodeficient individuals such as, for example, individuals who have undergone partial or total splenectomy.

blood related disorders

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to modulate hemostatic (stopping bleeding) or thrombolytic (clot dissolving) activity. For example, by enhancing hemostatic or thrombolytic activity, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the treatment or prevention of blood coagulation diseases, disorders and/or conditions (e.g., fibrinogen deficiency anemia, factor deficiency, hemophilia), platelet disease, disorder and/or condition (such as thrombocytopenia), or injury resulting from trauma, surgery, or other causes. Alternatively, fusion proteins of the invention capable of reducing hemostatic or thrombolytic activity and/or polynucleotides encoding albumin fusion proteins of the invention may be used to inhibit or dissolve clots. These molecules may play an important role in the treatment or prevention of heart attack (infarction), stroke, or scarring.

In a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the prevention, diagnosis, prediction and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, Thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful for preventing occlusion of saphenous vein grafts, reducing the occlusions that may accompany angioplasty Reduce the risk of perioperative thrombosis, reduce the risk of stroke in patients with atrial fibrillation, including non-rheumatic atrial fibrillation, and reduce the risk of embolism associated with prosthetic heart valves and/or mitral valve disease. Other uses of albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, the prophylaxis of extracorporeal devices (e.g., intravascular catheters, vascular access shunts in hemodialysis patients, blood Obstructions in dialysis units, and cardiopulmonary bypass units).

In another embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the prevention, diagnosis, prediction and/or treatment of diseases related to the tissue in which the polypeptide of the present invention is expressed. Diseases and disorders of the blood and/or blood-forming organs.

Fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to modulate hematopoietic activity (formation of blood cells). For example, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to increase all blood cells or a subset of blood cells such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g. The number of basocytes, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to reduce the number of blood cells or subsets of blood cells can be used in the prevention, detection, diagnosis and/or treatment of anemia and leukopenia as described below. Alternatively, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to reduce all blood cells or a subset of blood cells such as, for example, red blood cells, lymphocytes (B or T cells), myeloid cells (e.g. The number of basocytes, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to reduce the number of blood cells or subsets of blood cells is useful in the prevention, detection, diagnosis and/or treatment of leukocytosis such as, for example, eosinophilia.

The fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for preventing, treating or diagnosing blood dyscrasia.

Anemia is a condition in which the number of red blood cells or the amount of hemoglobin (the protein that carries oxygen) in them is lower than normal. Anemia may be caused by excessive bleeding, decreased production of red blood cells, or increased destruction of red blood cells (hemolysis). The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention and/or diagnosis of anemia. The anemia that can be treated, prevented or diagnosed with the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention includes iron deficiency anemia, hypohemochromic anemia, microcytic anemia, chlorosis (chlorosis) ), inherited sideroblastic anemia, idiopathic acquired sideroblastic anemia, erythrocyte aplasia, megaloblastic anemia (eg, pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia , Hemolytic anemia (such as autoimmune hemolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention and/or diagnosis of anemia associated with the following diseases, including but not limited to systemic lupus erythematosus, Cancer, lymphoma, chronic kidney disease, and anemia associated with enlarged spleen. The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention and/or diagnosis of anemia caused by drug treatment, such as with methyldopa, dapsone, and/or or sulfa drug-related anemia. In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention and/or diagnosis of anemia associated with abnormal red blood cell architecture, including but not limited to hereditary spherocytosis, hereditary Elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention and/or diagnosis of hemoglobin abnormalities (such as those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease related). In addition, albumin fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can be used for diagnosis, prediction, prevention and/or treatment of thalassemia, including but not limited to severe and α-thalassemia minor and β-thalassemia minor.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can be used for diagnosis, prediction, prevention and/or treatment of bleeding disorders, including but not limited to thrombocytopenia ( e.g. idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), von Willebrand's disease, hereditary platelet disorders (e.g., storage pool diseases, such as Chech-Sch's syndrome and Heck-Pull Schneider's syndrome, thromboxane A2 dysfunction, thrombocytopenia, and Burt-Surrey's syndrome), hemolytic uremic syndrome, hemophilias such as hemophilia A or factor VII deficiency, and Keith's disease (hemophilia B) or factor IX deficiency, hereditary hemorrhagic telangiectasia, also known as Lange-Ouer-Wayson syndrome, allergic purpura (Henner-Schönlein purpura) and disseminated intravascular coagulation.

The effects of albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention on blood clotting time can be monitored using any coagulation assay known in the art, including but not limited to whole blood partial procoagulant zymogenase time (PTT), activated partial thromboplastin time (aPTT), activated clotting time (ACT), recalcification activated clotting time, or Lee-White clotting time.

Several diseases and various drugs can cause platelet dysfunction. Therefore, in a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of acquired platelet dysfunction, such as accompanying Renal failure, leukemia, multiple myeloma, cirrhosis, and platelet dysfunction in systemic lupus erythematosus and associated with drug therapy, including high-dose aspirin, ticlopidine, nonsteroids Anti-inflammatory drugs (for arthritis, pain, and sprains), and penicillin.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, prediction, prevention and/or treatment of or are associated with increased or decreased white blood cell numbers diseases and disorders. Leukopenia occurs when the number of white blood cells decreases below normal levels. Leukopenia includes, but is not limited to, neutropenia and lymphopenia. An increase in the number of white blood cells relative to normal levels is called leukocytosis. During an infection, the body produces an increased number of white blood cells. Therefore, leukocytosis may simply be a normal physiological parameter reflecting infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leukocytosis includes, but is not limited to, eosinophilia and macrophage accumulation. In specific embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful for diagnosis, prediction, prevention and/or treatment of leukopenia. In other specific embodiments, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of leukocytosis.

Leukopenia can be a general decrease of all types of white blood cells, or it can be a specific loss of a particular type of white blood cells. Thus, in specific embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, prediction, prevention and/or treatment of a condition known as neutropenia Decreased number of neutrophils. The neutropenia that can be diagnosed, predicted, prevented and/or treated with the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, infantile hereditary agranulocytosis, family Chronic neutropenia, cyclic neutropenia, neutropenia resulting from or associated with dietary deficiency (eg, vitamin B12 deficiency or folic acid deficiency), resulting from drug therapy (eg, antibiotic regimens such as Penicillin therapy, sulfonamide drug therapy, anticoagulant therapy, anticonvulsant drugs, antithyroid drugs, and cancer chemotherapy) or associated neutropenia, and neutrophils resulting from increased neutrophil destruction Increased neutrophil destruction may be associated with some conditions in individuals with bacterial or viral infections, allergic disorders, autoimmune diseases, splenomegaly (eg, Felty's syndrome, malaria, and sarcoidosis), and some related to drug therapy.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of lymphopenia (decrease in the number of B and/or T lymphocytes), including but not Limitation due to stress, drug therapy (e.g., corticosteroid drug therapy, cancer chemotherapy, and/or radiation therapy), AIDS infection, and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infection, some viruses Lymphopenia in or associated with infection and/or hereditary disorders (eg, DiGeorge syndrome, Wilhelm-Auer syndrome, severe combined immunodeficiency, ataxia telangiectasia).

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to diagnose, predict, prevent and/or treat diseases and disorders related to the number of macrophages and/or the function of macrophages, These include, but are not limited to, Gaucher's disease, Nie-Peter's disease, Lay-Sey's disease, and Han-Schuh-Craig's disease.

In another embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment related to the number of eosinophils and/or eosinophils Diseases and disorders related to erythrocyte function, including but not limited to idiopathic hypereosinophilic syndrome, eosinophil-myalgia syndrome, and Han-Schücker syndrome sick.

In yet another embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, prediction, prevention and/or treatment of leukemia and lymphoma, including but not limited to acute lymphoma Cellular (lymphoblastic) leukemia (ALL), acute myeloid (myelocytic, myeloid, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (such as B-cell leukemia, T-cell leukemia, Sezary syndrome, and hairy cell leukemia), chronic myeloid (myeloid, myeloid, or granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma , Burkitt's lymphoma, and mycosis fungoides.

In other embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, prediction, prevention and/or treatment of diseases and disorders of plasma cells, including but not limited to plasma cells Cellular dyscrasia, monoclonal gammopathy, uncharacterized monoclonal gammopathy, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia , and Raynaud's phenomenon.

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can be used to treat, prevent and/or diagnose myeloproliferative disorders, including but not limited to polycythemia vera, relative Polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, unexplained myeloid metaplasia, thrombocytosis (both primary and secondary), and chronic myelogenous leukemia.

In other embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in preoperative treatments to increase hematopoiesis.

In other embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to enhance the migration, phagocytosis, and phagocytosis of neutrophils, eosinophils, and macrophages. action, superoxide generation, and antibody-dependent cytotoxicity.

In other embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful as agents to increase the number of stem cells in circulation prior to stem cell extraction. In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as agents to increase the number of circulating stem cells prior to platelet extraction.

In other embodiments, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful as agents that increase cytokine production.

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can be used for the prevention, diagnosis and/or treatment of primary hematopoietic disorders.

hyperproliferative disorder

In certain embodiments, fusion proteins of the invention and/or polynucleotides of albumin fusion proteins of the invention are useful in the treatment or detection of hyperproliferative disorders, including neoplasms. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can inhibit the proliferation of a disorder through direct or indirect interactions. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may enable the proliferation of other cells capable of inhibiting hyperproliferative disorders.

For example, hyperproliferative disorders can be treated by increasing the immune response, particularly increasing the quality of the antigen of the hyperproliferative disorder, or by allowing T cells to proliferate, differentiate, or mobilize. This immune response can be increased, either by enhancing an existing immune response, or by initiating a new immune response. Alternatively, reducing the immune response may also be a method of treating hyperproliferative disorders, such as chemotherapeutic agents.

Examples of hyperproliferative disorders that may be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, neoplasms located in the following locations: colon, abdomen, bone, breast, digestive tract System, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen , thoracic cavity, and genitourinary tract.

Similarly, other hyperproliferative disorders can also be treated or detected using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of these hyperproliferative disorders include, but are not limited to: acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) Hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin Chiggin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignancy, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, Brain tumor, breast cancer, renal pelvis and ureter cancer, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood (primary) liver cell carcinoma, childhood (primary) liver cancer, childhood severe acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brainstem glioma, childhood cerebellar astrocytoma, childhood brain Astrocytoma, extracranial germ cell tumor in childhood, Hodgkin's disease in childhood, Hodgkin's lymphoma in childhood, hypothalamic and optic pathway glioma in childhood, lymphoblastic leukemia in childhood, childhood medulloblastoma, non-Hodgkin's lymphoma in childhood, pineal and supratentorial primitive neuroectodermal tumor in childhood, primary liver cancer in childhood, rhabdomyosarcoma in severe childhood, soft tissue sarcoma in childhood, childhood Peripheral pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer, etympanoma, epithelial cancer, esophagus Carcinoma, Ewing's sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer, female breast cancer, Gaucher's disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumor, germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia , hypopharyngeal cancer, bowel cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lung cancer, lymphoproliferative disorders, Macroglobulinemia, female breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic occult primary squamous cell neck carcinoma, metastatic primary Squamous cell neck cancer, metastatic squamous cell neck cancer, multiple myeloma, multiple myeloma/plasma cell neoplasm, myelodysplastic syndrome, myeloid leukemia, myeloid leukemia, myeloproliferative disorder, Nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma of pregnancy, non-melanoma skin cancer, non-small cell lung cancer, occult primary metastatic squamous cell neck cancer , oropharyngeal carcinoma, bone-/malignant fibrosarcoma, osteosarcoma/malignant fibrous histiocytoma, osteosarcoma/malignant fibrous tissue of bone Cytoma, ovarian epithelial carcinoma, ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, paraproteinemia, purpura, parathyroid carcinoma, penile carcinoma, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple Myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoid sarcoma, Seza Li syndrome, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal and pineal tumors, T cell lymphoma, testicular cancer, thymoma, thyroid cancer , Transitional cell carcinoma of the renal pelvis and ureter, Transitional carcinoma of the renal pelvis and ureter, Trophoblastic tumor, Cell carcinoma of the ureter and renal pelvis, Urethral carcinoma, Uterine carcinoma, Uterine sarcoma, Vaginal carcinoma, Glioma of the optic pathway and hypothalamus, Vaginal carcinoma , Waldenstrom's macroglobulinemia, Wilms' neoplasm, and any other hyperproliferative disease other than neoplasms located in the organ systems listed above.

In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used for diagnosis, prediction, prevention, and/or treatment of premalignant conditions, and for the prevention of Development of a neoplastic or malignant state, including but not limited to those disorders described above. These uses are indicated by conditions known or suspected to occur in the aforementioned progression to neoplastic or cancerous growths, in particular hyperplasia, metaplasia, or most particularly dysplasia consisting of non-neoplastic cell growth (regarding A review of these abnormal growth conditions can be found in Robbins and Angell, 1976, "Basic Pathology", 2nd ed., W.B. Saunders Co., Philadelphia, pp. 68-79).

Hyperplasia is a form of controlled cell proliferation involving an increase in the number of cells in a tissue or organ without major changes in structure or function. Proliferative disorders that can be diagnosed, predicted, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, vascular follicular mediastinal lymph node hyperplasia, eosinophilia Increased angiolymphoid hyperplasia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign large lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture Hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of the prostate, nodules Nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.

Metaplasia is a form of controlled cell growth in which one type of mature or fully differentiated cell replaces another type of mature cell. Metaplastic disorders that can be diagnosed, predicted, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, idiopathic myeloid metaplasia, apocrine metaplasia , atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous cell metaplasia, amniotic squamous metaplasia, and symptomatic myeloid metaplasia.

Dysplasia is often a precursor to cancer and is found primarily in the epithelium; it is the most disordered form of nonneoplastic cell growth involving loss of individual cell identity and cellular architectural orientation. Dysplastic cells often have abnormally large, heavily stained nuclei and exhibit attenuation. Dysplasia characteristically occurs in the presence of chronic irritation or inflammation. Dysplastic disorders that can be diagnosed, predicted, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxia Dysplasia of the thorax, abnormal development of the atrium and fingers, abnormal development of the bronchopulmonary, abnormal development of the brain, abnormal development of the cervix, abnormal development of the cartilage ectoderm, abnormal development of the clavicle and skull, abnormal development of the congenital ectoderm, abnormal development of the cranial shaft, and development of the cranial carpal tarsus Abnormal, cranial metaphyseal dysplasia, dentine dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia, cerebral eyeball dysplasia, unilateral epiphyseal dysplasia, multiple epiphyseal dysplasia, punctate epiphysis Abnormal, epithelial dysplasia, facial digital (toe) genital dysplasia, familial fibrous dysplasia of jaw (jaw), familial white fold dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, vigorous bony development Abnormal, hereditary renal and retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphocytopenic thymic dysplasia, mammary gland dysplasia, mandibular and facial dysplasia, metaphyseal dysplasia, Mundi Neal dysplasia, monoskeletal fibrous dysplasia, mucosal epithelial dysplasia, multiple epiphyseal dysplasia, ocular and ear vertebral dysplasia, ocular vertebral dysplasia, odontogenic dysplasia, oculomandibular branch dysplasia, periapical dentin Dysplasia, bony fibrous dysplasia, pseudoachondropplasticspondyloepiphysial dysplasia, retinal dysplasia, septal eye dysplasia, spinal epiphyseal dysplasia, and ventricular radial dysplasia.

Other preneoplastic disorders that can be diagnosed, predicted, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrous cystic conditions, tissue hypertrophy, intestinal polyps, colonic polyps, and esophageal dysplasia), leukoplakia, keratosis, Bowen's disease, peasant's skin, solar cheilitis, and solar keratosis.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis and/or prediction of disorders associated with tissues in which the polypeptides of the invention are expressed.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to toxins or radioisotopes as described herein are useful in the treatment of cancers and neoplasms, including but Not limited to what is described herein. In yet another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to toxins or radioisotopes as described herein are useful in the treatment of acute myelogenous leukemia.

In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can affect apoptosis and thus can be used to treat many diseases associated with enhanced cell survival or inhibition of apoptosis. For example, diseases associated with enhanced cell survival or inhibition of apoptosis that can be diagnosed, predicted, prevented, and/or treated with the polynucleotides, polypeptides, and/or agonists or antagonists of the invention include carcinomas (such as follicular lymphoid Tumors, carcinomas with p53 mutations, and hormone-dependent tumors, including but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, bowel cancer, testicular cancer , gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer), autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis , systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpesviruses, poxviruses, and adenoviruses), inflammation, graft-versus-host disease, acute transplant exclusion, and chronic transplantation repel.

In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit the growth, progression, and/or metastasis of cancer, particularly those listed above.

Other diseases or conditions associated with enhanced cell survival that can be diagnosed, predicted, prevented, and/or treated using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, malignancies and related disorders The development and/or metastasis of leukemia (including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblasts, promyelocytes (promyelocytes), myelomonocytes, monocytes) and erythroleukemia)) and chronic leukemias (such as chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (such as Hodgkin's disease and non-Hodgkin's disease) , multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic Sarcomas, chordomas, angiosarcomas, endothelial sarcomas, lymphangiosarcomas, lymphangioendothelial sarcomas, synovial tumors, mesotheliomas, Ewing tumors, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer Carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, villi Membranous carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma craniopharyngioma, efferentoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with enhanced apoptosis that can be diagnosed, predicted, prevented, and/or treated with the fusion protein of the invention and/or the polynucleotide encoding the albumin fusion protein of the invention include AIDS; neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, and brain tumors or related diseases); autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis); myelodysplastic syndrome diseases (such as aplastic anemia), graft-versus-host disease, ischemic injury (such as that caused by myocardial infarction, stroke, and reperfusion injury), liver injury (such as hepatitis-related liver injury, ischemia/reperfusion injury, cholestasis (injury to the bile ducts), and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia, and anorexia.

Hyperproliferative diseases and/or disorders that can be diagnosed, predicted, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, neoplasms located at: Liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system , pelvis, skin, soft tissue, spleen, ribcage, and genitourinary tract.

Similarly, other hyperproliferative disorders can also be diagnosed, predicted, prevented, and/or treated using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of these hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorder, paraproteinemia, purpura, sarcoidosis, Sezary's syndrome, Waldenstrom's macroglobule Proteinemia, Gaucher's disease, histiocytosis, and any other hyperproliferative disorder in the organ systems listed above other than neoplasms.

Another preferred embodiment utilizes polynucleotides encoding albumin fusion proteins of the invention to inhibit abnormal cell division by using gene therapy and/or protein fusions or fragments thereof of the invention.

Thus, the present invention provides methods for treating cell proliferative disorders by inserting a polynucleotide encoding an albumin fusion protein of the invention into abnormally proliferating cells, wherein said polynucleotide represses said expression.

Another embodiment of the invention provides a method for treating a cell proliferative disorder in an individual comprising administering to abnormally proliferating cells one or more copies of an active gene of the invention. In a preferred embodiment, the polynucleotide of the present invention is a DNA construct comprising a recombinant expression vector that efficiently expresses the DNA sequence encoding said polynucleotide. In another preferred embodiment of the invention, the DNA construct encoding the fusion protein of the present invention is inserted into the cells to be treated using a retrovirus or more preferably an adenovirus vector (see G.J.Nabel et al., PNAS, 1999, 96: 324-326, which are incorporated herein by reference). In a most preferred embodiment, the viral vector is defective and does not transform non-proliferating cells, only proliferating cells. Furthermore, in a preferred embodiment, the polynucleotides of the present invention are inserted into proliferating cells either alone or in combination or fusion with other polynucleotides, which can then be induced by acting on a promoter upstream of said polynucleotides. External stimuli (ie, magnetism, specific small molecule, chemical, or drug administration, etc.) that encode protein product expression are regulated. As such, the beneficial therapeutic effects of the invention (ie, increased, decreased, or inhibited expression of the invention) can be specifically modulated in response to the external stimulus.

The polynucleotides of the invention can be used to suppress the expression of oncogenes or antigens. "Suppressing the expression of an oncogene" means repressing gene transcription, degrading gene transcripts (pre-messenger RNA), inhibiting splicing, disrupting messenger RNA, preventing post-translational processing of proteins, disrupting proteins, or inhibiting normal protein function.

For local administration to abnormally proliferating cells, the polynucleotides of the present invention can be administered by any method known to those skilled in the art, including but not limited to transfection of cells, electroporation, microinjection, or in the form of liposomes, etc. In vector, lipofection, or as naked polynucleotides, or any other method described throughout the specification. Polynucleotides of the present invention can be delivered by known gene delivery systems, such as but not limited to retroviruses (Gilboa, J., Virology, 44:845, 1982; Hocke, Nature, 320:275, 1986; Wilson et al. Human, Proc.Natl.Acad.Sci.U.S.A., 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol., 5:3403, 1985) or other efficient DNA delivery systems known to those skilled in the art ( Yates et al., Nature, 313:812, 1985). These references are illustrative only and are incorporated herein by reference. For specific delivery or transfection of abnormally proliferating cells while avoiding non-dividing cells, retroviral or adenoviral (as described in the art and elsewhere herein) delivery systems known to those skilled in the art are preferably employed. Since integration of retroviral DNA requires host DNA replication, and retroviruses cannot replicate themselves due to the lack of retroviral genes required for their life cycle, the use of this retroviral delivery system for polynucleotides of the invention will The genes and constructs are targeted to abnormally proliferating cells and avoid normal cells that do not divide.

The polynucleotides of the invention can be delivered directly to cell proliferative disorders/disease sites in internal organs, body cavities, etc. by using an imaging device to guide the injection needle exactly to the site of disease. The polynucleotides of the invention may also be administered to the site of disease during surgical intervention.

"Cell proliferative disorder" means an attack of any organ, cavity, or body part, or any combination thereof, characterized by single or multiple localized abnormal proliferations of cells, populations of cells, or tissues (whether benign or malignant) any human or animal disease or disorder.

Any amount of the polynucleotide of the invention may be administered so long as it has a biologically inhibitory effect on the proliferation of the cells being treated. Furthermore, it is possible to administer more than one polynucleotide of the invention simultaneously to the same site. "Biological inhibition" means partial or complete inhibition of growth, as well as reduction of cell proliferation or growth rate. Biological inhibitory doses can be determined by evaluating the effect of polynucleotides of the invention on target malignant or abnormal cell growth in tissue culture, tumor growth in animals, and cell culture, or any other method known to those of ordinary skill in the art.

In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to inhibit the angiogenesis of proliferating cells or tissues, either alone as a protein fusion, or directly or indirectly in combination with other polypeptides, As described elsewhere in this article. In a most preferred embodiment, the anti-angiogenic effect is achieved indirectly, for example, by inhibition of hematopoietic, tumor-specific cells such as tumor-associated macrophages (see Joseph, I.B. et al., J. Natl. Cancer Inst., 90(21):1648-53, 1998, which is incorporated herein by reference).

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to inhibit proliferative cells or tissues by inducing apoptosis. These fusion proteins and/or polynucleotides can act either directly or indirectly to induce apoptosis of proliferative cells and tissues, for example, in the activation of death domain receptors, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF receptor-related apoptosis-mediating protein (TRAMP), and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (see Schulze-Osthoff, K. et al. , Eur. J. Biochem., 254(3):439-59, 1998, which is incorporated herein by reference). Furthermore, in another preferred embodiment, these fusion proteins and/or polynucleotides can induce apoptosis by other mechanisms, such as in the activation of other proteins that activate apoptosis, or by stimulating the expression of these proteins, or Is alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectin, thioredoxin, anti-inflammatory protein (see for example Mutat.Res., 400 (1-2): 447-55, 1998; Med.Hypotheses, 50(5):423-33, 1998; Chem.Biol.Interact., April 24, 111-112:23-34, 1998; J.Mol.Med., 76(6):402 -12, 1998; Int. J. Tissue React., 20(1):3-15, 1998, all of which are incorporated herein by reference).

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to inhibit metastasis of proliferative cells or tissues. Inhibition can occur as a direct result of administration of these albumin fusion proteins and/or polynucleotides, or indirectly, such as activation of expression of proteins known to inhibit metastasis, such as α4 integrin (see, e.g., Curr. Top. Microbiol. Immunol., 231:125-41, 1998, which is incorporated herein by reference). This therapeutic effect of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

In another embodiment, the present invention provides delivery of a composition comprising an albumin fusion protein of the present invention and/or a polynucleotide encoding an albumin fusion protein of the present invention to a protein that expresses an albumin fusion protein of the present invention bound to, A method for targeting cells of a polypeptide which binds or associates. Albumin fusion proteins of the invention can associate with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic, and/or covalent interactions.

The albumin fusion proteins of the invention can be used to enhance the immunogenicity and/or antigenicity of proliferating cells or tissues, or directly, such as if "vaccination" of the albumin fusion proteins of the invention elicits immunity against proliferative antigens and This will occur either at the time, or indirectly, such as in activating the expression of proteins known to enhance the immune response to the antigens and immunogens, such as chemokines.

kidney disorder

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treating, preventing, diagnosing, and/or predicting disorders of the renal system. Renal disorders that can be diagnosed, predicted, prevented, and/or treated with the compositions of the present invention include, but are not limited to, renal failure, nephritis, renal vascular disorders, metabolic and congenital renal disorders, renal urinary disorders, autoimmune disorders , sclerosis and necrosis, electrolyte imbalance, and renal cancer.

Renal diseases that can be diagnosed, predicted, prevented, and/or treated using the compositions of the present invention include, but are not limited to, acute renal failure, chronic renal failure, atherosclerotic renal failure, end-stage renal disease, inflammatory diseases of the kidney (such as acute renal failure Glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis types I and II, mesangial proliferation chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute poststreptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis , interstitial nephritis, and poststreptococcal glomerulonephritis), renal vascular disorders (eg, renal infarction, atheroembolic nephropathy, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal hypoperfusion, renal Retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and renal disorders caused by urinary tract diseases (such as pyelonephritis, hydronephrosis, urolithiasis (kidney stones, nephrolithiasis), reflux nephropathy, urinary tract infection, urinary retention, and acute or chronic unilateral obstructive uropathy).

In addition, the composition of the present invention can be used to diagnose, predict, prevent, and/or treat renal metabolic and congenital disorders (such as uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal Diabetes mellitus, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteogenesis (renal rickets), Hartnap's disease, Bartter's syndrome, Liddell polycystic kidney disease, medullary cystic disease, medullary sponge kidney disease, Alpert syndrome, nail-patella syndrome, congenital nephrotic syndrome, crush syndrome, horseshoe kidney, diabetic nephropathy, renal Diabetes insipidus, analgesic nephropathy, nephrolithiasis, and membranous nephropathy), and renal autoimmune disorders (such as systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).

The composition of the present invention can be used to diagnose, predict, prevent, and/or treat sclerotic or necrotic disorders of the kidney (such as glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), Necrotizing glomerulonephritis, and renal papillary necrosis), renal cancer (eg, renal tumor, adrenal adenoid tumor, Wilms tumor, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma, squamous cell carcinoma, and Wilms tumor), and electrolyte imbalances (eg, nephrocalcinosis, pyuria, edema, hydronephrosis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia , hypercalcemia, hypophosphatemia, and hyperphosphatemia).

Compositions of the present invention may be administered using any method known in the art, including but not limited to needle injection directly at the delivery site, intravenous injection, topical application, catheter infusion, biolistic injectors, particle accelerators, gelatin sponge depots , other commercially available depot materials, osmotic pumps, solid pharmaceutical formulations for oral or suppository, pouring or topical application during surgery, aerosol delivery. These methods are known in the art. Compositions of the invention may be administered as part of a therapeutic agent, as described in more detail below. Methods for delivering polynucleotides of the invention are described in more detail herein.

Cardiovascular Disorders

The albumin fusion proteins of the invention and/or polynucleotides encoding the albumin fusion proteins of the invention can be used to treat, prevent, diagnose, and/or predict cardiovascular disorders, including but not limited to peripheral arterial disease, such as limb ischemia.

Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities such as arterial-arterial fistulas, arteriovenous fistulas, cerebral arterial malformations, congenital heart defects, pulmonary atresia, and machete syndrome. Congenital heart defects include but are not limited to aortic stenosis, three-chamber heart, coronary vascular anomalies, cruciform heart, dextrocardia, patent ductus arteriosus, Ebstein anomaly, Eisenmenger complex, left heart development Insufficiency syndrome, levocardia, tetralogy of Fallot, aortopulmonary malposition, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, cardiac septal defects such as aortopulmonary septal defect, endocardial cushion defect, Ruttenbach's syndrome, Triad of Fallot, Ventricular septal defect.

Cardiovascular disorders also include, but are not limited to, heart disease such as arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm , cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiogenic edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, cardiac rupture after infarction, ventricular septal rupture , valvular heart disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, cor pulmonale, rheumatic heart disease , ventricular dysfunction, congestion, cardiovascular pregnancy complications, machete syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Cardiac arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystoles, Adam-Stokes syndrome, bundle branch block, sinoatrial block, long QT syndrome, parallel systole, Lang-Gum-Leys syndrome, Maheimer-type pre-excitation syndrome, Waugh-Park-White syndrome, sick sinus syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia, supraventricular tachycardia, accelerated ventricular spontaneous rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junction zone Tachycardia, sinus nodal reentrant tachycardia, sinus tachycardia, torsades de pointes, and ventricular tachycardia.

Heart valve disease includes, but is not limited to, aortic insufficiency, aortic stenosis, heart murmur, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral stenosis, Pulmonary atresia, pulmonary insufficiency, pulmonary stenosis, tricuspid atresia, tricuspid insufficiency, and tricuspid stenosis.

Cardiomyopathy including but not limited to alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subaortic stenosis, subpulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial elastic fibers Hyperplasia, endomyocardial fibrosis, Karnes' syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes, but is not limited to, coronary artery disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary spasm, myocardial infarction, and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasias, hemangiomas, bacillary hemangiomas, Schilling-Linger's disease, Kreuter-Williams syndrome, Sturge-Weber syndrome, vascular Nervous edema, aortic disease, Taurin's arteritis, aortitis, Lerrisch syndrome, arterial occlusive disease, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic vascular disease, Diabetic retinopathy, embolism, thrombosis, erythematalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, machete syndrome, superior vena cava syndrome, telangiectasia, ataxia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer , vasculitis, and venous insufficiency.

Aneurysms include, but are not limited to, segmented aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary artery aneurysms, cardiac aneurysms, and iliac aneurysms.

Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vessel occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and thrombovasculitis obliterans.

Cerebrovascular disorders include but are not limited to carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral hypoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral arterial disease, cerebral embolism and thrombosis, carotid artery thrombosis, venous Sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, Periventricular leukomalacia, vascular headache, cluster headache, migraine, and spondylobasilar insufficiency.

Embolisms include, but are not limited to, air embolism, amniotic fluid embolism, cholesterol embolism, blue toe syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes, but is not limited to, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, venous sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and extremity ischemia (peripheral limb ischemia). Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's syndrome, Chow-Still's syndrome, mucocutaneous lymph node syndrome, thrombovasculitis obliterans, hypersensitivity vasculitis, Schulland-Henry Knoller's purpura, allergic cutaneous vasculitis, and Wagner's granulomatosis.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be administered using any method known in the art, including but not limited to needle injection directly at the site of delivery, intravenous injection, topical administration, Catheter infusions, biolistic injectors, particle accelerators, gelatin sponge depots, other commercially available storage materials, osmotic pumps, solid pharmaceutical formulations for oral or suppository, pouring or topical application during surgery, aerosol delivery. These methods are known in the art. Methods for delivering polynucleotides are described in more detail herein.

breathing disorder

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treating, preventing, diagnosing, and/or predicting diseases and/or disorders of the respiratory system.

Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (eg, acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps and sinusitis, juvenile angiofibromas, nasal Carcinoma and juvenile papilloma, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngocele, pharyngitis (eg, viral and bacterial), tonsillitis, tonsil cellulitis, parapharyngeal abscess, Laryngitis, laryngocele, and throat cancer (eg, nasopharyngeal carcinoma, tonsil carcinoma, laryngeal carcinoma), lung cancer (eg, squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), metastatic Allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (eg, exogenous allergic alveolitis, allergic interstitial pneumonia, organic pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wagner's Granuloma (granulomatous vasculitis, Goodpasque syndrome)), pneumonia (such as bacterial pneumonia (such as Streptococcus pneumoniae (pneumococcal pneumonia), Staphylococcus aureus (staphylococcal pneumonia) ), Gram-negative bacterial pneumonia (caused by, for example, Klebsiella and Pseudomonas), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumonia ( Legionnaires' disease), and Chlamydia psittaci (Psittacosis), and viral pneumonia (eg, influenza, fowl pox (varicella)).

Other respiratory diseases and disorders include, but are not limited to, bronchiolitis, poliomyelitis, croup, respiratory syncytial virus infection, mumps, erythema infectious (fifth disease), acute rash in infants, progressive rubella panencephalitis, German measles (rubella), and subacute sclerosing panencephalitis, fungal pneumonia (eg, histoplasmosis, coccidioidomycosis, blastomycosis, fungal infection (eg, caused by novel Cryptococcosis caused by Cryptococcus neoformans; Aspergillus caused by Aspergillus; Candida caused by Candida; and Mucormycosis)), Pneumocystis carinii (Pneumocystis carinii) pneumonia), atypical pneumonias (eg, mycoplasma and chlamydia), opportunistic pneumonia, nosocomial pneumonia, chemical pneumonia, and aspiration pneumonia, pleural disorders (eg, pleurisy, pleural effusion, and pneumothorax (eg, simple spontaneous pneumothorax, complex spontaneous pneumothorax, tension pneumothorax), obstructive airway disease (eg, asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung disease (eg, Silicosis, black lung disease (coal workers' pneumoconiosis), asbestosis, beryllium poisoning, occupational asthma, insinosis, and benign pneumoconiosis), infiltrating lung diseases (such as pulmonary fibrosis (such as fibrous alveolitis, common interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (such as Leittler-Siewe disease, Han-Xu - Ketosis, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and alveolar proteinosis), acute respiratory distress syndrome (also known as e.g. adult respiratory distress syndrome), edema, Pulmonary embolism, bronchitis (eg, viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused, for example, by Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.

anti-angiogenic activity

Inhibitory effects predominate in the naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis. Rastinejad et al., Cell, 56:345-355, 1989. In the rare instances where neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is tightly regulated and limited in space and time. In conditions of pathological angiogenesis, such as those exhibiting solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathological and supports the development of many neoplastic and non-neoplastic diseases. A number of serious diseases are governed by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of ophthalmic disorders, and psoriasis. See for example the following reviews: Moses et al., Biotech., 9:630-634, 1991; Folkman et al., N. Engl. J. Med., 333:1757-1763, 1995; Auerbach et al., J. Microvasc. Res ., 29:401-411, 1985; Folkman, "Advances in Cancer Research", edited by Klein and Weinhouse, Academic Press, New York, pp.175-203, 1985; Patz, Am.J.Opthalmol., 94:715- 743, 1982; and Folkman et al., Science, 221:719-725, 1983. In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, substantial evidence has accumulated that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science, 235:442-447, 1987.

The invention provides treatment of diseases or disorders associated with neovascularization by administering fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Malignant and metastatic conditions treatable with the polynucleotides and polypeptides of the invention or agonists or antagonists include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of these disorders see Fishman et al., "Medicine", 2nd Ed., J.B. Lippincott Co., Philadelphia, 1985). Thus, the present invention provides a method for treating angiogenesis-related diseases and/or disorders, comprising administering a therapeutically effective amount of an albumin fusion protein of the present invention and/or a polynucleoside encoding an albumin fusion protein of the present invention to an individual in need thereof acid. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used in a variety of other methods to therapeutically treat cancer or therapy. Cancers treatable with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, solid tumors, including prostate, lung, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver , parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanoma; glioblastoma; Kaposi's sarcoma; leiomyosarcoma ; non-small cell non-cancer; colorectal cancer; high-grade malignancy; and blood-borne neoplasms, such as leukemia. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be delivered locally to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

In other aspects, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat superficial forms of bladder cancer by, for example, intravesical administration. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be delivered directly into a tumor or near a tumor site by injection or catheter. Of course, the appropriate mode of administration will vary depending on the cancer being treated, as will be appreciated by one of ordinary skill. This article also discusses other delivery modes.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the treatment of disorders other than cancer involving angiogenesis. These disorders include, but are not limited to: benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granuloma; atherosclerotic plaques; angiogenic diseases of the eye such as diabetic retinopathy, premature birth Pediatric retinopathy, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolentic fibroplasia, redness, retinoblastoma, ocular uveitis, and pterygium (abnormal blood vessel growth); rheumatoid arthritis Psoriasis; Delayed wound healing; Endometriosis; Vasculogenesis; Granulation; Hypertrophic scars (keloids); Ununion fractures; Scleroderma; Trachoma; Vascular adhesion; Coronary artery collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Ouer-Williams syndrome; plaque neovascularization; telangiectasias; hemophilic joints; angiofibromas; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, in one aspect of the present invention, a method for treating hypertrophic scars and keloids is provided, comprising administering an albumin fusion protein of the present invention and/or a protein encoding an albumin fusion protein of the present invention to a hypertrophic scar or keloid. polynucleotide steps.

In one embodiment of the invention, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are injected directly into hypertrophic scars or keloids to prevent the development of these lesions. This therapy is particularly valuable in the prophylactic treatment of conditions known to lead to the development of hypertrophic scars and keloids (such as burns), and is preferred after the proliferative phase has had time to develop (approximately 14 days after the initial injury) but in hypertrophic scars or before the development of keloids. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including, for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolentic fibroplasia, and macular degeneration.

In addition, ophthalmic disorders associated with neovascularization that can be treated with albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to: neovascular glaucoma, diabetic retinopathy, adult Retinoblastoma, retrolentic fibroplasia, uveitis, retinopathy of prematurity, macular degeneration, corneal graft neovascularization, and ophthalmic inflammatory diseases, eye tumors, and diseases associated with choroidal or iris neovascularization. See, eg, the following reviews: Waltman et al., Am. J. Ophthal., 85:704-710, 1978; and Gartner et al., Surv. Ophthal., 22:291-312, 1978.

Thus, in one aspect of the invention, there is provided a method for treating ocular neovascular disease, such as corneal neovascularization (including keratoplasty neovascularization), comprising administering to a patient a therapeutically effective amount of a compound (such as The steps of the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention) inhibit the formation of blood vessels. Simply put, the cornea is tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries can extend into the cornea from the pericorneal vascular plexus at the junction of the cornea and the sclera. When the cornea becomes vascularized, it also becomes blurred, causing the patient's vision to decrease. If the cornea becomes completely opaque, vision may be completely lost. Corneal neovascularization can result from a wide variety of disorders, including, for example, corneal infections (such as trachoma, herpes simplex keratitis, leishmaniasis, and onchocerciasis), immunological processes (such as transplant rejection and Sioux-Johnson Syndrome), alkali burns, trauma, inflammation (any cause), poisoning and nutritional deficiencies, and as a complication of wearing contact lenses.

In a particularly preferred embodiment of the invention, it may be prepared for topical application in saline (in combination with any preservatives and antimicrobial agents commonly used in ophthalmic preparations) and administered in the form of eye drops. Solutions or suspensions can be prepared in their pure form and administered several times per day. Alternatively, anti-angiogenic compositions prepared as described above can also be applied directly to the cornea. In a preferred embodiment, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds the cornea. In other embodiments, anti-angiogenic factors or anti-angiogenic compositions may be used as adjunct therapy to conventional steroid therapy. Topical therapy can also be used prophylactically in corneal injuries known to have a high probability of inducing an angiogenic response such as chemical burns. In these cases, treatment can be started immediately, possibly with steroids, to help prevent subsequent complications.

In other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection will vary with the morphology of the individual lesion, but the goal of administration will be to place the composition at the leading edge of the vascular structures (ie, spread between the blood vessels and the normal cornea). In most cases this will involve corneal injection around the junction of the cornea and sclera to "protect" the cornea from advancing blood vessels. The method can also be used not only after corneal injury, so as to prophylactically prevent corneal neovascularization. In such cases, a substance may be injected into the cornea surrounding the corneal-scleral junction, interspersed between the corneal injury and its unwanted potential blood supply to the corneal-scleral junction. This approach can also be used in a similar manner to prevent capillary invasion of transplanted corneas. In the sustained release form, only 2-3 injections per year may be needed. Steroids may also be added to the injection to reduce inflammation caused by the injection itself.

In another aspect of the present invention, there is provided a method for treating neovascular glaucoma, comprising administering a therapeutically effective amount of an albumin fusion protein of the present invention and/or a polynucleotide encoding an albumin fusion protein of the present invention to the eye of a patient step, so that the formation of blood vessels is inhibited. In one embodiment, the compounds may be administered topically to the eye to treat early forms of neovascular glaucoma. In other embodiments, the compound may be infused into the anterior chamber angle region by injection. In other embodiments, the compound may also be placed at any site such that the compound is continuously released into the aqueous humor. In another aspect of the present invention, there is provided a method for treating proliferative diabetic retinopathy, comprising administering to a patient a therapeutically effective amount of the albumin fusion protein of the present invention and/or a multinuclear protein encoding the albumin fusion protein of the present invention. The step of nucleotide acid makes the formation of blood vessels inhibited.

In particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or vitreous to increase the local concentration of polynucleotides, polypeptides, antagonists, and/or agonists in the retina. Preferably, this treatment should be initiated before the development of serious disease requiring photocoagulation.

In another aspect of the present invention, there is provided a method for treating retrolentic fibrous tissue hyperplasia, comprising administering a therapeutically effective amount of the albumin fusion protein of the present invention and/or a polynucleoside encoding the albumin fusion protein of the present invention to the eye of the patient Acid step, so that the formation of blood vessels is inhibited. Compounds may be administered topically by intravitreal injection and/or intraocular implantation.

Additionally, disorders treatable with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, hemangiomas, arthritis, psoriasis, angiofibromas, atherosclerotic plaques, Delayed wound healing, granulation, hemophilic joints, hypertrophic scars, ununion fractures, O'Well syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

In addition, disorders and/or states that can be treated, prevented, diagnosed, and/or predicted with the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, solid tumors, blood-borne tumors Such as leukemia, tumor metastasis, Kaposi's sarcoma, benign tumors such as hemangioma, acoustic neuroma, neurofibroma, trachoma, and pyogenic granuloma, rheumatoid arthritis, psoriasis, ophthalmic angiogenic diseases such as diabetic Retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolentic fibroplasia, redness, retinoblastoma, and uveitis, delayed wound healing, endometriosis, blood vessels Neogenesis, granulation, hypertrophic scars (keloids), ununion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary artery collaterals, cerebral collaterals, arteriovenous malformations, ischemic extremity angiogenesis, -Weiller's syndrome, plaque neovascularization, telangiectasia, hemophilic joints, angiofibromas, fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, Contraceptives for the control of angiogenesis required for menstruation, diseases with angiogenesis as a pathological consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonella disease, and bacillary angiomatosis.

In one aspect of the birth control method, the compound is administered in an amount sufficient to block embryo implantation before or after intercourse and fertilization occurs, thereby providing an effective birth control method, possibly a "hangover" method. The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used to control menstruation, or in the treatment of endometriosis or as a peritoneal lavage fluid or as a peritoneal implant Enter and apply.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be incorporated into surgical sutures to prevent stitch granuloma.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (e.g., in the form of a spray or film) may be used to daub or spray a specific area prior to resection of a tumor, thereby separating normal surrounding tissue from malignant tissue, and/or preventing disease spread to surrounding tissues. In other aspects of the invention, the compositions (eg, in the form of a spray) can be delivered by endoscopic procedures to cover tumor or angiogenesis at the desired site of implantation. In other aspects of the invention, surgical meshes coated with anti-angiogenic compositions of the invention can be used in any procedure for which surgical meshes are available. For example, in one embodiment of the invention, a surgical mesh loaded with an anti-angiogenic composition of the invention can be used in abdominal cancer resection procedures (eg, after colectomy) to provide structural support and release an amount of anti-angiogenic factors.

In other aspects of the present invention, there is provided a method for treating a tumor resection site, comprising administering an albumin fusion protein of the present invention and/or a polynucleotide encoding an albumin fusion protein of the present invention to the resection margin of the tumor after resection , so that the recurrence of cancer in situ (local) and the formation of new blood vessels at the site are inhibited. In one embodiment of the invention, the anti-angiogenic composition is administered directly to the site of tumor resection (eg, by swabbing, brushing, or otherwise covering the resection margins of the tumor with the anti-angiogenic compound). Alternatively, anti-angiogenic compounds may be incorporated into known surgical patches prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy for malignancy and after neurosurgery.

In one aspect of the invention, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be administered to the resection margins of a wide variety of tumors, including, for example, breast, colon, brain, and liver tumors. For example, in one embodiment of the invention, an anti-angiogenic compound may be administered to the site of a neurological tumor following resection such that the formation of new blood vessels at the site is inhibited.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be administered with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasion factor, retinoic acid and its derivatives, paclitaxel (Paclitaxel), suramin, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, Plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and various forms of the lighter "group d" transition metals.

Lighter "group d" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum nuclides. These transition metal nuclides can form transition metal complexes. Suitable complexes of the above transition metal nuclides include oxo transition metal complexes.

Representative examples of vanadium complexes include oxovanadium complexes, such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrate.

Representative examples of tungsten and molybdenum complexes also include oxygen complexes. Suitable tungsten oxide complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten(IV) oxide and tungsten(VI) oxide. Suitable oxymolybdenum complexes include molybdate, molybdenum oxide, and oxymolybdenum complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum(IV) oxide, molybdenum(VI) oxide, and molybdic acid. A suitable copy of an oxymolybdenum complex is eg oxymolybdenum acetylacetonate. Other suitable tungsten and molybdenum complexes include coordinating hydroxy derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors can also be used in the context of the present invention. Representative examples include platelet factor 4; protamine sulfate; chitosan sulfate derivatives (prepared from snow crab shells) (Murata et al., Cancer Res., 51:22-26, 1991); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this complex can be enhanced by the presence of steroids, such as estrogen and tamoxifen citrate); staurosporine; regulators of substrate metabolism, including, for example, proline analogs, cis-hydroxyproline, d,L-3,4-dehydroproline, thiaproline, α,α-dipyridylaminopropionitrile fumarate, 4-propyl-5-(4- Pyridyl)-2(3H)-oxazolone; methotrexate; mitoxantrone; heparin; interferon; 2-macroglobulin-albumin; : 17321-17326, 1992); chymotrypsin (Tomkinson et al., Biochem.J., 286: 475-480, 1992); cyclodextrin tetradecyl sulfate; Eponemycin; camptothecin; fumagillin ( Ingber et al., Nature, 348:555-557, 1990); gold sodium thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest., 79:1440-1446, 1987); anti-collagenase serum ; α2-antiplasmin (Holmes et al., J.Biol.Chem., 262 (4): 1659-1664, 1987); )-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions, 36:312-316, 1992); Thalidomide; Angiogenesis-inhibiting steroids; AGM-1470; carboxyaminoimidazole; and metalloprotease inhibitors, such as BB94.

diseases at the cellular level

Diseases associated with enhanced cell survival or inhibition of apoptosis, including cancer (such as follicular lymphoid Tumors, carcinomas with p53 mutations, and hormone-dependent tumors, including but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, bowel cancer, testicular cancer , gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer); autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis , systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpesviruses, poxviruses, and adenoviruses), inflammation, graft-versus-host disease, acute transplant exclusion, and chronic transplantation repel.

In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit the growth, progression, and/or metastasis of cancer, particularly those listed above.

Other diseases or conditions associated with enhanced cell survival that may be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, the development and/or metastasis of malignancies and related disorders, Such as leukemia (including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic (promyelocytic), myelomonocytic, monocytic, and erythroleukemia)) and chronic Leukemias (such as chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (such as Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Val Denstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma , endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, Basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma , embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, Etympanoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases related to enhanced apoptosis that can be treated, prevented, diagnosed, and/or predicted using the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention include but are not limited to AIDS; Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or related diseases); autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis); bone marrow Dysplastic syndromes (such as aplastic anemia), graft-versus-host disease, ischemic injury (such as that caused by myocardial infarction, stroke, and reperfusion injury), liver injury (such as hepatitis-associated liver injury, ischemic /reperfusion injury, cholestasis (injury to the bile ducts), and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia, and anorexia.

Wound healing and epithelial cell proliferation

According to yet another aspect of the invention there is provided a method of using a fusion protein of the invention and/or a polynucleotide encoding an albumin fusion protein of the invention for therapeutic purposes, for example for stimulating epithelial cell proliferation for wound healing purposes and basal keratinocytes, and for stimulating hair follicle production and dermal wound healing. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used clinically to stimulate wound healing, including surgical wounds, excisional wounds, deep wounds involving dermal and epidermal lesions, ocular tissue Wounds, dental wounds, oral wounds, diabetic ulcers, dermal ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, burns caused by heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, Vitamin deficiencies, and complications related to steroids, radiotherapy, and systemic therapy with antineoplastic drugs and antimetabolites. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to promote dermal remodeling following dermal loss.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to improve the adhesion of skin grafts to the wound surface and to stimulate the re-epithelialization of the wound surface. The following are the types of grafts in which adhesion to wound surfaces can be improved using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention: autografts, artificial skin, allografts ( allograft), autologous dermal grafts, autologous epidermal grafts, avascular grafts, Brad-Brudner grafts, bone grafts, embryo grafts, cutis grafts, delayed grafts, dermal grafts ( dermic graft), epidermal graft, fascial graft, full-thickness skin graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, cornea Thin-layer grafts, mesh skin grafts, mucosal grafts, Otto-Tiger grafts, omentum grafts, repair grafts, pedicled grafts, full-thickness corneal grafts, layered skin grafts, thick Layered skin grafts. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to increase skin strength and improve the appearance of aging skin.

We believe that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention will also cause changes in hepatocyte proliferation and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can promote proliferation of epithelial cells, such as sebocytes, hair follicles, liver cells, type II pneumocytes, mucin-producing goblets cells, and other epithelial cells and their progenitors contained in the skin, lungs, liver, and gastrointestinal tract. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used to reduce gut toxicity side effects caused by radiotherapy, chemotherapy, or viral infection. The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention may have a cytoprotective effect on the small intestinal mucosa. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also stimulate healing of mucositis (mouth ulcers) caused by chemotherapy and viral infections.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are also useful for complete regeneration of full and partial thickness skin defects including burns (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), other skin Treatment of deficits such as psoriasis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the treatment of epidermolysis bullosa, i.e. defective adhesion of the epidermis to the underlying dermis, by accelerating the Cell regeneration results in frequent, open, and painful blisters. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are also useful in the treatment of gastric and duodenal ulcers and through scarring of the mucosal lining and glandular and duodenal mucosal linings The regeneration helps to heal faster. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, refer to diseases that cause destruction of the mucosal surface of the small or large intestine, respectively. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to promote resurfacing of mucosal surfaces to aid in better healing and prevent the development of inflammatory bowel disease. Treatment with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention is expected to have a significant effect on mucus production throughout the gastrointestinal tract and can be used to protect the intestinal mucosa from ingested noxious substances or surgery after the injury. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat diseases associated with underexpression.

In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to prevent and treat damage to the lung caused by various pathological conditions. The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention that can stimulate alveolar and bronchial epithelial proliferation and differentiation and promote repair can be used to prevent or treat acute or chronic lung injury. For example, emphysema leading to progressive loss of alveoli and inhalation injury causing necrosis of the bronchial epithelium and alveoli (ie, caused by smoke inhalation and burns) can be effectively treated using the polynucleotides or polypeptides, agonists or antagonists of the invention. Also, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to stimulate the proliferation and differentiation of type II pneumocytes, which is helpful in the treatment or prevention of diseases such as hyaline membrane disease, such as premature infants Infantile respiratory distress syndrome and bronchopulmonary dysplasia.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can stimulate the proliferation and differentiation of hepatocytes and are thus useful in alleviating or treating liver diseases and pathologies, such as fulminant hepatic cirrhosis Liver failure, liver injury caused by viral hepatitis and toxic substances (ie acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat or prevent the onset of diabetes. In patients recently diagnosed with type I and type II diabetes in which some islet cell function is preserved, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to maintain islet function, thereby alleviating, delaying , or prevent permanent manifestations of disease. Likewise, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used as an adjuvant therapy for islet cell transplantation to improve or promote islet cell function.

Neuroactivity and Neurological Disease

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the diagnosis and/or treatment of diseases, disorders, injuries, or damages of the brain and/or nervous system. Nervous system disorders that can be treated with compositions of the present invention (such as fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention) include, but are not limited to, nervous system injuries, and those that result in axonal disconnection, neuronal Depletion or degenerative, or demyelinating disease or disorder. Nervous system injuries that may be treated in patients (including human and non-human mammalian patients) according to the methods of the present invention include, but are not limited to, the following central (including spinal cord, brain) or peripheral nervous system injuries: (1) ischemic injury, In which partial nervous system hypoxia leads to neuronal damage or death, including cerebral infarction or ischemia or spinal cord infarction or ischemia; (2) traumatic injury, including injury caused by physical injury or related to surgery, such as severed part of the nerve Systemic injury, or crush injury; (3) Malignant injury, in which part of the nervous system is destroyed or injured by malignant tissue, which is either a nervous system-related malignancy or a malignant tumor derived from non-nervous system tissue; ( 4) Infectious injury in which part of the nervous system has been destroyed or damaged by infection, for example, through trachoma or associated with human immunodeficiency virus, herpes zoster, or herpes simplex virus infection or with Lyme disease, tuberculosis, or syphilis; (5) Degenerative injuries, in which part of the nervous system is destroyed or damaged due to degenerative processes, including but not limited to those associated with Parkinson's disease, Alzheimer's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS ) related degeneration; (6) damage associated with a nutritional disease or disorder in which part of the nervous system is destroyed or damaged by a nutritional disease or disorder of metabolism, including but not limited to vitamin B12 deficiency, folic acid deficiency, Wernicke (7) neurological damage associated with systemic diseases, including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) injury caused by toxic substances, including alcohol, lead, or certain neurotoxins; and (9) demyelinating injury, in which some nerves System disrupted or injured by demyelinating disorders including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and pons Central myelination.

In one embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to protect nerve cells from the damaging effects of hypoxia. In another preferred embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to protect nerve cells from the damaging effects of cerebral hypoxia. According to this embodiment, the composition of the invention is used for the treatment or prevention of neuronal cell damage associated with cerebral hypoxia. In a non-exclusive aspect of this embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neuronal cell damage associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neuronal cell damage associated with cerebral infarction.

In another preferred embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat or prevent neuronal cell damage associated with stroke. In a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat or prevent cerebral nerve cell damage related to stroke.

In another preferred embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat or prevent nerve cell damage associated with heart attack. In a specific embodiment, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used to treat or prevent brain nerve cell damage associated with heart attack.

Compositions of the invention useful for treating or preventing disorders of the nervous system can be selected by testing for biological activity in promoting neuronal survival or differentiation. For example, without limitation, compositions that elicit any of the following effects may be used in accordance with the invention: (1) increase the survival time of neurons in culture in the presence or absence of hypoxic or hypoxic conditions; (2) increase the survival time of neurons in culture; or in vivo to increase neuronal sprouting; (3) in culture or in vivo to increase the production of neuron-associated molecules, such as choline acetyltransferase or acetylcholinesterase in the case of motor neurons; or (4) in vivo Reduced neuronal dysfunction. These effects can be measured by any method known in the art. In a preferred non-limiting embodiment, increased neuronal survival can be routinely measured using methods enumerated herein or otherwise known in the art, such as, for example, Zhang et al., Proc. Natl. Acad. Sci. USA, 973637 -42, 2000 or Arakawa et al., J.Neurosci., 10:3507-15, 1990; increased neuronal sprouting can be measured by methods known in the art, such as, for example, Pestronk et al., Exp.Neurol., 70: 65-82, 1980 or methods enumerated in Brown et al., Ann.Rev.Neurosci., 4: 1742, 1981; increasing the production of neuron-associated molecules can be achieved by biological methods using techniques known in the art and depending on the molecule to be measured. Assays, enzymatic assays, antibody binding, Northern blot assays, etc.; whereas motor neuron dysfunction can be measured by assessing physical manifestations of motor neuron disturbances such as weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated in accordance with the present invention include, but are not limited to, disorders such as infarction, infection, exposure to toxins, trauma, surgical injury, that can affect motor neurons and other components of the nervous system degenerative diseases or malignancies, and disorders selectively affecting neurons such as amyotrophic lateral sclerosis, including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar palsy in childhood (Fazio-Lund syndrome), poliomyelitis and post-polio syndrome, and hereditary motor sensory neuropathy (Charcot-Marie-Tuse III disease).

In addition, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention may play a role in neuron survival, synapse formation, conduction, neural differentiation, and the like. Thus, compositions of the present invention (comprising fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention) are useful in the diagnosis and/or treatment or prevention of diseases or disorders associated with these effects, including but Not limited to learning and/or cognitive disorders. The compositions of the invention are also useful in the treatment or prevention of neurodegenerative disease states and/or behavioral disturbances. These neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, schizophrenia, mania, dementia, paranoia, Compulsive disorder, panic disorder, learning disabilities, ALS, psychosis, autism, and behavioral changes, including disturbances in eating, sleep patterns, balance, and perception. In addition, the compositions of the present invention may also be useful in the treatment, prevention, and/or detection of developmental or sex-linked disorders associated with developing embryos.

In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to protect neuronal cells from disease, injury, disorder, or damage associated with cerebrovascular disorders, including but not limited to carotid artery disease (eg, carotid thrombosis, carotid stenosis, or moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral hypoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral arterial disease, cerebral embolism, and thrombosis (eg, Carotid artery thrombosis, sinus thrombosis, or Wallenberg's syndrome), cerebral hemorrhage (eg, epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (eg, transient Cerebral ischemia, subclavian steal syndrome, or spondylobasilar insufficiency), vascular dementia (eg, multiinfarct), periventricular leukomalacia, and vascular headache (eg, cluster headache or migraine) .

According to another aspect of the invention, there are provided methods of utilizing fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention for therapeutic purposes, eg for stimulating neurological cell proliferation and/or differentiation. Therefore, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat and/or detect neurological diseases. In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as markers or detectors for particular neurological diseases or disorders.

Examples of neurological disorders that may be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include diseases of the brain, such as metabolic encephalopathy, including phenylketonuria such as maternal phenylketonuria , pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's encephalopathy, cerebral edema, brain neoplasms such as cerebellar neoplasms including medial tentorial neoplasms, ventricular neoplasms such as choroid plexus neoplasms, inferior Thalamic neoplasms, supratentorial neoplasms, Canavan's disease, cerebellar disorders such as cerebellar ataxias, including spinocerebellar degenerations such as ataxia telangiectasia, cerebellar dyssynergia, Friedreich's ataxia, horse- John's disease, olivopontocerebellar atrophy, cerebellar neoplasms such as intertentorial neoplasms, diffuse sclerosis such as periaxial encephalitis, globular cell leukodystrophy, metachromatic leukodystrophy, and subacute sclerosis panencephalitis.

Other neurological diseases that may be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include cerebrovascular disorders such as carotid artery disease, including carotid thrombosis, carotid stenosis, and moyamoya disease ), cerebral amyloid angiopathy, cerebral aneurysm, cerebral hypoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral arterial disease, cerebral embolism and thrombosis such as carotid thrombosis, venous sinus thrombosis, and Wallenberg Cerebral hemorrhage such as epidural hematoma, subdural hematoma, and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, subclavian steal syndrome, and spondylobasilar insufficiency , vascular dementias such as multi-infarct dementia, periventricular leukomalacia, vascular headaches such as cluster headache and migraine.

Other neurological diseases that can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include dementias such as AIDS dementia complex, Alzheimer's diseases such as Alzheimer's disease and Creutzfeldt-Jakob syndrome Alzheimer's syndrome, senile dementias such as Alzheimer's disease and progressive supranuclear palsy, vascular dementias such as multi-infarct dementia, encephalitis including periaxial encephalitis, viral encephalitis such as epidemic encephalitis, Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis, and West Nile fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveal meningoencephalitis syndrome, postencephalitic Parkinson's disease, and subacute sclerosis Panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy including infantile spasms, absence epilepsy, myoclonic epilepsy including MERRF syndrome, tonic-clonic epilepsy, focal epilepsy such as complex focal epilepsy, frontal and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as persistent focal epilepsy, and Hazard-Sert syndrome.

Other neurological diseases that can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hydrocephalus such as Dandy-Walker syndrome and normal pressure hydrocephalus, hypothalamic diseases such as Thalamic neoplasms, cerebral malaria, narcolepsy including cataplexy, bulbar poliomyelitis, pseudotumor cerebri, Reiter's syndrome, Reye's syndrome, thalamic disease, cerebral toxoplasmosis, cranial Tuberculosis and Zellweger's syndrome, central nervous system infection such as AIDS dementia complex, brain abscess, subdural empyema, encephalomyelitis such as equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis pneumonia, ovine demyelinating leukoencephalitis, and cerebral malaria.

Other neurological diseases that can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include meningitis such as arachnoiditis, aseptic meningitis such as viral meningitis including lymphocytic choroid Pleural meningitis, Bacterial meningitis including Haemophilus meningitis, Listeria meningitis, Meningococcal meningitis such as Waugh-Ferr syndrome, Pneumococcal meningitis and meningeal tuberculosis, Fungal meningitis such as Cryptococcal meningitis subdural effusion, meningoencephalitis such as uveomenoencephalitis syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes, poliomyelitis including bulbar poliomyelitis and post-polio syndrome , prion diseases (such as Creutzfeldt-Jakob syndrome, bovine spongiform encephalopathy, Gustav syndrome, kuru, scrapie), and cerebral toxoplasmosis.

Other neurological diseases that may be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include central nervous system neoplasms, such as brain neoplasms, including cerebellar neoplasms such as tentorial neoplasms, ventricles Neoplasms such as choroid plexus neoplasms, hypothalamic and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms including epidural neoplasms, demyelinating disorders such as Canavan disease, diffuse sclerosis including adrenoleukotrophs Dysfunction, periaxial encephalitis, globular cell leukodystrophy, diffuse sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy , multiple sclerosis, central pontine myelination, transverse myelitis, neuromyelitis optica, scrapie, concave back, chronic fatigue syndrome, sheep demyelinating leukoencephalitis, hypertensive syndrome, pseudomeningitis, Myelopathy such as congenital muscular atlasia, amyotrophic lateral sclerosis, spinal muscular atrophy such as Weidnig-Hoffmann disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, tabes dorsalis, Rigid syndrome, mental retardation such as Angelman syndrome, meowing syndrome, De Lange's syndrome, Down syndrome, gangliosidosis such as gangliosidosis G(M1), Morus De Hoffer's disease, Tay-Sachs' disease, Hartner's disease, homocystinuria, Lau-Mur-By's syndrome, Lay-Nee's syndrome, maple diabetes, mucolipidosis diseases such as fucosidosis, neuronal ceroid lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi syndrome, Reiter's syndrome, Rubinstein-Taybe syndrome, tuberous sclerosis, WAGR syndrome, neurological abnormalities such as holoprosencephaly, neural tube defects such as anencephaly including hydroanencephaly, A-Cha Twin's deformity, encephalocele, meningocele, myelomeningocele, spina bifida diseases such as spina bifida cysts and spina bifida occult.

Other neurological diseases that can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hereditary motor and sensory neuropathies, including Charcot-Marie disease, hereditary optic atrophy, Raytheon Fersum's disease, hereditary spastic paraplegia, Weidnig-Hoffmann disease, hereditary sensory and autonomic neuropathies such as congenital analgesia and familial dysautonomia, neurological manifestations (such as agnosia, including Sterman's syndrome, amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, social disturbances such as hearing disturbances including deafness, partial hearing loss, loudness restoration and tinnitus, speech disturbances such as aphasia Includes agraphia, anamia, Broca's aphasia and Wernicke's aphasia, dyslexia such as acquired dyslexia, language development disorders, language disorders such as aphasia including anamia, Broca's aphasia and Wernicke's aphasia Aphasia, dysmorphic speech, social disturbances such as language disturbances including dysarthria, imitation of speech, mutism and stuttering, development of disturbances such as aphonia and hoarseness, decerebrates, delirium, fasciculations, hallucinations, pseudomeningitis, Movement disorders such as Angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, convulsions, torticollis and tremor, muscle hypertonia such as muscle rigidity such as rigidity Body syndromes, muscle spasticity, paralysis such as facial palsy including otic shingles, gastroparesis, hemiplegia, ophthalmoplegia such as diplopia, Duane's syndrome, Horner's syndrome, chronic progressive external ophthalmoplegia Such as Keynes' syndrome, bulbar palsy, tropical spastic paraplegia, paraplegia such as Brown-Seccal syndrome, quadriplegia, respiratory and vocal cord paralysis, paresis, phantom limbs, taste disturbances such as anosmia and dysgeusia, visual disturbances Such as amblyopia, blindness, color vision defects, diplopia, hemianopia, blind spot and low normal vision, sleep disorders such as hypersomnia including Kline-Levin syndrome, insomnia and sleepwalking, convulsions such as trismus, unconsciousness such as coma, persistent Vegetative state and syncope and vertigo, neuromuscular disorders such as congenital muscle relaxation, amyotrophic lateral sclerosis, Lambert-Eaton myasthenic syndrome, motor neuron disease, muscle atrophy such as spinal muscular atrophy, Shy-Marie syndrome and Weidnig-Hoffmann disease, post-polio syndrome, muscular dystrophy, myasthenia gravis, atrophic myotonia, myotonia congenita, linear body myopathy, familial periodic paralysis, multiple Sexual (para)myoclonus, tropical spastic paraplegia and rigid body syndrome, peripheral nervous system disorders such as extremity pain, amyloid neuropathy, autonomic nervous system disorders such as Addison's syndrome, Barrett's syndrome , Familial Autonomic Disorders, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Disorders such as Acoustic Nerve Disorders such as Acoustic Neuroma including Neurofibromatosis 2 Facial Nerve Disorders such as Facial Nerve Pain, Melkson-Rosenthal syndrome, ocular motility disorder including amblyopia, nystagmus, eye movement Ophthalmoplegia, ophthalmoplegia such as Duane's syndrome, Horner's syndrome, chronic progressive external ophthalmoplegia including Keynes' syndrome, strabismus such as esotropia and exotropia, oculomotor nerve palsies, optic nerve disorders such as optic atrophy including Hereditary optic atrophy, optic disc drusen, optic nerve eyes such as neuromyelitis optica, optic disc edema, trigeminal neuralgia, vocal cord paralysis, demyelinating diseases such as neuromyelitis optica and concave back, and diabetic neuropathies such as diabetic foot.

Other neurological diseases that can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndromes such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervical and brachial neuralgia, facial and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculopathy Neuropathy and radiculitis such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia, and Weidnig-Hoff Mann disease, hereditary sensory and autonomic neuropathies include congenital analgesia and familial dysautonomia, POEMS syndrome, sciatica, gustatory sweating, and tetany.

hormone imbalance

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat, prevent, diagnose, and/or predict disorders and/or diseases related to hormone imbalance, and/or endocrine system disorder or disease.

Hormones secreted by the glands of the endocrine system control body growth, sexual function, metabolism, and other functions. Disorders can be classified in two ways: disturbances in hormone production and inability of tissues to respond to hormones. The etiology of these hormonal imbalances or endocrine system diseases, disorders or conditions can be genetic, physical (such as cancer and some autoimmune diseases), acquired (such as through chemotherapy, injury or toxins), or infectious. In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as markers or detectors for specific diseases or disorders related to endocrine system and/or hormonal imbalances.

Disorders and/or diseases of the endocrine system and/or hormonal imbalances cover disorders of uterine motility, including but not limited to: complications of pregnancy and childbirth (such as premature delivery, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and menstruation Periodic disorders and/or diseases (such as dysmenorrhea and endometriosis).

Disorders and/or diseases of the endocrine system and/or hormonal imbalances including those of the pancreas such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic hypoplasia, pheochromocytoma-islet cell tumor syndrome; disorders of the adrenal glands and/or diseases such as, for example, Addison's disease, corticosteroid deficiency, virilization disorders, hirsutism, Cushing's syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, Such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid gland, including but not limited to hyperthyroidism, thyroid Hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and resting lymphoid cellular thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic agenesis, Schüter's cell tumor of the thyroid, thyroid cancer, thyroid cancer, thyroid medulla Disorders and/or diseases of the parathyroid glands, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.

Additionally, disorders and/or diseases of the endocrine system and/or hormonal imbalances may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries also include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, runaway testicular syndrome (bilateral anorchia), Leydig cell congenital Anexogenous, cryptorchidism, Noonan syndrome, myotonic dystrophy, capillary hemangioma of the testis (benign), neoplasia of the testis, and neo-testis.

In addition, disorders and/or diseases of the endocrine system and/or hormonal imbalances may also include disorders and/or diseases such as, for example, polyglandular deficiency syndrome, pheochromocytoma, neuroblastoma, multiple endocrine neoplasia, and endocrine tissue disorders and/or cancer.

In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used in the diagnosis, prediction, prevention, and/or treatment of therapeutic effects with albumin fusion proteins of the invention Endocrine diseases and/or disorders associated with tissues in which the therapeutic protein corresponding to the protein moiety is expressed.

reproductive system disorder

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosing, treating, or preventing diseases and/or disorders of the reproductive system. Reproductive system disorders that may be treated with the compositions of the present invention include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders that result in infertility, complications of pregnancy, childbirth, or childbirth, and postpartum Difficulties (postpartum difficulties).

Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchidism (unilateral and bilateral), anorchia, ectopic testis, epididymitis, and orchitis ( Usually caused by infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, nodular vas deferens, germ cell tumors (eg, seminoma, embryogenic cell carcinoma, teratocarcinoma, choriocarcinoma, yolk sac tumor, and teratoma), stromal tumors (such as Leydig cell tumor), hydrocele, hemocele, varicocele, semimenocele, inguinal hernia, and disturbances of spermatogenesis (if not kinetocililiary syndrome, azoospermia, asthenospermia, azoospermia, hypospermia, and teratozoospermia).

Disorders of the reproductive system also include disorders of the prostate such as acute nonbacterial prostatitis, chronic nonbacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatic dystonia, prostatosis, granulomatous prostatitis, softening plaques, benign prostatic hypertrophy or regeneration, and neoplastic disorders of the prostate, including adenocarcinoma, transitional cell carcinoma, ductal carcinoma, and squamous cell carcinoma.

In addition, the compositions of the present invention are useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders such as glans posthitis, balanitis obliterans, phimosis, incarcerated phimosis, syphilis , herpes simplex virus, gonorrhea, nongonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, genital warts, genital warts, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including erythema of Keira, Bowen's disease, Bowenoid papulosis, Budhler's giant condyloma, and verrucous carcinoma; penile cancer, Includes squamous cell carcinoma, carcinoma in situ, verrucous carcinoma, and diffuse penile carcinoma; neoplastic disorders of the urethra, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders such as Priapism, Peyronie's disease, erectile dysfunction, and impotence.

In addition, diseases and/or disorders of the vas deferens include vasculitis and CBAVD (congenital bilateral absence of the vas deferens); in addition, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for Diagnosis, treatment, and/or prevention of seminal vesicle diseases and/or disorders, including cysticercosis, congenital chloride diarrhea, and polycystic kidney disease.

Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes, cystic fibrosis, Cartagena's syndrome, hyperthermia, multiple sclerosis, and Gynecomastia.

Additionally, polynucleotides, fusion proteins of the invention, and/or polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, treatment, and/or prevention of vaginal and vulvar diseases and/or disorders, including bacterial vaginosis , Candida vaginitis, Herpes simplex virus, Chancroid, Inguinal granuloma, Lymphogranuloma venereum, Scabies, Human papillomavirus, Vaginal trauma, Vaginal trauma, Adenosis, Chlamydia vaginitis, Gonorrhea, Trichomonas Vaginitis, genital warts, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis , and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanoma, bartholin adenocarcinoma, and vulvar intraepithelial neoplasia.

Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, vesicular mole (mole ), Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (eg, due to abnormal hormonal signaling), and neoplastic disorders such as adenocarcinoma, leiomyosarcoma, and sarcoma. In addition, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used as markers or detection materials for congenital uterine abnormalities, and for diagnosis, treatment, and/or prevention, such as Bicornuate, Septate, Simple Unicorate, Unicorate with Absolute Primitive Horns, Unicorate with Unconnected Primitive Horns, Unicorate with Connecting Luminal Horns, Arcuate, Gemini, and T-shaped uterus.

Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal II syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, Right ovarian venous syndrome, amenorrhea, hirutism, and ovarian cancer (including but not limited to primary and secondary cancerous growths, Sertoli-Leidig tumor, endometrioid carcinoma of the ovary, ovarian papilla Serous adenocarcinoma of the ovary, mucinous adenocarcinoma of the ovary, and Krukenberg tumor of the ovary).

Diseases and/or disorders of the cervix include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Narbert's cysts, cervical erosions, cervical incompetence, and cervical neoplasms (including, for example, cervical cancer , squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).

In addition, diseases and/or disorders of the reproductive system include pregnancy disorders and/or diseases, including miscarriage and stillbirth, such as early miscarriage, late miscarriage, spontaneous miscarriage, induced miscarriage, therapeutic miscarriage, threatened miscarriage, missed miscarriage, incomplete miscarriage, Complete miscarriage, recurrent miscarriage, missed miscarriage, and miscarriage infection; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes mellitus, intrauterine growth retardation, polyhydramnios, HELLP syndrome, early placenta Detachment, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. In addition, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the diagnosis, treatment, and/or prevention of diseases that may be complicated by pregnancy, including heart disease, heart failure, rheumatic heart disease , congenital heart disease, mitral valve prolapse, hypertension, anemia, kidney disease, infectious diseases (such as rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), Diabetes, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematosus, rheumatoid arthritis, Myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and intestinal obstruction.

Complications related to labor and delivery include premature rupture of the membranes, premature delivery, post-term pregnancy, overmaturity, labor progressing too slowly, fetal distress (eg, abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder Dystocia, prolapsed umbilical cord, amniotic fluid embolism, and abnormal uterine bleeding.

Additionally, diseases and/or disorders in the postpartum period include endometritis, uterine myositis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous vein thrombosis phlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.

Other disorders and/or diseases of the female reproductive system that can be diagnosed, treated, and/or prevented using the albumin fusion proteins of the invention and/or polynucleotides encoding the albumin fusion proteins of the invention include, for example, Turner's syndrome, pseudo Hermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (blood vessel congestion), aneromaia, anorgasmia, sexual displeasure, ruptured fallopian tubes, and menstrual pain.

infectious disease

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used in the treatment or detection of infectious agents. For example, infectious diseases can be treated by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells. The immune response can be enhanced, either by enhancing an existing immune response, or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also directly inhibit infectious agents without eliciting an immune response.

A virus is an example of an infectious agent that can cause a disease or condition that can be treated or detected with an albumin fusion protein of the invention and/or a polynucleotide encoding an albumin fusion protein of the invention. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and virus families: Arboviruses, Adenoviridae, Arenaviridae, Arteriviruses, Dirnaviridae (Birnaviridae), Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (hepatitis viruses), Herpesviridae (such as cytomegalovirus, herpes simplex virus, herpes zoster virus), Mononegavirus (such as Paramyxoviridae), measles virus, Rhabdoviridae), Orthomyxoviridae (such as influenza A, influenza B, and parainfluenza), papillomaviruses, Papovaviridae, Parvoviridae ), Picornaviridae, Poxviridae (such as smallpox or vaccinia), Reoviridae (such as rotavirus), Retroviridae (HTLV-I, HTLV-II, lentiviruses), and Togaviridae (eg rubella virus). Viruses belonging to these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, eye infections (eg, conjunctivitis, keratitis), chronic fatigue syndrome, Hepatitis (A, B, C, E, Chronic Active, D), Japanese Encephalitis B, Junin Fever, Chikungunya Fever, Rift Valley Fever, Yellow Fever, Meningitis, Opportunity Sexual infections (such as AIDS), pneumonia, Burkitt's lymphoma, fowl pox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, colds, poliomyelitis, leukemia, rubella, sexually transmitted diseases, skin diseases (such as Kaposi's, warts), and viremia. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect any of these conditions or diseases. In a specific embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used for the treatment of: meningitis, dengue fever, EBV, and/or hepatitis (such as hepatitis B). In another specific embodiment, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat patients who do not respond to one or more other commercially available hepatitis vaccines. In yet another specific embodiment, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used for the treatment of AIDS.

Similarly, bacterial and fungal agents that can cause diseases or symptoms that can be treated or detected with albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, the following Gram-negative and Gram-negative Lambert-positive bacteria, bacterial families, and fungi: Actinomyces (eg, Nocardia), Acinetobacter, Cryptococcus neoformans, Aspergillus ( Aspergillus), Bacillaceae (eg, Bacillus anthracis), Bacteroides (eg, Bacteroides fragilis), Blastomyces, Bordetella (Borrelia), Borrelia (such as Borrelia burgdorferi), Brucella, Candida, Campylobacter, Chlamydia ), Clostridium (such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani Clostridium tetani), Coccidioides, Corynebacterium (eg, Corynebacterium diphtheriae), Cryptococcus, Dermatocycoses, Escherichia coli Escherichia coli (such as enterotoxigenic Escherichia coli and enterohemorrhagic Escherichia coli), Enterobacter (such as Enterobacter aerogenes), Enterobacteriaceae (gram Klebsiella, Salmonella (eg, Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia Yersinia, Shigella), Erysipelothrix, Haemophilus (such as Haemophilus influenzae type B), Helicobacter, Legionella (such as Legionella pneumophila pneumophila), Leptospira, Listeria (e.g. Listeria monocytogenes), Mycoplasma, Mycobacterium ) (such as Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (such as Vibrio cholerae), Neisseriaceae (such as gonorrhea Neisseria gonorrheae, Neisseria meningilidis), Pasteurellaceae, Proteus, Pseudomonas (eg, Pseudomonas aeruginosa Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (such as Treponema spp., Leptospiraspp., Borrelia spp.) ), Shigella spp., Staphylococcus (eg Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (such as Streptococcus pneumoniae and group A, B, and C streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, sepsis, eye infections (eg, conjunctivitis), uveitis, tuberculosis , gingivitis, bacterial diarrhea, opportunistic infections (eg, AIDS-related infections), paronychia, prosthesis-related infections, dental caries, Reiter's disease, respiratory infections such as whooping cough or empyema, sepsis, Lyme disease , cat scratch disease, dysentery, paratyphoid, food poisoning, Legionnaires' disease, chronic and acute inflammation, erythema, yeast infection, typhoid, pneumonia, lymphatic, meningitis (eg, meningitis A and B), chlamydia, syphilis, diphtheria , leprosy, brucellosis, peptic ulcer, anthrax, spontaneous abortion, birth defects, pneumonia, lung infection, ear infection, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colon vaginitis, infertility, pelvic inflammatory disease, candida, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (such as cellulitis, skin mycoses), toxemia, urinary tract infection, wound infection, hospital infection. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect any of these conditions or diseases. In specific embodiments, the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention are used for the treatment of: tetanus, diphtheria, botulism, and/or meningitis B.

In addition, parasitic agents that cause diseases or symptoms that can be treated, prevented, and/or diagnosed with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, the following families or species: Babesia (disease), Babesia (disease), Coccidiosis (disease), Cryptosporidium (disease), Dinuclear amoeba (disease), Equine equine disease, External parasites (disease), Giardia Worm (disease), Helminth (disease), Leishmania (disease), Schistosomiasis (schistosomiasis) disease, Pyoplasma tyrleri (disease), Toxoplasma gondii (disease), Trypanosoma (disease), and Trichomonas (disease) Plasmodium (disease) and sporozoites (such as Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or conditions, including but not limited to: scabies, chigger mange, eye infections, enteric diseases (eg, dysentery, giardiasis), liver disease, lung disease, opportunistic infections (eg, AIDS-related ), malaria, pregnancy complications, and toxoplasmosis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat, prevent, and/or diagnose any of these conditions or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used for the treatment, prevention, and/or diagnosis of malaria.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be administered by either administering an effective amount of the albumin fusion protein of the present invention to the patient, or collecting cells from the patient, giving the cells The polynucleotides of the invention are supplied and the engineered cells are returned to the patient (ex vivo therapy). In addition, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as antigens in vaccines for eliciting immune responses against infectious diseases.

regeneration

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used for differentiation, proliferation, and attraction of cells, leading to tissue regeneration (see Science, 276:59-87, 1997). Tissue regeneration can be used for repair, replacement, or replacement due to birth defects, trauma (wounds, burns, cuts, or ulcers), aging, disease (such as osteoporosis, osteoarthritis, periodontal disease, liver failure), Surgery includes tissue damaged by cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine injury.

Tissues that can be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, or cardiac muscle), vasculature (including blood vessels and lymphatic vessels), nerves, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissues. Preferably, hyperplasia occurs without or with reduced scarring. Regeneration can also include angiogenesis.

In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can enhance regeneration of refractory tissues. For example, improving tendon/ligament regeneration will speed up recovery time after injury. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be used prophylactically in efforts to avoid damage. Specific conditions that may be treated include tendonitis, carpal tunnel syndrome, and other tendon or ligament defects. Another example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds.

Similarly, nerve and brain tissue can also be regenerated using the fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention, so that nerve cells can proliferate and differentiate. Diseases that may be treated using this method include central and peripheral nervous system diseases, neurological diseases, or mechanical and traumatic disorders (eg, spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, with peripheral nerve injury, peripheral neuropathy (such as caused by chemotherapy or other medical therapy), localized neuropathy, and central nervous system disease (such as Alzheimer's disease, Parkinson's disease, Huntington's ALS, Amyotrophic Lateral Sclerosis, and Shy-Drager Syndrome) can be treated using the albumin fusion protein of the present invention and/or polynucleotides encoding the albumin fusion protein of the present invention.

Gastrointestinal disorders

Albumin fusion proteins of the present invention and/or polynucleotides encoding albumin fusion proteins of the present invention can be used to treat, prevent, diagnose, and/or predict gastrointestinal disorders, including inflammatory diseases and/or conditions, infection, cancer ( Such as intestinal neoplasms (carcinoid tumors of the small intestine, non-Hodgkin's lymphoma of the small intestine, lymphoma of the small intestine)), and ulcers, such as peptic ulcers.

Gastrointestinal disturbances include dysphagia, odynophagia, esophageal inflammation, peptic esophagitis, gastric reflux, submucosal fibrosis and strictures, Malory-Weiss lesions, leiomyomas, lipomas, epidermal carcinomas, adenocarcinomas, Gastric retention disorders, gastroenteritis, gastric atrophy, gastric cancer, gastric polyps, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic , plasmacytic, and Menitriere's), and peritoneal disorders (eg, chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasm, peritonitis, pneumoperitoneum, bubphrenic abscess).

Gastrointestinal disorders also include disorders related to the small intestine such as malabsorption syndrome, bloat, irritable bowel syndrome, glucose intolerance, celiac disease, duodenal ulcer, duodenitis, tropical sprue, HP Ear disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, ileal obstruction, Meckel's diverticulum, multiple diverticulum, complete failure of the small and large bowel, lymphoma, and bacterial and parasitic diseases (such as traveler's diarrhea , typhoid and paratyphoid, cholera, roundworms (Ascariasis lumbricoides), hookworms (Ancylostoma duodenale), nematodes (Enterobius vermicularis), tapeworms (Taenia saginata, granulosus Echinococcus granulosus, Diphyllobothrium spp., and T. solium) infection.

Liver diseases and/or disorders including intrahepatic cholestasis (Alagir syndrome, biliary cirrhosis), fatty liver (alcoholic fatty liver disease, Reye's syndrome), hepatic vein thrombosis, hepatolenticular degeneration , hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), cirrhosis (alcoholic, biliary, and experimental), alcohol Liver disease (fatty liver, hepatitis, cirrhosis), parasites (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, Portal hypertension, hepatomegaly, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, C, D, drug-induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, Hepatitis D, Hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varicose vessels, hepatic encephalopathy, primary biliary cirrhosis, primary cirrhosis Hepatic cholangitis, hepatocellular adenoma, hemangioma, gallstones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, Epithelial tumors, fibrolamellar liver cancer, focal nodular hyperplasia, hepatic adenocarcinoma, hepatic gallbladder adenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, hepatic hemangioendothelioma, mesenchymal hamartoma, Mesenchymal tumors of the liver, nodular regenerative hyperplasia, benign liver tumors (hepatic cysts [simple cysts, polycystic liver disease, hepatic and gallbladder adenomas, choledochal cysts], mesenchymal tumors [mesenchymal hamartoma, Infant hemangioendothelioma, hemangioma, purpuric liver disease, lipoma, inflammatory pseudotumor, mixed type], epithelioma [cholangioepithelium (cholangiohamartoma, bile duct adenoma), hepatocellular (adenoma, focal nodule hyperplasia, nodular regenerative hyperplasia)], malignant liver tumors [hepatocytes, hepatoblastoma, hepatocellular carcinoma, cholangiocellular carcinoma, cholangiocarcinoma, cystadenocarcinoma, vascular tumors, angiosarcoma, Kaposi's sarcoma, vascular Endothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, primary lymphoma]), purpuric liver disease, erythropoietic liver disease porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).

Diseases and/or disorders of the pancreas include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasm, islet cell neoplasm, pancreatoblastoma), and other pancreatic diseases (eg, cystic fibrosis, cysts (pancreatic pseudocyst, pancreatic fistula, insufficiency)).

Gallbladder disorders include gallstones (cholelithiasis and choledocholithiasis), post-cholecystectomy syndrome, gallbladder diverticular disease, acute cholecystitis, chronic cholecystitis, bile duct neoplasms, and mucocele.

Diseases and/or disorders of the large bowel include antibiotic-resistant colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (eg, anal fissures, hemorrhoids), diseases of the colon (colitis , colonic neoplasia [colon cancer, adenomatous polyps of the colon (such as villous adenoma), colon cancer, colorectal cancer], colonic diverticulitis, colonic diverticular disease, megacolon [Hillsprung's disease, toxic giant colon]; sigmoid colon disease [proctocolitis, sigmoid neoplasm]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal disease (duodenal neoplasm, duodenal obstruction , duodenal ulcer, duodenitis), enteritis (enterocolitis), FIN enteropathy, ileal disease (ileal neoplasm, ileitis), immunoproliferative small bowel disease, inflammatory bowel disease (ulcerative colon inflammatory disease, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, baguillosis, yeast infection (blastocystis infection), cryptosporidiosis, binuclear amoebiasis, amoebic dysentery , giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (entering loop syndrome, duodenal obstruction, fecal obstruction, pseudo-obstructive rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, bleeding, perforation, gastric ulcer, Zollinger- Ellison syndrome), postgastrectomy syndrome (dumping syndrome), stomach disorders (eg, achlorhydria, duodenogastric reflux (bile reflux), antral vasodilation, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, gastric dilatation, gastric diverticulum, gastric neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyps), gastric rupture, gastric ulcer, gastric torsion), tuberculosis, visceral , vomiting (such as hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting), and hemorrhagic colitis.

Other diseases and/or disorders of the gastrointestinal system include biliary disorders such as gastroschisis, fistulas (eg, biliary, esophageal, gastric, intestinal, pancreatic), neoplasms (eg, biliary neoplasms, esophageal neoplasms such as esophagus Adenocarcinoma, squamous cell carcinoma of the esophagus, gastrointestinal neoplasms, pancreatic neoplasms such as adenocarcinoma of the pancreas, mucinous cystic neoplasms of the pancreas, cystic neoplasms of the pancreas, pancreatic blastoma, and peritoneal neoplasms), esophagus Disorders (eg, candidiasis, candidiasis, glycogenic acanthosis, ulcers, Barrett's esophageal varices, atreses, cysts, diverticula (eg, Zenker's diverticulum), fistulas (eg, tracheoesophageal fistula) , motility disorders (eg, CREST syndrome, dysphagia, achalasia, spasticity, gastroesophageal reflux), vegetations, perforations (eg, Bullhaver syndrome, Mallory-Weiss syndrome), strictures, esophagitis, Diaphragmatic hernia (such as hiatal hernia); gastrointestinal diseases such as gastroenteritis (such as pseudocholera, Norwalk virus infection), bleeding (such as hematemesis, melena, peptic ulcer bleeding), gastric vegetations (gastric cancer, gastric polyps, gastric adenocarcinoma, gastric cancer), hernias (eg, congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal disorders (eg, cecal disease (appendicitis, cecal neoplasm)).

Chemotaxis

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may have chemotactic activity. Chemotactic molecules attract or mobilize cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells, and/or endothelial cells) to specific sites in the body, such as Inflamed, infected, or hyperproliferative sites. The mobilized cells can then fight and/or treat a particular trauma or abnormality.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can improve the chemotactic activity of specific cells. These chemotactic molecules can thus be used to treat inflammation, infection, hyperproliferative disorders, or any disorder of the immune system by increasing the number of cells targeted to specific sites in the body. For example, chemotactic molecules can be used to treat wounds or other initiations to tissue by attracting immune cells to the site of injury. The chemoattractant molecules of the invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit chemotactic activity. These molecules are also useful in the treatment of disorders. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful as inhibitors of chemotaxis.

binding activity

Albumin fusion proteins of the invention can be used to screen for molecules that bind the Therapeutic protein portion of the fusion protein or to which the Therapeutic protein portion of the fusion protein binds. Binding of the fusion protein to the molecule can stimulate (agonist), increase, inhibit (antagonist), or decrease the activity of the fusion protein or the bound molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (such as receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the Therapeutic protein portion of the fusion protein of the invention, such as a fragment of the ligand, or a natural substrate, ligand, structural or functional mimic (see Coligan et al., Current Protocols in Immunology, 1(2): Chapter 5, 1991). Similarly, the molecule may be closely related to the native receptor bound by the Therapeutic protein portion of the albumin fusion protein of the invention, or at least a receptor fragment (e.g., active site point). In either case, the molecule can be rationally designed using known techniques.

Preferably, the selection of these molecules involves the generation of suitable cells expressing the albumin fusion proteins of the invention. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.

Assays may simply test the binding of candidate compounds to albumin fusion proteins of the invention, where binding is detected by a label or in an assay involving competition with a labeled competitor. In addition, the assay can test candidate compounds for signal generation through binding to the fusion protein.

Alternatively, assays can be performed using cell-free preparations, fusion proteins/molecules attached to solid supports, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of: admixing the candidate compound with an albumin-containing fusion protein solution; measuring the fusion protein/molecule activity or binding; and comparing the fusion protein/molecule activity or binding to a standard.

Preferably, the ELISA assay can use monoclonal or polyclonal antibodies to measure the level or activity of the fusion protein in a sample, such as a biological sample. Either by direct or indirect binding to the albumin fusion protein, or by competing with the albumin fusion protein for a substrate, the antibody measures fusion protein level or activity.

Additionally, the receptors to which the Therapeutic protein moiety of an albumin fusion protein of the invention binds can be identified by a number of methods known to those skilled in the art, such as ligand panning and FACS sorting (Coligan et al., Current Protocols in Immun., 1(2), Chapter 5, 1991). For example, where the Therapeutic protein portion of the fusion protein corresponds to FGF, expression cloning can be employed in which polyadenylated RNA is produced from cells responding to albumin fusion proteins, such as various receptors known to contain FGF family proteins NIH3T3 cells and SC-3 cells, and cDNA libraries constructed from this RNA were divided into several pools and used to transfect COS cells or other cells that did not respond to albumin fusion proteins. Transfected cells cultured on slides were exposed to albumin fusion proteins of the invention, which were previously labeled. Albumin fusion proteins can be labeled by a variety of methods, including iodination or introduction of recognition sites for site-specific protein kinases.

After fixation and incubation, slides were subjected to autoradiographic analysis. Positive pools are identified, and an iterative molecular pooling and rescreening process is used to prepare subsets and retransfect, resulting in single clones encoding putative receptors.

As an alternative to receptor identification, a labeled albumin fusion protein can be photoaffinity linked to a cell membrane or extract preparation of a receptor molecule expressing the Therapeutic protein portion of the albumin fusion protein of the invention, and the linked material can be passed through PAGE analysis resolves and exposes X-ray film. Tagged complexes containing fusion protein receptors can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequences obtained from the microassays will be used to design a set of degenerate oligonucleotide probes for screening cDNA libraries to identify genes encoding putative receptors.

In addition, gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling") techniques may be employed to manipulate fusion proteins and/or Therapeutic protein portions of albumin fusion proteins of the invention or activity of the albumin component, thereby efficiently producing agonists and antagonists of the albumin fusion protein of the present invention. See generally U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458 and Patten, P.A. et al., Curr. Opinion Biotechnol., 8:724-33, 1997; Harayama, S., Trends Biotechnol., 16(2): 76-82, 1998; Hansson, L.O. et al., J. Mol. Biol., 287:265-76, 1999; and Lorenzo, M.M. and Blasco, R., Biotechniques, 24(2):308-13, 1998; Each of these patents and publications is incorporated herein by reference. In one embodiment, alterations of polynucleotides encoding albumin fusion proteins of the invention, and thus albumin fusion proteins encoded thereby, can be accomplished by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into desired molecules by homologous or site-specific recombination. In another embodiment, polynucleotides encoding albumin fusion proteins of the invention, and thus albumin fusion proteins encoded thereby, may be altered by performing random mutagenesis by performing error-prone PCR prior to recombination. , random nucleotide insertion, or other methods. In another embodiment, one or more components, motifs, components, parts, domains, fragments, etc. of an albumin fusion protein of the invention may be combined with one or more of one or more heterologous molecules. Recombination of components, motifs, components, parts, domains, fragments, etc. In preferred embodiments, the heterologous molecule is a family member. In a more preferred embodiment, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF ), fibroblast growth factor (FGF), TGF-β, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activators A and B, decapentaplegic (dpp ), 60A, OP-2, dorsin, growth differentiation factor (GDF), nodal, MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3, TGF-β5, and glial cell-derived neuronal Nutritional factor (GDNF).

Other preferred fragments are Therapeutic protein portions of albumin fusion proteins of the invention and/or biologically active fragments of albumin components. A biologically active fragment refers to a fragment that exhibits an activity similar, but not necessarily identical, to that of the Therapeutic protein portion and/or the albumin component of an albumin fusion protein of the invention. The biological activity of a fragment may include increased desired activity or decreased undesired activity.

In addition, the invention provides methods of screening compounds to identify compounds that modulate the action of albumin fusion proteins of the invention. An example of such an assay involves mixing mammalian fibroblasts, an albumin fusion protein of the invention, and a compound to be screened ^and3 [H]thymidine under cell culture conditions in which fibroblasts will proliferate normally. Control assays can be performed in the absence of the compound to be screened and determined whether the compound stimulates proliferation by measuring ³ [H]thymidine uptake in each case compared to the amount of fibroblast proliferation in the presence of the compound. The amount of fibroblast proliferation was measured by liquid scintillation chromatography, which measures ³ [H]thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

In another approach, a mammalian cell or membrane preparation expressing a receptor for a Therapeutic protein component of a fusion protein of the invention is incubated with a labeled fusion protein of the invention in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured. Alternatively, the response of a known second messenger system following the interaction of the compound to be screened with the receptor is measured, and the ability of the compound to bind the receptor and elicit a second messenger response is measured to determine if the compound is a potential fusion protein. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels, or phosphoinositide hydrolysis.

All of these aforementioned assays can be used as diagnostic or predictive markers. By activating or inhibiting the fusion protein/molecule, molecules discovered using these assays can be used to treat a disease or produce a specific outcome (such as blood vessel growth) in a patient. In addition, these assays enable the discovery of agents that inhibit or enhance the production of albumin fusion proteins of the invention by suitably manipulated cells or tissues.

Accordingly, the invention includes a method of identifying compounds that bind to an albumin fusion protein of the invention comprising the steps of: (a) incubating a candidate binding compound with an albumin fusion protein of the invention; and (b) determining whether binding occurs . In addition, the invention includes methods for identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with an albumin fusion protein of the invention, (b) measuring biological activity, and (c) measuring fusion Whether the biological activity of the protein has changed.

targeted delivery

In another embodiment, the invention provides a method of delivering a composition to a target cell expressing a recipient of a component of an albumin fusion protein of the invention.

As discussed herein, fusion proteins of the invention can be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic, and/or covalent interactions. In one embodiment, the invention provides a method of specifically delivering a composition of the invention to a cell by administering a fusion protein of the invention (including an antibody) associated with a heterologous polypeptide or nucleic acid. In one embodiment, the invention provides a method for delivering a therapeutic protein into a target cell. In another embodiment, the invention provides methods for delivering single-stranded nucleic acid (such as antisense or ribozyme) or double-stranded nucleic acid (such as DNA capable of integrating into the genome of a cell or replicating episomally and capable of transcription) to Methods in Target Cells.

In another embodiment, the invention provides methods for specifically destroying cells (such as destroying tumor cells) by administering an albumin fusion protein of the invention (such as a polypeptide of the invention or an antibody of the invention) associated with a toxin or a cytotoxic agent. method.

"Toxin" means a compound, radioisotope, holotoxin, modified toxin, catalytic subunit of a toxin, or one not normally found in a cell or on a cell surface, under defined conditions, that binds to and activates an endogenous cytotoxic effector system. any molecule or enzyme that causes cell death. Toxins that may be used in accordance with the present invention include, but are not limited to, radioactive isotopes, compounds such as, for example, antibodies (or complement-fixing moieties thereof) that bind intrinsic or induced endogenous cytotoxic effector systems, thymidine kinase, and the like known in the art. , endonuclease, RNase, α-toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saponin, charantin, gelonin, American Pokeweed antiviral protein, α-baunomycin, and cholera toxin. "Cytotoxic prodrug" means a non-toxic compound that is converted into a cytotoxic compound by an enzyme normally present in a cell. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, arabinosin Glycine, daunorubicin, and phenoxyacetamide derivatives of doxorubicin.

drug screening

It is also contemplated that albumin fusion proteins of the invention or polynucleotides encoding these fusion proteins are used to screen for activity in modifying albumin fusion proteins of the invention or proteins corresponding to the Therapeutic protein portion of an albumin fusion protein. The use of the molecule. Such methods would involve exposing the fusion protein to a selected compound suspected of having antagonist or agonist activity and determining the activity of the fusion protein upon binding.

The invention is particularly useful for screening therapeutic compounds by using the albumin fusion proteins of the invention, or binding fragments thereof, in a variety of drug screening techniques. Albumin fusion proteins employed in this assay can be attached to a solid support, expressed on the cell surface, free in solution, or localized within the cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells stably transformed with a recombinant nucleic acid expressing an albumin fusion protein. Drugs are screened against such transformed cells or supernatants obtained by culturing such cells in competitive binding assays. For example, complex formation between a test agent and an albumin fusion protein of the invention can be measured.

Thus, the invention provides methods of screening for drugs or any other agent that affects the activity mediated by the albumin fusion proteins of the invention. These methods include contacting the reagent with an albumin fusion protein or fragment thereof of the invention by methods well known in the art, and determining the presence of a complex between the reagent and the albumin fusion protein or fragment thereof. In such competitive binding assays, the reagents to be screened are usually labeled. Following incubation, free reagent is separated from reagent present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular reagent to bind to an albumin fusion protein of the invention.

Another drug screening technique provides high-throughput screening of compounds with suitable binding affinities for the albumin fusion proteins of the invention and is described in great detail in European Patent Application 84/03564, published September 13, 1984 , which is incorporated herein by reference. Briefly, a large number of different small peptide test compounds are synthesized on a solid substrate such as a plastic pin or some other surface. Peptide test compounds are reacted with albumin fusion proteins of the invention and washed. Bound peptide is then detected by methods well known in the art. Purified albumin fusion proteins can be directly coated on plates for use in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture and immobilize peptides on solid supports.

The invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding an albumin fusion protein of the invention compete specifically with a test compound for binding to an albumin fusion protein or fragment thereof. Thus, antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with an albumin fusion protein of the invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifying polypeptides and non-polypeptides that bind albumin fusion proteins of the invention, and binding molecules thus identified. These binding molecules are useful, for example, as agonists and antagonists of the albumin fusion proteins of the invention. Such agonists and antagonists may be used in accordance with the invention in the therapeutic embodiments described in detail below.

The method comprises the steps of: contacting an albumin fusion protein of the invention with a plurality of molecules; and identifying molecules that bind the albumin fusion protein.

The step of contacting an albumin fusion protein of the invention with various molecules can be performed in a variety of ways. For example, it is conceivable to immobilize an albumin fusion protein on a solid support and to contact a solution of various molecules with the immobilized polypeptide. Such a procedure would be analogous to the affinity chromatography process in which the affinity matrix consists of immobilized albumin fusion proteins of the invention. Molecules with selective affinity for albumin fusion proteins can then be purified by affinity selection. The nature of the solid support, the procedures used to attach albumin fusion proteins to the solid support, solvents, and conditions for affinity separation or selection are extremely convenient and are well known to those of ordinary skill in the art.

Alternatively, the plurality of polypeptides may also be separated into substantially separate fractions comprising individual polypeptides or subsets thereof. For example, multiple polypeptides can be separated by gel electrophoresis, column chromatography, or similar methods known to those of ordinary skill in the art for separating polypeptides. Individual polypeptides can also be produced from transformed host cells in such a way that they are expressed on or around their outer surface (eg recombinant phage). Individual isolates can then be "probed" with the albumin fusion protein of the invention, optionally in the presence of the inducer required for expression, to determine whether there are any selective affinity interactions between the albumin fusion protein and the individual clone . For additional convenience, the polypeptides may first be transferred to a solid support prior to contacting the albumin fusion protein with each fraction comprising the individual polypeptides. This solid support may simply be a piece of filter membrane, such as made of nitrocellulose or nylon. Thus, positive clones can be identified from transformed host cell collections expressing libraries comprising a DNA construct encoding a polypeptide having selective affinity for an albumin fusion protein of the invention. In addition, the amino acid sequence of a polypeptide having selective affinity for an albumin fusion protein of the present invention can be directly determined by conventional methods, or it is often more convenient to determine the coding sequence of the DNA encoding the polypeptide. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined by the polypeptide itself, micro-sequencing technology can be used. Sequencing techniques can include mass spectrometry.

In some cases, it may be desirable to wash away any unbound polypeptide from the mixture of the albumin fusion protein of the invention and the plurality of polypeptides prior to attempting to assay or detect the presence of a selective affinity interaction. This washing step may be particularly desirable when binding albumin fusion proteins or polypeptides of the invention to a solid support.

The plurality of molecules provided according to this method can be provided by means of a diverse library, such as a random or combinatorial peptide or non-peptide library that can be used to screen for molecules that specifically bind to an albumin fusion protein of the invention. Many available libraries are known in the art, such as chemically synthesized libraries, recombinant libraries (such as phage display libraries), and libraries based on in vitro translation. Fodor et al., Science, 251:767-773, 1991; Houghten et al., Nature, 354:84-86, 1991; Lam et al., Nature, 354:82-84, 1991; Medynski, Bio/Technology, 12: 709-710, 1994; Gallop et al., J. Medicinal Chemistry, 37(9): 1233-1251, 1994; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90: 10922-10926, 1993; Erb et al. USA, 91:11422-11426, 1994; Houghten et al., Biotechniques, 13:412, 1992; Jayawickreme et al., Proc.Natl.Acad.Sci.USA, 91:1614- 1618, 1994; Salmon et al., Proc. Examples of chemically synthesized libraries are described in 5381-5383, 1992.

Scott et al., Science, 249:386-390, 1990; Devlin et al., Science, 249:404-406, 1990; Christian et al., J.Mol.Biol., 227:711-718, 1992; Lenstra, J Examples of phage display libraries are described in Immunol. Meth., 152:149-157, 1992; Kay et al., Gene, 128:59-65, 1993; and PCT Publication No. WO94/18318, August 18, 1994 .

In vitro translation-based libraries include, but are not limited to, those described in PCT Publication No. WO91/05058, April 18, 1991; and Mattheakis et al., Proc.

As an example of a non-peptide library, a benzodiazepine library (see eg Bunin et al., Proc. Natl. Acad. Sci. USA, 91:4708-4712, 1994) may be suitable for this use. Peptoid libraries may also be used (Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371, 1992). Ostresh et al., Proc. Natl. Acad. Sci. USA, 91: 11138-11142, 1994, describe another example of a useful library in which the amide functionality of the peptides has been permethylated to generate a chemically transformed combinatorial library.

The variety of non-peptide libraries that can be used in the present invention is extremely wide. For example, Ecker and Crooke, Bio/Technology, 13:351-360, 1995 lists benzodiazepines, hydantoins, diketopiperazines, biphenyls, Sugar analogues, β-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminated imides, and oxazolones.

Non-peptide libraries can be roughly divided into two categories: decorated monomers and oligomers. The decorative monomer library adopts a relatively simple scaffold structure on which a variety of functional groups are added. Scaffolds are often molecules with known useful pharmacological activities. For example, the scaffold can be a benzodiazepine structure.

Non-peptidic oligomer libraries utilize large numbers of monomers and assemble them together to generate new shapes based on the order of the monomers. Among the monomer units that have been employed are carbamate, pyrrolinone, and morpholino. Peptoids, i.e. peptide-like oligomers in which the side chains are attached to the α-amino group rather than the α-carbon, form the basis of another form of non-peptidic oligomer library. The original non-peptidic oligomer libraries utilized a single type of monomer and thus contained repeating backbones. More recent libraries have utilized more than one monomer, allowing for increased library flexibility.

Screening libraries can be performed by various methods that are generally known. See, for example, the following references, which disclose screening of peptide libraries: Parsley et al., Adv. Exp. Med. Biol., 251:215-218, 1989; Scott et al., Science, 249:386-390, 1990; et al., BioTechniques, 13:422-427,1992; Oldenburg et al., Proc.Natl.Acad.Sci.USA, 89:5393-5397,1992; Yu et al., Cell, 76:933-945,1994; et al., Science, 241:577-580, 1988; Bock et al., Nature, 355:564-566, 1992; Tuerk et al., Proc.Natl.Acad.Sci.USA, 89:6988-6992, 1992; Ellington et al., Nature, 355:850-852, 1992; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223,409, and U.S. Patent No. 5,198,346, all to Ladner et al.; Rebar et al., Science, 263:671-673, 1993; and PCT Publication No. WO94/18318.

In a specific embodiment, screening to identify molecules that bind albumin fusion proteins of the invention can be performed by contacting library members with albumin fusion proteins of the invention immobilized on a solid support and harvesting molecules that bind to albumin fusion proteins. of those library members to execute. For example, Parmley et al., Gene, 73:305-318, 1988; Fowlkes et al., BioTechniques, 13:422-427, 1992; PCT Publication No. WO94/18318; and references cited therein describe what is known as "panning". Examples of these screening methods for the technique

In another embodiment, a two-hybrid system for screening interacting proteins in yeast (Fields et al., Nature, 340:245-246, 1989; Chien et al., Proc.Natl.Acad.Sci.USA, 88 : 9578-9582, 1991) can be used to identify molecules that specifically bind to the polypeptides of the invention.

When the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term "preference" as used herein means that the method used to construct the library operates with constraints on one or more parameters that determine the resulting collection of molecules (peptides in this case) diversity.

Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position in a peptide is the same for all 20 amino acids. However, bias can be introduced into the library by specifying, for example, that a lysine occurs every 5 amino acids or that positions 4, 8, and 9 of the decapeptide library are fixed to contain only arginine. Clearly, many types of preferences are conceivable, and the invention is not limited to any particular preference. Furthermore, the present invention contemplates certain types of peptide libraries, such as phage display peptide libraries and peptide libraries employing DNA constructs comprising lambda phage vectors and DNA inserts.

As noted above, where the binding molecule is a polypeptide, the polypeptide may have from about 6 to less than about 60 amino acid residues, preferably from about 6 to about 10 amino acid residues, and most preferably from about 6 to about 22 amino acid residues. amino acid. In another embodiment, the binding polypeptide has 15-100 amino acids or 20-50 amino acids.

Selected binding polypeptides can be obtained by chemical synthesis or recombinant expression.

Other activities

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to stimulate new blood vessels in ischemic tissue resulting from various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions Formation (re-vascularization) treatment. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to stimulate angiogenesis and limb regeneration, as discussed above.

The albumin fusion proteins of the invention and/or polynucleotides encoding the albumin fusion proteins of the invention are also useful in the treatment of wounds caused by injuries, burns, postoperative tissue repair, and ulcers because they promote a variety of cells of different origin mitotic cells, such as fibroblasts and skeletal muscle cells, and thereby promote the repair or replacement of damaged or diseased tissues.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to stimulate neuronal growth, as well as to treat and prevent neuronal damage that occurs in certain neuronal disorders or neurodegenerative conditions. Injuries, such as Alzheimer's disease, Parkinson's disease, and AIDS-related complexes. The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention may have the ability to stimulate the growth of chondrocytes, so they can be used to enhance bone and periodontal regeneration, and provide help.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used to prevent skin aging caused by sunburn by stimulating the growth of keratinocytes.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to prevent hair loss. Likewise, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to stimulate the growth and differentiation of hematopoietic and myeloid cells when used in combination with other cytokines.

Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be used in cell cultures to maintain organs or support primary tissues prior to transplantation. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also be used to induce differentiation of tissues of mesoderm origin in early embryos.

In addition to the hematopoietic lineage discussed above, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can also increase or decrease differentiation or proliferation of embryonic stem cells.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used to regulate the characteristics of mammals, such as height, weight, coat color, eye color, skin, proportion of adipose tissue, pigmentation, Body shape, and body shape (such as cosmetic surgery). Similarly, the albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to regulate the metabolism of mammals, affecting catabolism, anabolism, energy processing, utilization and storage.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can be used to affect biological rhythm, heart rhythm, depression (including depression), violent tendency, pain tolerance level, reproductive ability (preferably by activin or inhibin-like activity), hormone or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities to alter the mental or physical state of a mammal.

The albumin fusion protein of the present invention and/or the polynucleotide encoding the albumin fusion protein of the present invention can also be used as a food additive or preservative, such as for improving or reducing storage capacity, fat content, lipid, protein, carbohydrate, Microorganisms, minerals, radiation factors, or other nutrients.

The applications described above are applicable to an extremely wide variety of hosts. These hosts include, but are not limited to, humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, minipigs, chickens, goats, cows, sheep, dogs, cats, non-human primates ,and people. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog, or cat. In preferred embodiments, the host is a mammal. In the most preferred embodiment, the host is human.

Having generally described the present invention, it will be more readily understood with reference to the following examples. These examples are provided by way of illustration and are not intended to be limiting.

Without further elaboration, we believe that one of ordinary skill in the art, given the preceding description and the following illustrative examples, can make and use the variations found in the present invention and practice the claimed method. Accordingly, the following working examples specify preferred embodiments of the invention and are not to be construed as limiting the remainder of the disclosure in any way.

Example

Example 1: Generation of pScNHSA and pScCHSA

The vectors pScNHSA (ATCC Deposit No. PTA-3279) and pScCHSA (ATCC Deposit No. PTA-3276) are derivatives of pPPC0005 (ATCC Deposit No. PTA-3278) and are used as cloning vectors for inserting therapeutic proteins or fragments thereof or mutants. The polynucleotide of the body is adjacent to and in the same translation frame as the polynucleotide encoding human serum albumin "HSA". pScCHSA can be used to generate Therapeutic protein-HSA fusions, while pScNHSA can be used to generate HSA-Therapeutic protein fusions.

Generation of pScCHSA: albumin fusion with the albumin moiety C-terminal to the therapeutic moiety

A vector that facilitates cloning of the DNA encoding the Therapeutic protein into the N-terminus of the DNA encoding the mature albumin protein was prepared by altering the nucleic acid sequence encoding the chimeric HSA signal peptide in pPPC0005 to include Xho I and Cla I restriction sites.

In the first step, the inherent XhoI and Cla I sites of pPPC0005 (located at the 3' end of the ADH1 terminator sequence) were removed by digesting pPPC0005 with Xho I and Cla I, the sticky ends were filled in with T4 DNA polymerase, and the blunt ends were religated to generate pPPC0006.

In the second step, Xho I and Cla I restriction sites were added to the nucleic acid sequence encoding the HSA signal peptide in pPPC006 (chimera of the HSA leader sequence and the kex2 site from mating factor α "MAF") using two rounds of PCR. In the first round of PCR, amplification was performed with primers shown in SEQ ID NO: 36 and SEQ ID NO: 37. The primer whose sequence is shown as SEQ ID NO: 36 comprises the nucleic acid sequence encoding part of the HSA signal peptide sequence, the kex2 site from the mating factor α leader sequence and the amino terminal part of the mature form of HSA. Four point mutations were introduced in the sequence, creating Xho I and Cla I sites found at the junction of the chimeric signal peptide and the mature form of HSA. These four mutations are underlined in the sequence shown below. In pPPC0005, the nucleotides at these four positions are T, G, T and G from 5' to 3'.

5'-GC C T C GA G AAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG A TTTAAAGATTTGGG-3' (SEQ ID NO: 36) and

5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3' (SEQ ID NO: 37). Then with the upstream flanking primer,

5'-TACAAACTTAAGAGTCCAATTAGC-3'(SEQ ID NO:38) and downstream flanking primer 5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-3'(SEQ ID NO:39)

Second round of PCR. The PCR product thus generated was then purified, digested with Afl II and Xba I, and ligated into the same sites of pPPC0006 to generate pScCHSA. The plasmid thus generated has Xho I and Cla I sites introduced into the signal sequence. The presence of the Xho I site produced a single amino acid change in the end of the signal sequence from LDKR to LEKR. A nucleic acid sequence comprising a polynucleotide encoding an albumin fusion protein therapeutic portion having a 5'Sal I site (which is compatible with the XhoI site) and a 3'Cla I site is linked to Xho I and Cla of pScCHSA After the I site, the D to E change will not be present in the final albumin fusion protein expression plasmid. Ligation of Sal I to Xho I restores the original amino acid sequence of the signal peptide sequence. The DNA encoding the therapeutic portion of the albumin fusion protein can be inserted after the Kex2 site (Kex2 cuts after the dibasic amino acid sequence KR at the end of the signal peptide) and before the Cla I site.

Generation of pScNHSA: albumin fusion with the albumin moiety N-terminal to the therapeutic moiety

A vector that facilitates cloning of the DNA encoding the Therapeutic protein moiety into the C-terminus of the DNA encoding the mature albumin protein was prepared by adding three eight base pair restriction sites to pScCHSA. Asc I, Fse I and Pme I restriction sites were added between the Bsu36I and Hind III sites at the end of the nucleic acid sequence encoding the mature HSA protein. This was achieved using two complementary synthetic primers (SEQ ID NO: 40 and SEQ ID NO: 41 ) containing underlined Asc I, Fse I and Pme I restriction sites.

5'-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAAC TAAGCTTAATTCT -3' (SEQ ID NO: 40) and

5'- AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT -3' (SEQ ID NO: 41). These primers were annealed, digested with Bsu36 I and Hind III, and ligated into the same sites in pScCHSA to generate pScNHSA.

Example 2: Generation of general constructs for yeast transformation

The vectors pScNHSA and pScCHSA can be used as cloning vectors for the insertion of a polynucleotide encoding a Therapeutic protein or a fragment or variant thereof, adjacent to a polynucleotide encoding mature human serum albumin "HSA". pScCHSA is used to generate Therapeutic protein-HSA fusions, while pScNHSA can be used to generate HSA-Therapeutic protein fusions.

Generation of Albumin Fusion Constructs Comprising HSA-Therapeutic Protein Fusion Products

GGCGCGCC

(N)₁₅-3’(SEQ IDNO：43)，其中斜体序列是Pme I位点，双下划线序列是Fse I位点，单下划线序列是Ase I位点，方框核苷酸是两个串联终止密码子的反向互补序列，而(N)₁₅与编码目的治疗性蛋白质的最后15个核苷酸的反向互补序列相同。一旦扩增得到PCR产物，它可以用Bsu36I和(Asc I、Fse I或Pme I)之一进行切割，并连接到pScNHSA中。 DNA encoding a Therapeutic protein (as shown in SEQ ID NO: X or a sequence known in the art) can use primers that facilitate generation of fusion constructs (e.g., by adding restriction sites, encoding seamless fusions, encoding linkers sequence, etc.) for PCR amplification. For example, one skilled in the art can design a polynucleotide that encodes the last four amino acids of the mature form of HSA (and contains a Bsu36I site) to a 5' primer that is added to the 5' end of the DNA encoding a Therapeutic protein; and a stop codon A 3' primer added to the 3' end of the Therapeutic protein coding sequence with sub and suitable cloning sites. For example, a forward primer used to amplify DNA encoding a Therapeutic protein can have the sequence, 5'-aagctG CCTTAGG CTTA(N) ₁₅ -3' (SEQ ID NO: 42), where the underlined sequence is the Bsu36I site, capitalized The nucleotides of (N) encode the last four amino acids (ALGL) of the mature HSA protein, while (N) ₁₅ are identical to the first 15 nucleotides encoding the therapeutic protein of interest. Similarly, a reverse primer used to amplify DNA encoding a Therapeutic protein may have the sequence, 5'-GCGCGC

GGCGCGCC

(N) ₁₅ -3' (SEQ IDNO: 43), wherein the italicized sequence is the Pme I site, the double underlined sequence is the Fse I site, the single underlined sequence is the Ase I site, and the box nucleotides are two tandem The reverse complement of the stop codon and (N) ₁₅ is identical to the reverse complement of the last 15 nucleotides encoding the therapeutic protein of interest. Once the PCR product is amplified, it can be cut with either Bsu36I and (Asc I, Fse I or Pme I) and ligated into pScNHSA.

The presence of an Xho I site in the HSA chimeric leader sequence produced a single amino acid change in the chimeric signal sequence, the end of the HSA-kex2 signal sequence, from LDKR (SEQ ID NO: 44) to LEKR (SEQ ID NO: 45 ).

Generation of Albumin Fusion Constructs Containing Gene-HSA Fusion Products

GAGCAACCTCACTCTTGTGTGCATC(N)₁₅-3′(SEQ IDNO：47)，其中斜体序列是Cla I位点，下划线核苷酸是编码HSA成熟形式的最初9个氨基酸(DAHKSEVAH，SEQ ID NO：48)的DNA的反向互补序列，而(N)₁₅与编码目的治疗性蛋白质的最后15个核苷酸的反向互补序列相同。一旦扩增得到PCR产物，它可以用Sal I和Cla I进行切割，并连接到经Xho I和Cla I消化的pScCHSA中。可能需要不同的信号或前导序列，例 如可通过本领域已知的标准方法将转化酶“INV”(Swiss-Prot AccessionP00724)、交配因子α“MAF”(Genbank Accession AAA18405)、MPIF(GeneseqAAF82936)、纤蛋白B(Swiss-Prot Accession P23142)、簇蛋白(Swiss-ProtAccession P10909)、胰岛素样生长因子结合蛋白4(Swiss-Prot AccessionP22692)和HSA前导序列的变换(permutation)亚克隆到合适载体中。 Similar to the method described above, DNA encoding a Therapeutic protein can be PCR amplified using the following primers: A polynucleotide containing a SalI site and encoding the last three amino acids DKR of the HSA leader sequence is added to the DNA encoding a Therapeutic protein and a 3' primer that adds a polynucleotide containing a ClaI site encoding the first few amino acids of mature HSA to the 3' end of the DNA encoding the therapeutic protein. For example, a forward primer used to amplify DNA encoding a Therapeutic protein can have the sequence, 5'-aggagcg tcGAC AAAAGA(N) ₁₅ -3' (SEQ ID NO: 46), where the underlined sequence is the Sal I site, Capitalized nucleotides encode the last three amino acids (DKR) of the HSA leader sequence, while (N) ₁₅ are identical to the first 15 nucleotides encoding the therapeutic protein of interest. Similarly, a reverse primer used to amplify DNA encoding a Therapeutic protein may have the sequence, 5'-CTTTAA

GAGCAACCTCACTCTTGTGTGCATC (N) _15-3 ' (SEQ ID NO: 47), wherein the sequence in italics is the Cla I site, and the underlined nucleotides are the DNA encoding the first 9 amino acids of the mature form of HSA (DAHKSEVAH, SEQ ID NO: 48) The reverse complement sequence, and (N) ₁₅ is identical to the reverse complement sequence encoding the last 15 nucleotides of the therapeutic protein of interest. Once the PCR product is amplified, it can be cut with Sal I and Cla I and ligated into pScCHSA digested with Xho I and Cla I. Different signal or leader sequences may be required, for example invertase "INV" (Swiss-Prot AccessionP00724), mating factor alpha "MAF" (Genbank Accession AAA18405), MPIF (GeneseqAAF82936), fiber Protein B (Swiss-Prot Accession P23142), clusterin (Swiss-Prot Accession P10909), insulin-like growth factor binding protein 4 (Swiss-Prot Accession P22692) and permutations of the HSA leader were subcloned into appropriate vectors.

Generation of albumin fusion constructs suitable for expression in Saccharomyces cerevisiae

A Not I fragment containing DNA encoding an N- or C-terminal albumin fusion protein was generated from pScNHSA or pScCHSA and can then be cloned into the Not I site of pSAC35 with the LEU2 selectable marker. The vector thus produced was then used for transformation of the S. cerevisiae expression system.

Example 3: General expression in Saccharomyces cerevisiae

Expression vectors compatible with yeast expression can be transformed by lithium acetate, electroporation, or those known in the art and/or Sambrook, Fritsch and Maniatis, 1989, "Molecular Cloning: A Laboratory Manual", 2nd Edition, Vols 1-3 and Other methods described in Ausubel et al., 2000, Massachusetts General Hospital and Harvard Medical School, "Current Protocols in Molecular Biology", volumes 1-4, were transformed into Saccharomyces cerevisiae. Expression vectors are introduced into Saccharomyces cerevisiae strains DXY1, D88 or BXP10 by transformation, and individual transformants can be for example prepared in 10 ml YEPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) at 30°C. Cells were cultured for 3 days, and the cells were harvested in the stationary phase after 60 hours of culture. The supernatant was collected by centrifuging the cells at 3000g for 10 minutes.

In addition to the LEU2 selection marker, pSAC35 (Sleep et al., 1990, Biotechnology 8:42 and see Figure 3) contains the entire yeast 2 μm plasmid providing replication function, the PRB1 promoter and the ADH1 termination signal.

Example 4: General purification of albumin fusion proteins expressed from albumin fusions in Saccharomyces cerevisiae

In preferred embodiments, albumin fusion proteins of the invention comprise a mature form or portion thereof fused to a Therapeutic protein (e.g., a mature form of a Therapeutic protein listed in Table 1, or listed in Table 2 as SEQ ID NO: The mature form of HSA at the N- or C-terminus of the Therapeutic protein indicated by Z). In one embodiment of the invention, the albumin fusion protein of the invention further comprises a signal sequence directing the nascent fusion polypeptide in the secretory pathway of the host for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is cleaved and the mature albumin fusion protein is secreted directly into the culture medium. Albumin fusion proteins of the invention preferably comprise a heterologous signal sequence (e.g., a non-native signal sequence for a particular therapeutic protein), including but not limited to MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4 , variant HSA leaders including but not limited to chimeric HSA/MAF leaders, or other heterologous signal sequences known in the art. Especially preferred are those signal sequences listed in Table 2 and/or signal sequences listed in the "Expression of Fusion Proteins" and/or "Other Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" sections of the description above. In a preferred embodiment, the fusion protein of the invention further comprises an N-terminal methionine residue. The invention also encompasses polynucleotides encoding these polypeptides, including fragments and/or variants.

Albumin fusion proteins expressed in yeast as described above can be purified on a Dyax peptide affinity column on a small scale as follows. Supernatants from yeast expressing albumin fusion proteins were osmolarized with 3 mM phosphate buffer pH 6.2, 20 mM NaCl, and 0.01% Tween 20 to reduce volume and remove pigment. The solution was then filtered through a 0.22 μm device. The filtrate was loaded onto a Dyax peptide affinity column. The column was eluted with 100 mM Tris/HCl pH 8.2 buffer. Protein-containing peak fractions were collected and analyzed on SDS-PAGE after 5-fold concentration.

For large-scale purification, the methods described below can be used. Over 2 liters of the supernatant were osmolarized in 20 mM Tris/HCl pH 8.0 and concentrated to 500 ml. The concentrated protein solution was loaded onto a pre-equilibrated 50 ml DEAE-Sepharose Fast Flow column, the column was washed, and the protein was eluted with a linear NaCl gradient from 0 to 0.4 M NaCl in 20 mM Tris/HCl pH 8.0. Fractions containing protein were pooled and adjusted to pH _6.8 with 0.5M sodium phosphate ( _NaH2PO4 ). (NH ₄ ) ₂ SO ₄ at a final concentration of 0.9M was added to the protein solution, and the whole solution was loaded onto a pre-equilibrated 50 ml Butyl650S column. Proteins were eluted with a linear gradient of ammonium sulfate (0.9 to OM in (NH ₄ ) ₂ SO ₄ ). Fractions containing the albumin fusion were pooled again, osmolarized with 10 mM Na ₂ HPO ₄ /citrate buffer pH 5.75, and loaded onto a 50 ml pre-equilibrated SP-Sepharose Fast Flow column. Proteins were eluted with a linear gradient of NaCl from 0 to 0.5M. Fractions containing the protein of interest were pooled and buffer exchanged to 10 mM Na ₂ HPO ₄ /citric acid pH 6.25, conductivity < 2.5 mS/cm using an Amicon concentrator. This protein solution was loaded onto a 15ml pre-equilibrated Q-Sepharose high-efficiency column, the column was washed, and the protein was eluted with a linear gradient of NaCl from 0 to 0.15M NaCl. The purified protein can then be formulated into a specific buffer composition by buffer switching.

Example 5: Generation of general constructs for transfection of mammalian cells

Generation of albumin fusion constructs suitable for expression in mammalian cell lines

Albumin fusion constructs can be produced in expression vectors for use in mammalian cell culture systems. DNA encoding a Therapeutic protein can be cloned into the N- or C-terminus of HSA in a mammalian expression vector by standard methods known in the art (eg, PCR amplification, restriction digest, and ligation). Once the expression vector is constructed, transfection to a mammalian expression system can be performed. Suitable vectors are known in the art and include, but are not limited to, eg, pC4 vectors and/or commercially available vectors from Lonza Biologics, Inc. (Portsmouth, NH).

DNA encoding human serum albumin has been cloned into the pC4 vector suitable for mammalian culture systems, resulting in plasmid pC4:HSA (ATCC Accession No. PTA-3277). This vector has a dihydrofolate reductase, "DHFR" gene, allowing selection in the presence of methotrexate.

The pC4:HSA vector is suitable for expressing albumin fusion proteins in CHO cells. For expression in other mammalian cell culture systems, it may be desirable to subclon fragments comprising or consisting of DNA encoding albumin fusion proteins into candidate expression vectors. For example, a fragment comprising or consisting of DNA encoding a mature albumin fusion protein can be subcloned into another expression vector, including but not limited to any of the mammalian expression vectors described herein.

In a preferred embodiment, the DNA encoding the albumin fusion construct is subcloned by procedures known in the art into a vector provided by Lonza Biologics, Inc. (Portsmouth, NH) for expression in NSO cells.

Generation of Albumin Fusion Constructs Comprising HSA-Therapeutic Protein Fusion Products

Using pC4:HSA (ATCC Accession No. PTA-3277), albumin fusion constructs can be generated in which the Therapeutic protein moiety is C-terminal to the mature albumin sequence. For example, DNA encoding a Therapeutic protein or a fragment or variant thereof can be cloned into the vector between the Bsu 36I and Asc I restriction sites. When cloning into Bsu 36I and Asc I, the same primers (SEQ ID NO: 42 and 43) designed for cloning into the yeast vector system can be used (see Example 2).

Generation of Albumin Fusion Constructs Containing Gene-HSA Fusion Products

Using pC4:HSA (ATCC Accession No. PTA-3277), albumin fusion constructs can be generated in which the Therapeutic protein portion is cloned N-terminal to the mature albumin sequence. For example, DNA encoding a Therapeutic protein with its own signal sequence can be cloned between the Bam HI (or HindIII) and Cla I sites of pC4:HSA. When cloning into Bam HI or Hind III sites, it is preferred to include a Kozak sequence (CCGCCACC ATG , SEQ ID NO: 49) before the translation initiation codon of the DNA encoding the Therapeutic protein. If the Therapeutic protein does not have a signal sequence, then the DNA encoding the Therapeutic protein can be cloned into pC4:HSA between the Xho I and Cla I sites. When using an Xho I site, the following 5' (SEQ ID NO: 50) and 3' (SEQ ID NO: 51 ) exemplary PCR primers can be used:

5'-CCGCCG CTCGAG GGGTGTGTTTCGTCGA(N) ₁₈ -3' (SEQ ID NO: 50)

5'-AGTCCC ATCGAT GAGCAACCTCACTCTTGTGTGCATC(N) _18-3 ' (SEQ ID NO: 51)

In the 5' primer (SEQ ID NO: 50), the underlined sequence is the Xho I site; and the Xho I site and the DNA following the Xho I site encode the last seven amino acids of the native human serum albumin leader sequence. In SEQ ID NO: 50, "(N) ₁₈ " is identical to the first 18 nucleotides encoding the therapeutic protein of interest. In the 3' primer (SEQ ID NO: 51), the underlined sequence is the Cla I site; and the Cla I site and the following DNA is the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1) reverse complementary sequence. In SEQ ID NO: 51, "(N) ₁₈ " is the reverse complement of the last 18 nucleotides of the DNA encoding the Therapeutic protein of interest. Using these two primers, the therapeutic protein of interest can be PCR amplified, the PCR product purified, digested with Xho I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pC4:HSA vector.

If a candidate leader sequence is desired, the native albumin leader sequence can be replaced by a chimeric albumin leader sequence, ie, the HSA-kex2 signal peptide, or other alternative leader sequences, by standard methods known in the art. (For example, one skilled in the art can routinely PCR amplify the replacement leader sequence and subclone the PCR product into an albumin fusion construct to replace the albumin leader sequence while maintaining the reading frame).

Example 6: General expression in mammalian cell lines

It can be obtained by calcium phosphate precipitation, lipofectamine reagent, electroporation, or known in the art and/or Sambrook, Fritsch and Maniatis, 1989, "Molecular Cloning: A Laboratory Manual", 2nd edition and in Ausubel et al., 2000 Other transfection methods described in Current Protocols in Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Volumes 1-4 transfect albumin fusion constructs produced in expression vectors suitable for expression in mammalian cell lines. into appropriate cell lines. Transfected cells are then selected by the presence of a selection agent determined by the selectable marker in the expression vector.

The pC4 expression vector (ATCC Accession No. 209646) is a derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146). pC4 contains the strong promoter long terminal repeat "LTR" of Rous sarcoma virus (Cullen et al., March 1985, Molecular and Cellular Biology, 438-447) and a fragment of the cytomegalovirus "CMV" enhancer (Boshart et al. , 1985, Cell 41:521-530). This vector also contains the 3' intron of the rat preproinsulin gene, polyadenylation and termination signals, and the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary "CHO" cells or other cell lines lacking an active DHFR gene were used for transfection. Transfection of the albumin fusion construct in pC4 into CHO cells by methods known in the art will allow expression of the albumin fusion protein in CHO cells, followed by cleavage of the leader sequence, and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant.

The pEE12.1 expression vector was provided by Lonza Biologics, Inc. (Portsmouth, NH), which is a derivative of pEE6 (Stephens and Cockett, 1989, Nucl. Acids Res. 17:7110). This vector contains the promoter, enhancer and complete 5' untranslated region of human cytomegalovirus major immediate early gene "hCMV-MIE" (International Publication No. WO89/01036), the upstream region of the target sequence, and glutamine Synthetase gene (Murphy et al., 1991, Biochem J.227:277-279; Bebbington et al., 1992, Bio/Technology 10:169-175; U.S. Pat. No. 5,122,464), whose purpose is to select Transfected cells were selected in medium containing methionine sulphoximine. Transfection of the albumin fusion construct produced in pEE12.1 into NSO cells by methods known in the art (International Publication No. WO86/05807) allows expression of the albumin fusion protein in NSO cells, followed by cleavage of the leader sequence, and secreted into the supernatant. The albumin fusion protein can then be further purified from the supernatant using techniques described herein or otherwise known in the art.

Expression of albumin fusion proteins can be analyzed by, for example, SDS-PAGE and Western blotting, reverse phase HPLC analysis, or other methods known in the art.

Stable CHO and NSO cell lines transfected with albumin fusion constructs were generated and selected by methods known in the art (e.g. lipofectamine reagent transfection), e.g., with 100 nM methotrexate to select for the presence of dihydrofolate reductase The "DHFR" gene was used as a vector for selection marker or by cultivation in the absence of glutamine. Expression levels can be checked, for example, by immunoblotting, firstly with anti-HSA serum as the primary antibody, or secondly with sera containing antibodies directed against the Therapeutic protein moiety of a given albumin fusion protein as the primary antibody.

Using anti-HSA serum as the primary antibody, the expression level was checked by western blotting. Specific productivity is determined by ELISA, where the capture antibody can be a monoclonal antibody directed against the therapeutic protein moiety of the albumin fusion, and the detection antibody can be a monoclonal anti-HSA biotinylated antibody (or vice versa), followed by A commercial protocol was used to combine horseradish peroxidase/streptavidin and analyze.

Example 7: Expression of Albumin Fusion Proteins in Mammalian Cells

Albumin fusion proteins of the invention can be expressed in mammalian cells. A typical mammalian expression vector contains a promoter element that mediates the initiation of transcription of the mRNA, a protein coding sequence, and signals required for transcription termination and transcript polyadenylation. Other elements include enhancers, Kozak sequences, and intervening sequences flanked by RNA splice donor and acceptor sites. Efficient transcription was achieved with early and late promoters from SV40, long terminal repeats (LTRs) from retroviruses such as RSV, HTLVI, HIVI, and early promoters from cytomegalovirus (CMV). However, cellular elements (such as the human actin promoter) can also be used.

Expression vectors suitable for practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0 and pCMVSport 3.0. Mammalian host cells that can be used include, but are not limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO )cell.

Alternatively, the albumin fusion protein can be expressed in a stable cell line containing a polynucleotide encoding the albumin fusion protein integrated into the chromosome. Co-transfection with selectable markers such as DHFR, gpt, neomycin or hygromycin allows identification and isolation of transfected cells.

Transfection polynucleotides encoding fusion proteins can also be amplified to express large amounts of the encoded fusion protein. The DHFR (dihydrofolate reductase) marker can be used to establish cell lines carrying hundreds or even thousands of copies of the gene of interest (see e.g. Alt et al., J. Biol. Chem. 253:1357-1370, 1978; Hamlin et al. People, Biochem. et Biophys. Acta 1097: 107-143, 1990; Page et al., Biotechnology 9: 64-68, 1991). Another useful selectable marker is the enzyme glutamine synthetase (GS) (Murphy et al., Biochem J. 227:277-279, 1991; Bebbington et al., Bio/Technology 10:169-175, 1992). Using these markers, mammalian cells are grown in selective media and the most resistant cells are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are commonly used for protein production.

A derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146), expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR) of Rous sarcoma virus (Cullen et al. , Molecular and Cellular Biology, 438-447, March, 1985) plus a fragment of the CMV enhancer (Boshart et al., Cell 41:521-530, 1985). Multiple cloning sites, such as BamHI, XbaI and Asp718 with restriction enzyme sites, facilitate the cloning of target genes. The vector also contains the 3' intron of the rat preproinsulin gene, polyadenylation and termination signals, and the mouse DHFR gene under the control of the SV40 early promoter.

Specifically, for example, plasmid pC6 is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector was then isolated from a 1% agarose gel.

A polynucleotide encoding an albumin fusion protein of the invention is generated using techniques known in the art, and the polynucleotide is amplified using PCR techniques known in the art. If a naturally occurring signal sequence is used to produce the fusion protein of the invention, the vector does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, International Publication No. WO96/34891).

The amplified fragment encoding the fusion protein of the present invention was isolated from a 1% agarose gel using a commercial kit ("Geneclean", BIO 101 Inc., La Jolla, Ca.). This fragment was then digested with appropriate restriction enzymes and purified again on a 1% agarose gel.

The amplified fragment encoding the albumin fusion protein of the present invention was then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1Blue cells are subsequently transformed, and bacteria containing the fragment inserted into plasmid pC6 are identified, for example, by restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene were used for transfection. 5 μg of expression plasmid pC6 or pC4 were co-transfected with 0.5 μg of plasmid pSVneo using lipofection reagent (Felgner et al., supra). Plasmid pSV2-neo contains a dominant selectable marker, neo gene from Tn5, encoding an enzyme that confers resistance to a group of antibiotics including G418. Cells were seeded in α-MEM supplemented with 1 mg/ml G418. Two days later, cells were trypsinized and plated on hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with 10, 25 or 50 ng/ml methotrexate plus 1 mg/ml G418. After approximately 10-14 days, individual clones were trypsinized and seeded into 6-well petri dishes or 10 ml flasks with different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones grown in the highest concentration of methotrexate were subsequently transferred to new 6-well dishes containing even higher concentrations of methotrexate (1 μΜ, 2 μΜ, 5 μΜ, 10 mM, 20 mM). The same procedure was repeated until clones growing at concentrations of 100-200 μΜ were obtained. Expression of the desired fusion protein is analyzed by, for example, SDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 8: General Purification of Albumin Fusion Proteins Expressed from Albumin Fusion Constructs in Mammalian Cell Lines

In preferred embodiments, albumin fusion proteins of the invention comprise a mature form or portion thereof fused to a Therapeutic protein (e.g., a mature form of a Therapeutic protein listed in Table 1, or listed in Table 2 as SEQ ID NO: The mature form of HSA at the N- or C-terminus of the mature form of the Therapeutic protein indicated by Z). In one embodiment of the invention, the albumin fusion protein of the invention further comprises a signal sequence directing the nascent fusion polypeptide in the secretory pathway of the host for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is cleaved and the mature albumin fusion protein is secreted directly into the culture medium. Albumin fusion proteins of the invention preferably comprise a heterologous signal sequence (e.g., a non-native signal sequence for a particular therapeutic protein), including but not limited to MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4 , variant HSA leaders including but not limited to chimeric HSA/MAF leaders, or other heterologous signal sequences known in the art. Especially preferred are those signal sequences listed in Table 2 and/or signal sequences listed in the "Expression of Fusion Proteins" and/or "Other Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" sections of the description above. In a preferred embodiment, the fusion protein of the invention further comprises an N-terminal methionine residue. The invention also encompasses polynucleotides encoding these polypeptides, including fragments and/or variants.

Purification of albumin fusion proteins from mammalian cell line supernatants uses different protocols depending on the expression system used.

Purified from CHO and 293T cell lines

Purification of albumin fusion proteins from CHO cell supernatants or transiently transfected 293T cell supernatants may involve first capture with anionic HQ resin using sodium phosphate buffer and elution with a phosphate gradient followed by purification on a Blue Sepharose FF column Affinity chromatography was performed, eluting with a salt gradient. BlueSepharose FF removes major BSA/fetuin contaminants. Further purification on Poros PI50 resin using a phosphate gradient removes and reduces endotoxin contamination and concentrates the albumin fusion protein.

Purified from NSO cell line

Purification of albumin fusion proteins from NSO cell supernatants may involve Q-Sepharose anion exchange chromatography, followed by step-eluted SP-sepharose purification, followed by step-eluted Phenyl-650M purification, and finally permeability .

The purified protein can then be formulated by buffer switching.

Example 9: Bacterial Expression of Albumin Fusion Proteins

The polynucleotide encoding the albumin fusion protein of the invention comprising the bacterial signal sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence to synthesize the insert. In order to clone the amplified product into an expression vector, the primer used to amplify the polynucleotide encoding the insert preferably contains a restriction site such as BamHI and XbaI at the 5' end of the primer. For example, BamHI and Xbal correspond to restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Ampr), bacterial origin of replication (ori), IPTG regulatable promoter/operator (P/O), ribosome binding site (RBS), hexahistidine tag (6-His ) and restriction enzyme cloning sites.

The pQE-9 vector was digested with BamHI and Xbal, and the amplified fragment was ligated into the pQE-9 vector, maintaining the reading frame starting from the bacterial RBS. The ligation mix was then used to transform E. coli strain M15/rep4 (Qiagen, Inc.), which contains multiple copies of plasmid pREP4, which expresses the lacI repressor and confers kanamycin resistance (Kanr). Transformants were identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis.

Clones containing the desired construct were grown overnight (O/N) in liquid culture in LB medium supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). O/N cultures were used to inoculate large cultures at a ratio of 1:100 to 1:250. Cells were grown to an optical density 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG (isopropyl-BD-thiogalactoside) was then added to a final concentration of 1 mM. IPTG induces P/O clearance by inactivating the lacI repressor, leading to increased gene expression.

Cells were incubated for an additional 3-4 hours. Cells were then harvested by centrifugation (6000Xg, 20 minutes). The cell pellet was dissolved in the chaotropic agent 6M guanidine-HCl or preferably 8M urea and 2-mercaptoethanol at a concentration exceeding 0.14M by agitation at 4°C for 3-4 hours (see e.g. Burton et al., Eur. J. Biochem. 179:379-387, 1989). Cell debris was removed by centrifugation, and the polypeptide-containing supernatant was loaded onto a nickel-nitrilotriacetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6xHis tag bind Ni-NTA resins with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist, 1995, QIAGEN, Inc., supra).

Briefly, supernatants were loaded onto columns in 6M guanidine-HCl pH8. The column was first washed with 10 times the volume of 6M guanidine-HCl pH8, then washed with 10 times the volume of 6M guanidine-HCl pH6, and finally the peptide was eluted with 6M guanidine-HCl pH5. the

The purified protein was then refolded by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA column. Exemplary conditions are as follows: Refolding uses a linear 6M-1M urea gradient in 500mM NaCl, 20% glycerol, 20mM Tris/HCl pH 7.4, containing protease inhibitors. Renaturation should be performed for 1.5 hours or longer. After renaturation, the protein was eluted by adding 250 mM imidazole. Imidazole was removed by a final dialysis step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. Purified proteins were stored at 4°C or frozen at -80°C.

In addition to the expression vectors described above, the present invention also includes an expression vector called pHE4a (ATCC Accession Number 209645, deposited on February 25, 1998), which contains a phage operably linked to a polynucleotide encoding an albumin fusion protein of the present invention The operator and promoter element, designated pHE4a (ATCC Accession Number 209645, deposited on February 25, 1998). This vector contains: 1) neomycin phosphotransferase gene as selectable marker, 2) E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Delgarno sequence, and 6) Lactose operon repressor gene (lacIq). The origin of replication (oriC) was derived from pUC19 (LTI, Gaithersburg, MD). Promoter and operator sequences are synthetic.

DNA can be inserted into pHE4a by restriction digesting the vector with NdeI and XbaI, BamHI, XhoI or Asp718, electrophoresis of the restriction products on a gel, and separation of larger fragments (stuffer fragments should be about 310 base pairs). DNA inserts were generated according to PCR protocols described herein or otherwise known in the art using PCR primers with restriction sites for NdeI (5' primer) and XbaI, BamHI, XhoI or Asp718 (3' primer). PCR inserts were gel purified and restricted with compatible enzymes. Insert and vector are ligated according to standard protocols.

The engineered vectors in the above schemes can be replaced to express proteins in bacterial systems.

Example 10: Isolation of selected cDNA clones from deposited samples

As shown in Table 3, a number of albumin fusion constructs of the invention are deposited with the ATCC. The albumin fusion construct may comprise any of the following expression vectors: Saccharomyces cerevisiae expression vector pSAC35, mammalian expression vector pC4, or mammalian expression vector pEE12.1.

pSAC35 (Sleep et al., 1990, Biotechnology 8:42), pC4 (ATCC Accession No.209646; Cullen et al., Molecular and Cellular Biology, 438-447, 1985; Boshart et al., Cell 41:521-530, 1985) and pEE12.1 (Lonza Biologies, Inc.; Stephens and Cockett, Nucl. Acids Res. 17:7110, 1989; International Publication No. WO89/01036; Murphy et al., Biochem J.227:277-279, 1991; Bebbington et al. Human, Bio/Technology 10: 169-175, 1992; U.S. Patent No. 5,122,464; International Publication No. WO86/05807) The vector contains an ampicillin resistance gene for cultivation in bacterial cells. These vectors and/or albumin fusion constructs containing them can be transformed using techniques described in the art, such as Hanahan, plated on Luria-Broth agar plates containing 100 μg/ml ampicillin and incubated overnight at 37°C. into an E. coli strain such as Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037).

Deposited material in samples designated by ATCC deposit numbers referenced in Table 3 may also contain one or more additional albumin fusion constructs, each encoding a different albumin fusion protein. Accordingly, deposits sharing the same ATCC accession number contain at least one albumin fusion construct identified in the corresponding row of Table 3.

There are two methods that can be used to isolate a particular albumin fusion construct from the deposit of plasmid DNA referenced in Table 3 for that albumin fusion construct.

Method 1: Screening

First, albumin fusion constructs can be isolated directly by screening deposited plasmid DNA samples using methods known in the art using a polynucleotide probe corresponding to SEQ ID NO: X of the individual construct ID numbers in Table 1. For example, an Applied Biosystems DNA synthesizer can be used to synthesize a specific polynucleotide of 30-40 nucleotides based on the reported sequence. Oligonucleotides can be labeled with ³² P-γ-ATP using, for example, T4 polynucleotide kinase and purified according to conventional methods (e.g., Maniatis et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring, NY, 1982). Using techniques known to those skilled in the art, such as those provided by the vector supplier or in the relevant publications or patents cited above, the albumin fusion constructs from the indicated ATCC deposits were transformed into the above-described in a suitable host (such as XL-1Blue, Stratagene). Transformants are spread onto 1.5% agar plates (containing a suitable selection agent such as ampicillin) at a density of approximately 150 transformants (colonies) per plate. Nylon was used according to conventional methods of bacterial colony screening (e.g. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd Edition, 1989, Cold Spring Harbor Laboratory Press, pp 1.93-1.104) or other techniques known to those skilled in the art. Membrane screen these plates.

Method 2: PCR

Alternatively, DNA encoding a given albumin fusion protein can be amplified from a sample of the deposited albumin fusion construct having SEQ ID NO: X, e.g. Two primers specifying 17-20 nucleotides to hybridize to the DNA of the albumin fusion protein. Polymerase chain reaction is carried out under conventional conditions, for example in 25 μl reaction mixture containing 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM _MgCl2 , 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol each of primers, and 0.25 units of Taq polymerase. A Perkin-Elmer Cetus automatic thermal cycler was used for 35 PCR cycles (denaturation at 94°C for 1 minute; annealing at 55°C for 1 minute; extension at 72°C for 1 minute). Amplified products were analyzed by agarose gel electrophoresis, and DNA bands with expected molecular weights were excised and purified. The PCR product was confirmed to be the selected sequence by subcloning and sequencing of the DNA product.

Several methods are available to identify 5' or 3' non-coding portions of genes that may not be present in the deposited clones. These methods include, but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or equivalent to the 5' and 3' "RACE" protocols known in the art. For example, a method similar to 5' RACE can be used to generate deleted 5' ends of desired full-length transcripts (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684, 1993).

Briefly, specific RNA oligonucleotides are ligated to the 5' ends of RNA populations that may contain RNA transcripts of full-length genes. Primer sets containing primers specific to the ligated RNA oligonucleotide and primers specific to the known sequence of the gene of interest are used to PCR amplify the 5' portion of the desired full-length gene. This amplified product can then be sequenced and used to generate a full-length gene.

This above method starts with total RNA isolated from the desired source, but polyA+ RNA can also be used. If desired, the RNA preparation can then be treated with phosphatase to remove 5' phosphate groups on degraded or damaged RNA, which may interfere with the subsequent RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase to remove the cap at the 5' end of the messenger RNA. This reaction leaves a 5' phosphate group at the 5' end of the uncapped RNA, which can then be ligated to RNA oligonucleotides using T4 RNA ligase.

Using this modified RNA preparation as a template, the first strand cDNA is synthesized using gene-specific oligonucleotides. Using the first strand synthesis reaction as a template, the desired 5' end is PCR amplified using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest. The product thus generated was then sequenced and analyzed to confirm that the 5' sequence belonged to the expected gene.

Example 11: Multifusion Fusions

Albumin fusion proteins (eg, comprising a Therapeutic protein (or a fragment or variant thereof) fused to albumin (or a fragment or variant thereof) may additionally be fused to other proteins to create a "multifusion protein." These multi-fusion proteins can be used in a wide variety of applications. For example, fusion of albumin fusion proteins of the invention to His-tag, HA-tag, Protein A, IgG domains and maltose-binding protein will facilitate purification (see e.g. EPA 394,827; Traunecker et al., Nature 331:84-86 , 1988). Nuclear localization signals fused to polypeptides of the invention can target proteins to specific subcellular locations, while covalent heterodimers or homodimers can increase or decrease the activity of albumin fusion proteins. In addition, the fusion of additional protein sequences to the albumin fusion proteins of the invention can further enhance the solubility and/or stability of the fusion proteins. The fusion proteins described above can be generated using or routinely modifying techniques known in the art and/or by modifying the following scheme outlining the fusion of polypeptides to IgG molecules.

Briefly, the Fc portion of a human IgG molecule can be PCR amplified using primers spanning the 5' and 3' ends of the sequences described below. These primers should also have convenient restriction enzyme sites to facilitate cloning into expression vectors, preferably mammalian or yeast expression vectors.

For example, if pC4 (ATCC Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site should be disrupted. Next, the vector containing the human Fc portion is re-restricted with BamHI, and the linearized vector and the polynucleotide encoding the albumin fusion protein of the invention (generated and isolated using techniques known in the art) are ligated into this BamHI site . Note that the polynucleotide encoding the fusion protein of the invention was cloned without a stop codon, otherwise the Fc-containing fusion protein would not be produced.

If a naturally occurring signal sequence is used to produce the albumin fusion proteins of the invention, pC4 does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, International Publication No. WO 96/34891).

Human IgG Fc region:

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGT

GCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCC

CAAAACCCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACAT

GCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG

GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG

TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC

TCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATTCTCAAAGCC

AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCG

GGATGAGCTGACCAAGAACCAGGTCAAGCCTGACCTGCCTGGTCAAAG

GCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC

CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC

TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG

CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC

CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGA

CGGCCGCGACTCTAGAGGAT (SEQ ID NO: 52)

Example 12: Production of Antibodies from Albumin Fusion Proteins

hybridoma technology

Antibodies that bind albumin fusion proteins of the invention and portions of albumin fusion proteins of the invention (e.g., the Therapeutic protein portion or the albumin portion of the fusion protein) can be prepared in a variety of ways (see Current Protocols, p. 2 chapter). As an example of these methods, a preparation of an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to generate polyclonal antisera with higher specific activity.

Monoclonal antibodies specific for an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention are prepared using hybridoma technology (Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6 :511,1976; Kohler et al., Eur.J.Immunol.6:292,1976; Hammering et al., in "Monoclonal Antibodies and T-Cell Hybridomas", Elsevier, N.Y., pp.563-681, 1981). Typically, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention. Splenocytes from these mice were extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, it is preferred to use the parental myeloma cell line (SP20) available from the ATCC. After fusion, the hybridoma cells thus produced are selectively maintained in HAT medium and subsequently cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232, 1981). Hybridoma cells obtained by this selection are then assayed to identify clones secreting antibodies capable of binding an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention.

Alternatively, additional antibodies capable of binding an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention can be prepared in a two-step procedure using anti-idiotypic antibodies. This method takes advantage of the fact that antibodies are themselves antigens, so it is possible to obtain a second antibody that binds to a certain antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. Spleen cells from this animal are then used to generate hybridoma cells, and the hybridoma cells are screened to identify clones that produce antibodies that bind to an albumin fusion protein of the invention (or a portion of an albumin fusion protein of the invention) The capacity for specific antibodies can be blocked by fusion proteins of the invention or parts of albumin fusion proteins of the invention. These antibodies comprise anti-idiotypic antibodies directed against antibodies specific for fusion proteins of the invention (or portions of albumin fusion proteins of the invention) and are used to immunize animals to induce additional fusion proteins of the invention (or portions of albumin fusion proteins of the invention) ) Formation of specific antibodies.

An antibody is "humanized" for in vivo use of the antibody in humans. These antibodies can be produced using genetic constructs derived from monoclonal antibody-producing hybridoma cells. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (reviewed in Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Cabilly et al. People, U.S. Patent No. 4,816,567; People such as Taniguchi, EP171496; People such as Morrison, EP173494; People such as Neuberger, WO8601533; People such as Robinson, International Publication No. WO8702671; 314:268, 1985).

Antibody fragments directed against an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention are isolated from a scFv library. A library of antibody fragments reactive against an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention was constructed from naturally occurring V genes isolated from human PBL, wherein the donor may or may not have been exposed to it ( See, eg, US Patent 5,885,793, which is incorporated herein by reference in its entirety).

Library recovery (rescue). A scFv library was constructed from RNA of human PBL as described in International Publication No. WO92/01047. To recover phages displaying antibody fragments, approximately 10 ⁹ E. coli containing phagemids were used to inoculate 50 ml of 2xTY (2xTY-AMP-GLU) containing 1% glucose and 100 μg/ml ampicillin, and cultured with shaking until the OD reached 0.8. 5ml of this culture was used to inoculate 50ml of 2xTY-AMP-GLU, and 2×10 ⁸ TU of helper phage with gene 3 deleted (M13 with gene 3 deleted, see International Publication No. WO92/01047) were added, and the culture was grown in Incubate at 37°C for 45 minutes with shaking, and then at 37°C for 45 minutes with shaking. The culture was centrifuged at 4000 rpm for 10 minutes, the pellet was resuspended in 2 liters of 2xTY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin, and incubated overnight. Phage were prepared as described in International Publication No. WO92/01047.

M13 with gene 3 deleted was prepared as follows: M13 helper phage with gene 3 deleted does not encode gene 3 protein, so phage (phagemid) displaying antibody fragments have higher affinity for antigen binding. Infectious gene 3-deleted M13 particles were prepared by culturing helper phage in cells containing a pUC19 derivative that provides the wild-type genome 3 protein during phage morphogenesis. The cultures were incubated at 37°C for 1 hour without shaking and then at 37°C for 1 hour with shaking. Centrifuge the pellet (IEC-Centra 8, 400rpm for 10 minutes), resuspend in 300ml 2xTY medium (2xTY-AMP-KAN) containing 100μg/ml ampicillin and 25μg/ml kanamycin, and culture overnight at 37°C with shaking . Phage particles were purified and concentrated from culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius) to yield about ¹⁰ transducing units /ml (ampicillin-resistant clones) final concentration.

Library panning. Immunotubes (Nunc) were coated overnight in PBS with 4 ml of 100 μg/ml or 10 μg/ml albumin fusion proteins of the invention or fractions of albumin fusion proteins of the invention. The tubes were blocked with 2% Marvel-PBS at 37°C for 2 hours, and then washed 3 times with PBS. Approximately 10 ¹³ TU of phage were applied to the tube and incubated over and under turntable for 30 minutes at room temperature, then allowed to stand for an additional 1.5 hours. Tubes were washed 10 times with PBS, 0.1% Tween-20 and 10 times with PBS. To elute the phage, 1 ml of 100 mM triethylamine was added and rotated on an inverting turntable for 15 minutes, after which the solution was immediately neutralized with 0.5 ml of 1.0 M Tris-HCl pH 7.4. The phages were then used to infect 10 ml of mid-log E. coli TG1 by incubating the eluted phages with the bacteria for 30 minutes at 37°C. E. coli were then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The bacterial pool thus generated was then recovered as described above with helper phage deleted for gene 3 to prepare phage for the next round of selection. This process was then repeated for a total of 4 rounds of affinity purification, where in rounds 3 and 4 the washing tubes were increased to 20 washes with PBS, 0.1% Tween-20 and 20 washes with PBS.

Binder identification. Phage eluted in rounds 3 and 4 of selection were used to infect E. coli HB2151 and soluble scFv (Marks et al., 1991 ) were produced from single colonies for the assay. ELISA was performed using microtiter plates coated with 10 pg/ml of the albumin fusion protein of the invention or a fraction of the albumin fusion protein of the invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA were then identified by PCR fingerprinting (see eg International Publication No. WO92/01047) and subsequently by sequencing. These ELISA-positive clones can also be detected by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signaling, ability to block or competitively inhibit antibody/antigen binding, and competitive antagonistic or antagonistic activity. Further identification.

Example 13: [ ³ H]-2-deoxyglucose uptake test

Fat, skeletal muscle, and liver are insulin-sensitive tissues. Insulin facilitates glucose uptake/transport into these tissues. In the case of fat and skeletal muscle, insulin initiates signaling that culminates in the translocation of the glucose transporter 4 molecule, GLUT4, from a specialized intracellular compartment to the cell surface. Once on the cell surface, GLUT4 allows the uptake/transport of glucose.

[ ³ H]-2-deoxyglucose uptake

A number of adipose and muscle-related cell lines are available for testing glucose uptake/transport activity in the absence or presence of any one or more combinations of therapeutic drugs listed for the treatment of diabetes. Specifically, 3T3-L1 murine fibroblasts and L6 murine skeletal muscle cells can be differentiated into 3T3-L1 adipocytes and myotubes, respectively, as suitable in vitro models for [ ³ H]-2-deoxyglucose uptake assays (Urso et al. People, J Biol Chem 274(43):30864-73, 1999; Wang et al., J Mol Endocrinol 19(3):241-8, 1997; Haspel et al., J Membr Biol 169(1):45-53, 1999; Tsakiridis et al., Endocrinology 136(10):4315-22, 1995). Briefly, 2 x ¹⁰⁵ cells/100 μl of adipocytes or differentiated L6 cells were transferred to 96-well tissue culture dishes or “TC” coated with 50 μg/ml poly-L-lysine. in post-differentiation medium and cultured overnight at 37°C in 5% CO ₂ . Cells were first washed once with serum-free low-glucose DMEM medium, then with 100 μl/well of the same medium and with 100 μl/well of buffer or any combination of one or more therapeutic drugs listed for the treatment of diabetes, For example, increasing concentrations of therapeutic agents of the invention (e.g. specific fusions disclosed in SEQ ID NO: Y and fragments and variants thereof), ie 1 nM, 10 nM and 100 nM, starve at 37°C in the absence or presence of 1 nM insulin 16 hours. Plates were washed three times with 100 μl/well HEPES buffered saline. Insulin was added at 1 nM in HEPES buffered saline at 37°C for 30 minutes in the presence of 10 μM labeled [ ³ H]-2-deoxyglucose (Amersham, #TRK672) and 10 μM unlabeled 2-deoxyglucose (SIGMA, D-3179) . As a control, the same conditions were practiced, but lacking insulin. Cytochalasin B (SIGMA, C6762) was added at a final concentration of 10 μM at 100 μl/well in different wells to measure non-specific uptake. Cells were washed three times with HEPES buffered saline. Labeled, i.e. 10 μM [ ³ H]-2-deoxyglucose and unlabeled, i.e. 10 μM 2-deoxyglucose were added at room temperature for 10 minutes. Cells were washed three times with cold phosphate buffered saline, "PBS". Cells were lysed by the addition of 150 [mu]l/well 0.2N NaOH followed by incubation at room temperature for 20 minutes with shaking. Samples were then transferred to scintillation vials with 5 ml of scintillation fluid. Count the tubes in a beta-scintillation counter. Uptake in repeated conditions, differing in the absence or presence of insulin, was determined by the following equation: [(counts per minute "cpm" of insulin - non-specific cpm)/(cpm without insulin - non-specific cpm)]. Mean responses were within the limits of approximately 5-fold and 3-fold that of adipocyte and myotube controls, respectively.

cell differentiation

Make cells fully confluent in a T-75 ^cm flask. The medium was removed and replaced with 25ml pre-differentiation medium for 48 hours. Cells were cultured at 37°C in 5% _CO2 , 85% humidity. After 48 hours, the pre-differentiation medium was removed and replaced with 25 ml of differentiation medium for 48 hours. Cells were cultured again at 37°C in 5% CO ₂ , 85% humidity. After 48 hours, the medium was removed and replaced with 30 ml post-differentiation medium. The medium was maintained for 14-20 days after differentiation, or until complete differentiation was achieved. Change the medium every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC (#SA-1096).

Example 14: In vitro assay of [ ³ H]-thymidine incorporation into pancreatic cell lines

GLP-1 was recently demonstrated to induce differentiation of the rat pancreatic ductal epithelial cell line ARIP in a time- and dose-dependent manner, which was associated with increased islet duodenal homeobox-1 (IDX-1) and insulin mRNA levels (Hui et al., 2001, Diabetes 50(4):785-96). IDX-1 in turn increases mRNA levels of the GLP-1 receptor.

Cell Types Tested

RIN-M cells : These cells are available from the American Type Tissue Culture Collection (ATCC cell line number CRL-2057). The RIN-M cell line is derived from a radiation-induced transplantable rat islet cell tumor. This line was established from tumor xenografts in nude mice. The cells produce and secrete islet polypeptide hormones and produce L-dopa decarboxylase (a marker for cells with amine precursor uptake and decarboxylation or APUD activity).

ARLP cells : These are pancreatic exocrine cells of epithelial morphology available from the American Type Tissue Culture Collection (ATCC cell line number CRL-1674). See also references: Jessop, NW and Hay, RJ, "Characteristics of two rat pancreatic exocrine cell lines derived from transplantable tumors", In Vitro 16:212, 1980; Cockell, M. et al., "Identification of a cell-specific DNA-binding activity that interacts with transcriptional activator of genes expressed in the acinar pancreas", Mol. Cell. Biol. 9: 2464-2476, 1989; Roux, E. et al., "The cell-specific transcription factor PTF1 contains two differential subunits that interact with the DNA", Genes Dev.3:1613-1624, 1989; and Hui, H. et al., "Glucagon-ike peptide 1 induces differentiation of islet duodenal homcobox-1-positive pancreatic ductal cells into insulin-secreting cells" , Diabetes 50:785-796, 2001.

cell preparation

The RIN-M cell line was cultured in RPMI1640 medium (HyClone, #SH300027.01) containing 10% fetal bovine serum (HyClone, #SH30088.03), and was mixed with 1:3 to 1:6 every 6-8 days. Ratio subculture. Change the medium every 3-4 days.

ARIP (ATCC #CRL-1674) cell line in Ham's F12K medium (ATCC, #30-2004) containing 2 mM L-glutamine adjusted to 1.5 g/L sodium bicarbonate and 10% fetal calf serum nourish. ARIP cell lines were subcultured twice a week at a ratio of 1:3 to 1:6. Change the medium every 3-4 days.

Assay protocol

Cells were seeded into 96-well plates at 4000 cells/well and cultured for 48-72 hours to 50% confluency. Cells were switched to serum-free medium at 100 μl/well. After 48-72 hours of incubation, serum and/or therapeutic agents of the invention (such as albumin fusion proteins of the invention and fragments and variants thereof) are added to the wells. The cultivation was continued for another 36 hours. [ ³ H]-thymidine (5-20 Ci/mmol) (Amersham Pharmacia, #TRK120) was diluted to 1 μCi/5 μl. After 36 hours of incubation, 5 microliters were added to each well and incubated for an additional 24 hours. The reaction was terminated by gently washing the cells once with cold phosphate-buffered saline, "PBS". Cells were then fixed with 100 microliters of 10% ice-cold TCA for 15 minutes at 4°C. Remove PBS and add 200 µl of 0.2N NaOH. The plates were incubated with shaking at room temperature for 1 hour. Transfer the solution to a scintillation vial and add 5 ml of scintillation fluid compatible with aqueous solution and mix vigorously. The tubes were counted in a beta-scintillation counter. As a negative control, buffer only was used. As a positive control, fetal bovine serum was used.

Example 15: Determination of diabetes

Glycosuria (ie, excess sugar in the urine) can be readily measured to provide a disease state index of diabetes. Excess urine in patient samples compared to normal patient samples is a symptom of IDDM and NIDDM. The therapeutic effect of such patients with IDDM and NIDDM is manifested in the resulting reduction in the amount of excess glucose in the urine. In a preferred embodiment of IDDM and NIDDM monitoring, a patient's urine sample is assayed for the presence of glucose using techniques known in the art. Diabetes in humans is defined as a urinary glucose concentration exceeding 100 mg/100 ml. Excessive sugar levels in those patients showing glycosuria can be measured even more accurately by obtaining a blood sample and measuring serum glucose.

Example 16: Assays to Detect Stimulation or Inhibition of B Cell Proliferation and Differentiation

The generation of a functional humoral immune response requires soluble and associated signals between B-lineage cells and their microenvironment. Signals can deliver positive stimuli that cause B-lineage cells to continue their programmed development, or negative stimuli that instruct cells to stop their current developmental pathway. To date, a number of stimulatory and inhibitory signals have been found to affect B cell responsiveness, including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14, and IL-15 . Interestingly, these signals are weak effectors by themselves, but are able to bind multiple co-stimulatory proteins to induce activation, proliferation, differentiation, homing, tolerance and death in B cell populations.

The best studied class of B-cell co-stimulatory proteins is the TNF superfamily. Within this family, CD40, CD27 and CD30, together with their corresponding ligands CD154, CD70 and CD153, have been found to regulate various immune responses. Assays that can be used to detect and/and observe the proliferation and differentiation of these B cell populations and their precursors are useful tools for determining the effect that various proteins may have on the proliferation and differentiation of these B cell populations. Listed below are two assays designed to detect differentiation, proliferation or inhibition of B cell populations and their precursors.

In Vitro Assays - Albumin fusion proteins of the invention (including fusion proteins comprising fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) can be assessed in B cell populations and their precursors ability to induce activation, proliferation, differentiation, or inhibition and/or death. Qualitatively measured activity of albumin fusion proteins of the invention on purified human tonsil B cells over a dose range from 0.1 to 10000 ng/ml was assessed in a standard B-lymphocyte co-stimulation assay in the presence of formalin Purified tonsil B cells were cultured with immobilized Staphylococcus aureus Cowan 1 (SAC) or immobilized anti-human IgM antibody as priming agent. Secondary signals such as IL-2 and IL-15 act synergistically with SAC and IgM crosslinking to elicit B cell proliferation, as measured by tritiated thymidine incorporation. Novel synergists can be readily identified using this assay. The assay involves the isolation of human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. Based on the expression of CD45R(B220), it was estimated that more than 95% of the cell population thus generated were B cells.

Multiple dilutions of each sample were placed in each well of a 96-well plate, and suspending medium (RPMI 1640 containing 10% FBS, 5X10 ^-5 M 2ME, 100 U/ml penicillin, 10 μg/ml streptomycin and 10 ^-5 dilutions of SAC) in a total volume of 150 μl of 10 ⁵ B cells. Proliferation or inhibition was quantified by a 20 hour pulse (luCi/well) of3H-thymidine (6.7Ci/mM) starting 72 hours after factor addition. Positive and negative controls were IL2 and culture medium, respectively.

In vivo assay - BALB/c mice were injected (i.p.) twice daily with buffer alone, or 2 mg/Kg of albumin fusion proteins of the invention (including fragments or variants containing therapeutic proteins and/or albumin or albumin) fusion proteins of fragments or variants of proteins). Mice received this treatment for 4 consecutive days, at which time the mice were sacrificed and various tissues and sera were collected for analysis. Comparison of H&E sections from normal spleens and spleens treated with albumin fusion proteins of the invention identified consequences of the activity of the fusion proteins on splenocytes, such as diffusion of periarterial lymphatic sheaths and/or marked nucleated cell structures in the red pulp region. Increase, which may indicate activation of differentiation and proliferation of B cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R (B220), were used to determine whether any physiological changes in splenocytes, such as spleen disorganization, were due to infiltration into the loosely defined B cell compartment of the established T cell compartment B cell expression (representation) increased.

Flow cytometric analysis of spleens from mice treated with albumin fusion proteins was used to indicate whether albumin fusion proteins specifically increased the proportion of ThB+, CD45R (B220) double (dull) B cells beyond that seen in control mice. Observed.

Likewise, the expected consequence of increasing the expression of mature B cells in vivo is a corresponding increase in serum Ig titers. Therefore, serum IgM and IgA levels were compared between buffer and fusion protein treated mice.

Example 17: T Cell Proliferation Assay

A CD-3 induced proliferation assay was performed on PBMCs and measured by uptake ^of3H -thymidine. The assay was performed as follows. 96-well plates were coated with 100 μl/well mAb against CD3 (HIT3a, Pharmingen) or an isotype-matched control mAb (B33.1) overnight at 4°C (1 μg/ml in 0.5M bicarbonate buffer pH9 .5), and then washed three times with PBS. PBMCs were isolated from human peripheral blood by F/H gradient centrifugation and added to four wells (5×10 ⁴ /well) of mAb-coated plates containing 10% FCS and P/S in the presence of different concentrations of albumin fusion of the present invention Proteins (including fusion proteins comprising fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) in RPMI (total volume 200 μl). The relevant protein buffer and media alone served as controls. After 48 hours of incubation at 37°C, the plates were spun at 1000 rpm for 2 minutes, and 100 μl of the supernatant was removed and stored at -20°C to measure IL-2 (or other cytokines), if a proliferative effect was observed. 100 μl of medium containing 0.5 μCi ³ H-thymidine was added to the wells, and incubated at 37° C. for 18-24 hours. Wells were harvested and ³ H-thymidine incorporation was used as a measure of proliferation. Anti-CD3 alone was used as a positive control for proliferation. IL-2 (100 U/ml) was also used as a control to enhance proliferation. A control antibody that does not induce T cell proliferation is used as a negative control for the effect of the fusion protein of the present invention.

Example 18: Effects of the fusion protein of the present invention on the expression of MHC class II, co-stimulatory molecules and adhesion molecules and cell differentiation of monocytes and monocyte-derived human dendritic cells

Dendritic cells are generated by expanding proliferative precursors found in peripheral blood: Adherent PBMC or elutriated monocyte fractions are incubated with GM-CSF (50ng/ml) and IL-4 (20ng/ml)7 -10 days. These dendritic cells have a characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activators such as TNF-α leads to rapid changes in surface phenotypes (increased expression of MHC class I and II, co-stimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes are associated with increased antigen-presenting capacity and functional maturation of dendritic cells.

FACS analysis of surface antigens was performed as follows. Cells were treated with increasing concentrations of albumin fusion protein of the present invention or LPS (positive control) for 1-3 days, washed with PBS containing 1% BSA and 0.02mM sodium azide, and then diluted with 1:20 appropriate FITC- or PE -Incubate the labeled monoclonal antibody at 4°C for 30 minutes. After further washing, labeled cells were analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effects on cytokine production. Cytokines produced by dendritic cells, particularly IL-12, are important in the initiation of T cell-dependent immune responses. IL-12 strongly affects the occurrence of Thl helper T cell immune response and induces cytotoxic T and NK cell functions. ELISA was used to measure IL-12 release as follows. Dendritic cells (10 ⁶ /ml) were treated with increasing concentrations of albumin fusion proteins of the invention for 24 hours. LPS (100 ng/ml) was added to the cell culture as a positive control. Cell culture supernatants are then collected and analyzed for IL-12 content using a commercial ELISA kit (eg, R&D Systems, Minneapolis, MN). Use the standard protocol provided with the kit.

Effects on expression of MHC class II, co-stimulatory molecules and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptors. Modulation of the expression of MHC class II antigens and other co-stimulatory molecules such as B7 and ICAM-1 can lead to changes in the ability of monocytes to present antigens and induce T cell activation. Increased expression of Fc receptors may be associated with improved monocyte cytotoxic activity, cytokine release, and phagocytosis.

Surface antigens were tested using FACS analysis as follows. Monocytes were treated with increasing concentrations of the albumin fusion protein of the present invention or LPS (positive control) for 1-5 days, washed with PBS containing 1% BSA and 0.02mM sodium azide, and then mixed with the appropriate FITC diluted 1:20 - or PE-labeled monoclonal antibodies were incubated at 4°C for 30 minutes. After further washing, labeled cells were analyzed by flow cytometry on a FACScan (Becton Dickinson).

Increased monocyte activation and/or survival. Assays for molecules that activate (or inactivate) monocytes and/or increase monocyte survival (or decrease monocyte survival) are known in the art and can be routinely used to determine whether a molecule of the invention has Function of monocyte suppressor or activator. Albumin fusion proteins of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) were purified from single donor leukopacks (American Red Cross, Baltimore, MD) by centrifugation through a Histopaque gradient (Sigma). Monocytes were isolated from PBMCs by countercurrent centrifugation.

Monocyte Survival Assay. Human peripheral blood mononuclear cells gradually lose viability when cultured in the absence of serum or other stimuli. Their death results from an internally regulated process (apoptosis). Addition of activating factors such as TNF-[alpha] to the cultures significantly improved cell survival and prevented DNA fragmentation. Apoptosis was measured using propidium iodide (PI) staining as follows. Monocytes were cultured for 48 hours in the presence of 100ng/ml TNF-α in serum-free medium (positive control) (negative control) and in the presence of different concentrations of fusion proteins in polypropylene tubes . Cells were suspended at a concentration of 2×10 ⁶ /ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. In this experimental paradigm, PI uptake has been shown to correlate with DNA fragmentation.

Effects on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cell populations of the immune system through stimulated cytokine release. ELISA to measure cytokine release was performed as follows. Human monocytes were cultured at a density of 5 x ¹⁰⁵ cells/ml with increasing concentrations of the albumin fusion protein of the invention and under the same conditions but in the absence of the fusion protein. For IL-12 production, cells were primed overnight with IFN (100 U/ml) in the presence of the fusion protein. LPS (10 ng/ml) was then added. Conditioned medium was collected after 24 hours and kept frozen until use. Measurements of TNF-[alpha], IL-10, MCP-1, and IL-8 are then performed using commercial ELISA kits (eg, R&D Systems, Minneapolis, MN) and applying the standard protocols provided with the kits.

Oxidative burst. Purified monocytes were placed in 96-well dishes at 2-1×10 ⁵ cells/well. In a total volume of 0.2 ml medium (RPMI 1640 + 10% FCS, glutamine and antibiotics), increasing concentrations of albumin fusion proteins of the invention were added to the wells. After 3 days of culture, the plates were centrifuged and the medium was removed from the wells. Add 0.2ml per well of phenol red solution (140mM NaCl, 10mM potassium phosphate buffer pH 7.0, 5.5mM dextrose, 0.56mM phenol red and 19U/ml HRPO) to the macrophage monolayer, and the stimulus (200nM PMA) . The plates were incubated at 37°C for 2 hours and the reaction was terminated by adding 20 µl of 1N NaOH to each well. Absorbance was read at 610 nm. To calculate the amount of _H2O2 produced by _macrophages , a standard curve of _H2O2 solutions of known molarity was performed for _each experiment.

Example 19: Effect of the albumin fusion protein of the present invention on the growth of vascular endothelial cells

On the first day, human umbilical vein endothelial cells (HUVEC) were seeded at a density of 2-5×10 ⁴ cells/35 mm dish containing 4% fetal bovine serum (FBS), 16 units/ml heparin and 50 units/ml endothelial cell growth Supplement (ECGS, Biotechnique, Inc.) in M199 medium. On day 2, the medium was changed to M199 containing 10% FBS, 8 units/ml heparin. Albumin fusion proteins of the invention and positive controls such as VEGF and basic FGF (bFGF) were added at different concentrations. On days 4 and 6, the medium was changed. On day 8, cell numbers were measured with a Coulter counter.

An increase in the number of HUVEC cells indicates that the fusion protein can proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the fusion protein inhibits vascular endothelial cells.

Example 20: Rat corneal wound healing model

This animal model shows the effect of the albumin fusion proteins of the invention on neovascularization. The experimental protocol includes:

A 1-1.5 mm long incision is made from the center of the cornea into the stroma.

Insert the spatula under the edge of the incision, facing the outer corner of the eye.

A bag was made (with the bottom 1-1.5 mm from the edge of the eye).

Particles containing 50ng-5[mu]g of the albumin fusion protein of the invention are placed in the bag.

Treatment with the albumin fusion protein of the invention may also be applied topically to corneal wounds at a dose ranging from 20 mg to 500 mg (daily treatment for five days).

Example 21: Diabetic Mice and Glucocorticoid Impaired Wound Healing Model

Diabetic db+/db+ mouse model

To demonstrate that the albumin fusion proteins of the invention accelerate the healing process, a genetic diabetic mouse model of wound healing was used. The full thickness skin wound healing model in db+/db+ mice is a well characterized, clinically relevant, and reproducible model of impaired wound healing. Healing of diabetic wounds relies on granulation tissue formation and re-epithelialization rather than contraction (Gartner, M.H. et al., J. Surg. Res. 52:389, 1992; Greenhalgh, D.G. et al., Am. J. Pathol. 136: 1235, 1990).

Diabetic animals have many of the characteristic features observed in type 2 diabetes. Homozygous (db+/db+) mice are obese than their normal heterozygous (db+/+m) siblings. Mutant diabetic (db+/db+) mice harbor a single autosomal recessive mutation (db+) on chromosome 4 (Coleman et al., Proc. Natl. Acad. Sci. USA 77:283-293, 1982). Animals exhibited polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, elevated or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375, 1978; Debray-Sachs , M. et al., Clin. Exp. Immunol. 51 (1): 1-7, 1983; Leiter et al., Am. J. of Pathol. 114: 46-55, 1985). Peripheral neuropathy, myocardial complications and microvascular injury, basement membrane hypertrophy and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232, 1984; Robertson et al., Diabetes 29(1):60-67, 1980; Giacomelli et al., Lab Invest. 40(4):460-473, 1979; Coleman, D.L., Diabetes 31(Suppl):1-6, 1982) . These homozygous diabetic mice develop insulin-resistant hyperglycemia similar to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377, 1978).

The features observed in these animals suggest that healing in this model may be similar to that observed in human diabetes (Greenhalgh et al., Am. J. of Pathol. 136:1235-1246, 1990).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their nondiabetic (db+/+m) hybrid siblings (Jackson Laboratories) were used in this study. Animals were purchased at 6 weeks of age and 8 weeks of age at the start of the study. Animals were housed individually and had access to food and water ad libitum. All manipulations were performed using aseptic technique. Experiments were performed in accordance with the regulations and guidelines of the Human Genome Sciences, Inc. Institutional Animal Care and Use Committee (Institutional Animal Care and Use Committee) and guidelines for the care and use of laboratory animals.

The trauma protocol was performed as previously reported (Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172:245-251, 1990). Briefly, on the day of wounding, animals were anesthetized by intraperitoneal injection of avertin (0.01 mg/ml), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal area of the animal was shaved and the skin was washed with 70% ethanol solution and iodine. The surgical area was dried with sterile gauze prior to wounding. An 8mm full-thickness skin wound was then created with a Keyes tissue punch. Immediately after the wound, the surrounding skin is gently stretched to eliminate wound expansion. The wound was kept open during the experiment. Topical treatment was applied for 5 consecutive days starting from the day of wounding. Before treatment, the wound was gently cleaned with sterile saline and gauze.

Wounds were inspected visually and photographed at a fixed distance on the day of surgery and every two days thereafter. Wound closure was determined by daily measurements on days 1-5 and day 8. Wounds were measured horizontally and vertically using graduated Jameson calipers. Wounds were considered healed if granulation tissue was no longer visible and the wound was covered by continuous epithelium.

The albumin fusion protein of the present invention was administered in vehicle daily for 8 days at doses ranging from 4 mg to 500 mg per wound. For the vehicle control group, 50 ml of vehicle solution was obtained.

Animals were euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg/kg). Wounds and adjacent skin were then harvested for histology and immunohistochemistry. Tissue samples were placed in 10% neutral buffered formalin between biopsy sponges in tissue cassettes for further processing.

Three groups of 10 animals per group (5 diabetic and 5 non-diabetic controls) were evaluated: 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure was analyzed by measuring area in horizontal and vertical axes and obtaining the total planar area of the wound. Contraction was then assessed by determining the difference between the initial wound area (day 0) and the wound area after treatment (day 8). The wound area on day 1 was 64 mm ² , the corresponding size of the skin punch. Use the following formula to calculate:

a.[Open area on day 8]-[Open area on day 1]/[Open area on day 1]

The samples were fixed in 10% buffered formalin, and the paraffin-embedded blocks were sliced (5 mm) perpendicular to the wound surface, and cut with a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining was performed on cross-sections of bisected wounds. Histological examination of wounds was used to assess whether the healing process and morphological appearance of the repaired skin was altered by treatment with albumin fusion proteins of the invention. This assessment includes verification of the presence of cellular aggregates, inflammatory cells, capillaries, fibroblasts, re-epithelialization, and epidermal maturation (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235, 1990). A calibrated lens micrometer is used by a blinded observer.

Tissue sections were also immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin was used as a positive tissue control, while non-immunized IgG was used as a negative control. Keratinocyte outgrowth was determined by assessing the extent of wound re-epithelialization using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin samples was visualized by using anti-PCNA antibody (1:50) with the ABC Elite detection system. Human colon carcinoma was used as a positive tissue control, while human brain tissue was used as a negative control. Each sample included sections in which the primary antibody was omitted and replaced with non-immunized mouse IgG. Grading of these sections was based on the extent of proliferation on a scale of 0-8, with a low-end grade reflecting mild proliferation to a high-end scale reflecting intense proliferation.

Experimental data were analyzed using an unpaired t-test. A p-value less than 0.05 was considered significant.

Steroid Impairment Rat Model

Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, "Glucocorticoids and Wound healing", in Anti-Inflammatory Steroid Action: Basic and Clinical Aspects, 280-302, 1989; Wahl et al., J. Immunol. 115:476-481, 1975; Werb et al., J. Exp. Med. 147:1684-1694, 1978). Glucocorticoids inhibit angiogenesis, reduce vascular permeability (Ebert et al., An.Intern.Med.37:701-705, 1952), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors5:295 -304, 1991; Haynes et al., J.Clin.Invest.61: 703-797, 1978) and a transient reduction in the production of circulating monocytes (Haynes et al., J.Clin.Invest.61: 703-797, 1978 ; Wahl, "Glucocorticoids and wound healing", in "Antiinflammatory Steroid Action: Basic and Clinical Aspects", Academic Press, New York, pp.280-302, 1989) to delay wound healing. It is a well-documented phenomenon that systemic steroid administration impairs wound healing in rats (Beck et al., Growth Factors 5:295-304, 1991; Haynes et al., J.Clin.Invest.61:703-797, 1978; Wahl, "Glucocorticoids and wound healing," in Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp.280-302, 1989; Pierce et al., Proc.Natl.Acad.Sci.USA86:2229-2233 , 1989).

To demonstrate that the albumin fusion proteins of the present invention are capable of accelerating the healing process, the effect of multiple topical applications of the fusion proteins on full-thickness excisional skin wounds in rats in which healing was impaired by systemic administration of methylprednisolone was evaluated.

Young adult male Sprague Dawley rats (Charles River Laboratories) weighing 200-300 g were used in this example. Animals were purchased at 8 weeks of age and 9 weeks of age at the start of the study. Healing responses in rats were impaired by systemic administration of methylprednisolone (17 mg/kg/rat intramuscular) at the time of wounding. Animals were housed individually and had access to food and water ad libitum. All manipulations were performed using aseptic technique. This study was performed in accordance with the regulations and guidelines of the Human Genome Sciences, Inc. Institutional Animal Care and Use Committee (Institutional Animal Care and Use Committee) and guidelines for the care and use of laboratory animals.

Follow the trauma protocol described above. On the day of wounding, the animals were anesthetized by intraperitoneal injection of cobrazine (50 mg/kg) and xylazine (5 mg/kg). The dorsal area of the animal was shaved and the skin was washed with 70% ethanol solution and iodine solution. The surgical field was dried with sterile gauze prior to wounding. An 8mm full-thickness skin wound was then created with a Keyes tissue punch. The wound was kept open during the experiment. Topical application of the test material was carried out once a day for 7 consecutive days starting from the day of wounding and subsequent application of methylprednisolone. Before treatment, the wound was gently cleaned with sterile saline and gauze.

Wounds were inspected visually and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure was determined by daily measurements on days 1-5 and day 8. Wounds were measured horizontally and vertically using graduated Jameson calipers. Wounds were considered healed if granulation tissue was no longer visible and the wound was covered by continuous epithelium.

The albumin fusion protein of the present invention was administered in vehicle daily for 8 days at doses ranging from 4 mg to 500 mg per wound. For the vehicle control group, 50 ml of vehicle solution was obtained.

Animals were euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg/kg). Wounds and adjacent skin were then harvested for histology. Tissue samples were placed in 10% neutral buffered molar ethanol between biopsy sponges in tissue cassettes for further processing.

Three groups of 10 animals per group (5 diabetic and 5 non-diabetic controls) were evaluated: 1) untreated group, 2) vehicle placebo control, 3) treated group.

Wound closure was analyzed by measuring area in horizontal and vertical axes and obtaining the total planar area of the wound. Closure was subsequently assessed by determining the difference between the initial wound area (Day 0) and the wound area after treatment (Day 8). The wound area on day 1 was 64 mm ² , the corresponding size of the skin punch. Use the following formula to calculate:

b.[Open area on day 8]-[Open area on day 1]/[Open area on day 1]

The samples were fixed in 10% buffered molar ethanol, and the paraffin-embedded blocks were sliced (5 mm) perpendicular to the wound surface, and cut with an Olympus microtome. Routine hematoxylin-eosin (H&E) staining was performed on cross-sections of bisected wounds. Histological examination of wounds enables assessment of whether the healing process and morphological appearance of the repaired skin is improved by treatment with albumin fusion proteins of the invention. A calibrated lens micrometer was used by a blinded observer to determine the distance to the wound breach.

Experimental data were analyzed using an unpaired t-test. A p-value less than 0.05 was considered significant.

Example 22: Animal model of lymphedema

The purpose of this experimental method is to create a suitable and reliable lymphedema model for testing the therapeutic effect of the albumin fusion protein of the present invention in the lymphangiogenesis and remodeling of the lymphatic circulatory system of the rat hindlimb. Effects were measured by swollen volume of the affected limb, quantification of the amount of lymphatic vasculature, total plasma protein, and histopathology. Acute lymphedema was observed for 7-10 days. Perhaps more importantly, the chronic course of edema was followed for up to 3-4 weeks.

Before starting surgery, blood samples were collected for protein concentration analysis. Pentobarbital was administered to male rats weighing approximately 350 g. Subsequently, the right leg is shaved from knee to hip. The depilated area was scrubbed with gauze soaked in 70% EtOH. Blood samples were collected for serum total protein testing. After marking 2 measurement levels (0.5 cm above the heel, the middle of the dorsal paw), circumference and volume measurements were taken and the paw was then injected with dye. 0.05 ml of 1% Evan's blue was injected intradermally into the dorsum of the right and left paws. Circumference and volume measurements were then taken after the dye was injected into the paw.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially so that the femoral vessel can be located. Dissect and separate the flap using forceps and hemostats. After locating the femoral vessels, locate the lymphatic vessels that run laterally beneath the vessels. The major lymphatic vessels in this area are then electrically coagulated or suture ligated.

The muscles of the dorsum of the leg (adjacent to the semitendinosus and adductor muscles) were dissected directly using a microscope. Lymph nodes in the crook of the leg are then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the crooked lymph nodes were then ligated by sutures. The crural lymph nodes and any accompanying fatty tissue are then removed by cutting the connective tissue.

Take care to control any minor bleeding caused by this manipulation. After lymphatic occlusion, the flap was closed with hquid skin (Vetbond) (AJ Buck). The separated skin edges were closed under the musculature, leaving an approximately 0.5 cm gap around the leg. The skin can also be anchored by suturing under the muscle when needed.

To avoid infection, animals were housed individually in netting (no bedding). Recovering animals are checked daily and pass the peak of optimal edema, which generally occurs on days 5-7. A stable edema peak was subsequently observed. To assess the intensity of lymphedema, the circumference and volume of 2 designated locations on each paw were measured before surgery and daily for 7 days. The effect of plasma protein on lymphedema was determined, and it was also investigated whether protein analysis was a valid testing perimeter. Weights of control and edematous limbs were assessed at 2 locations. Analysis is performed in a blinded fashion.

Circumference measurement: Under brief gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a cloth tape measure. Measurements were taken at the ankle and dorsal paw by 2 different people and the two readings were averaged. Readings were obtained from control and edematous limbs.

Volume measurements: On the day of surgery, animals were anesthetized with pentobarbital and examined prior to surgery. For daily volume determinations, animals were briefly anesthetized with halothane (rapid immobilization and rapid recovery), both legs were shaved and the legs were similarly marked with waterproof markers. The legs were submerged first in water and then in the instrument to the level of each marker and then measured by Buxco edema software (Chen/Victor). Data is recorded by one person while another dips the limb into the marked area.

Blood-Plasma Protein Measurements: Blood was collected, centrifuged, and serum separated before surgery, then at the end, for comparison of total protein and Ca2 ⁺ .

Limb Weight Comparison: Following blood collection, animals were prepared for tissue collection. Limbs were excised with quillitine, and experimental and control legs were cut at the ligature and weighed. A second weighing is performed after tibio-root detachment and the foot is weighed.

Histological preparation: Dissect the transverse muscle located behind the knee (leg bend) area and arrange it in a metal mold, fill with freeze gel (freezeGel), immerse in cold methylbutane, place in a labeled room at -80°C in sample bag until sectioned. Immediately after sectioning, muscles were observed under a fluorescent microscope for lymph.

Example 23: Inhibition of TNFα-induced expression of adhesion molecules by albumin fusion protein of the present invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between lymphocytes and cell surface adhesion molecules (CAMs) on the vascular endothelium. The adhesion process follows a multistep cascade in both normal and pathological conditions that involves intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression. The expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and extravasate into local tissues during an inflammatory response. Local concentrations of cytokines and growth factors are involved in the regulation of the expression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent pro-inflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often leading to pathological consequences.

The potential of albumin fusion proteins of the invention to mediate inhibition of TNF-α-induced CAM expression can be tested. A modified ELISA assay using EC as a solid phase absorber was used to measure CAM expression on TNF-α-treated EC following costimulation with a member of the FGF protein family.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord harvests and maintained in a humidified incubator at 37°C with 5% _CO2 supplemented with 10% FCS and 1 % Penicillin/Streptomycin growth medium (EGM-2; Clonetics, San Diego, CA). HUVECs were seeded into 96-well dishes at a concentration of 1 × ¹⁰⁴ cells/well and incubated at 37°C for 18-24 hours or until confluent. Cell monolayers were then washed 3 times with RPMI-1640 serum-free solution supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin and treated with the indicated cytokines and/or growth factors for 24 hours at 37°C. After culture, cells were assessed for CAM expression.

Human umbilical vein endothelial cells (HUVEC) were cultured to confluence in standard 96-well dishes. The growth medium was removed from the cells and replaced with 90 [mu]l 199 medium (10% FBS). Samples for testing and positive or negative controls were added to plates in triplicate (10 [mu]l volume). Plates were incubated at 37°C for 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). The plates were aspirated to remove the medium and 100 [mu]l of 0.1% paraformaldehyde-PBS (containing Ca ⁺⁺ and Mg ⁺⁺ ) was added to each well. The plates were kept at 4°C for 30 minutes.

The fixative was then removed from the wells, the wells were washed once with PBS (+Ca, Mg) + 0.5% BSA, and drained. Do not let the hole dry out. Add 10 μl of diluted primary antibody to test and control wells. Anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-selectin-biotin were used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml antibody stock). Cells were incubated for 30 minutes at 37°C in a humidified environment. Wells were washed 3 times with PBS (+Ca, Mg)+0.5% BSA.

Then 20 μl of diluted ExtrAvidin-alkaline phosphatase (1:5,000 dilution) was added to each well and incubated at 37°C for 30 minutes. Wells were washed 3 times with PBS (+Ca, Mg)+0.5% BSA. Dissolve 1 piece of p-Nitrophenol Phosphate (p-Nitrophenol Phosphate, pNPP) in 5ml glycine buffer (pH10.4). 100 μl of pNPP substrate in glycine buffer was added to each test well. Standard wells in triplicate were prepared from working dilutions of ExtrAvidin-alkaline phosphatase in glycine buffer: 1:5,000 (10 ⁰ ) > 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 . 5 μl of each dilution was added to triplicate wells, so that AP contents in each well were 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. Then 100 μl of pNNP reagent must be added to each standard well. The plates must be incubated at 37°C for 4 hours. A volume of 50 μl of 3M NaOH was added to all wells. Results were quantified at 405nm on a plate reader. Use the background subtraction option on blank wells filled with glycine buffer only. The template was set to show the concentration of AP-conjugate [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng] in each standard well. Results will be displayed as the amount of bound AP-conjugate in each sample.

Example 24: Construction of GAS reporter constructs

One signal transduction pathway involved in cell differentiation and proliferation is called the Jaks-STAT pathway. The proteins activated in the Jaks-STAT pathway bind the gamma activation site "GAS" element or interferon sensitive response element ("ISRE"), which are located in the promoters of many genes. Binding of proteins to these elements alters the expression of associated genes.

GAS and ISRE elements are recognized by a class of transcription factors known as signal transducers and activators of transcription, or "STATs." The STAT family has six members. Stat1 and Stat3 are present in many cell types, as is Stat2 (prevalent as a response to IFN-α). Stat4 is more restricted and absent in many cell types, although it is found in class I T helper cells, cells treated with IL-12. Stat5 was originally called mammary growth factor, but it has been found in higher concentrations in other cells, including bone marrow cells. It can be activated by many cytokines in tissue culture cells.

STATs are translocated from the cytoplasm to the nucleus upon activation of tyrosine phosphorylation by a group of kinases known as the Janus kinase ("Jaks") family. Jaks represent a distinct family of soluble tyrosine kinases, including Tyk2, Jak1, Jak2 and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in quiescent cells.

Jaks are activated by a variety of receptors summarized in the table below. (Adapted from the review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51, 1995). The family of cytokine receptors capable of activating Jaks is divided into two groups: (a) Class 1 includes IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL- 12. Receptors for IL-IS, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and Thrombopoietin; and (b) Class 2 including IFN-a, IFN-g, and IL-10 Class 1 receptors share a conserved cysteine motif (a group of 4 conserved cysteines and 1 tryptophan) and a WSXWS motif (encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO :53) near the center of the membrane)

Thus, upon ligand binding to the receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind GAS elements. This whole process is involved in the Jaks-STAT signal transduction pathway. Thus, activation of the Jaks-STAT pathway as reflected by the binding of GAS or ISRE elements can be used to indicate proteins involved in cell proliferation and differentiation. For example, growth factors and cytokines are known to activate the Jaks-STAT pathway (see Table 5 below). Thus, by using GAS elements linked to receptor molecules, activators of the Jaks-STAT pathway can be identified.

table 5

To construct synthetic GAS containing the promoter element for use in the biological assays described in Examples 27-29, a PCR-based strategy was employed to generate the GAS-SV40 promoter sequence. The 5' primer contained four tandem copies of the GAS binding site found in the IRF1 promoter and previously shown to bind STAT upon induction by various cytokines (Rothman et al., Immunity 1:457-468, 1994 ), although other GAS or ISRE elements could be used instead. The 5' primer also contained an 18 bp sequence complementary to the SV40 early promoter sequence, flanked by Xho I sites. The sequence of the 5' primer is:

5'-GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG-3' (SEQ ID NO: 54)

Downstream primers are complementary to the SV40 promoter and flanked by Hind III sites:

5'-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3' (SEQ ID NO: 55)

PCR amplification was performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The PCR fragment thus obtained was digested with Xho I/Hind III and subcloned into BLSK2-(Stratagene). Sequencing with forward and reverse primers confirmed that the insert contained the following sequence:

5'- CTCGAG ATTTCCCCGAAATCTAGATTTCCCCCGAAATGATTTCCCCGA

AATGATTTCCCCGAAATATCTGCCATCTCAAATTAGTCAGCAACCATAGTC

CCGCCCCTAACTCCGCCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC

ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG

GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTT

GGAGGCCTAGGCTTTTGCAAAAAGCTT -3' (SEQ ID NO: 56)

With this GAS promoter element linked to the SV40 promoter, the GAS:SEAP2 reporter construct was then engineered. Here, the reporter is secreted alkaline phosphatase, or "SEAP". However, it is clear that any reporter molecule can be substituted for SEAP in this or any other embodiment. Well-known reporters that can be used in place of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody. protein.

The above sequence confirms that the synthetic GAS-SV40 promoter element was subcloned into the pSEAP-promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, creating GAS-SEAP carrier. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

Therefore, to generate a stable mammalian cell line expressing the GAS-SEAP reporter, the GAS-SEAP cassette was removed from the GAS-SEAP vector using SalI and NotI, and these restriction sites in the multiple cloning site were used to insert the new A backbone vector for the mycin resistance gene, such as pGFP-1 (Clontech), creates the GAS-SEAP/Neo vector. Once transformed into mammalian cells, this vector can be used as a GAS-binding reporter as described in Examples 27-29.

Other constructs can be made using the description above and substituting a different promoter sequence for GAS. For example, the construction of reporters containing EGR and NF-KB promoter sequences is described in Examples 27-31. However, many other promoters can be substituted using the protocols described in these examples. For example, the SRE, IL-2, NFAT or osteocalcin promoters can be replaced alone or in combination (eg, GAS/NF-KB/EGR, GAS/NF-KB, I1-2/NFAT or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelium), HUVEC (endothelium), Reh (B-cells), Saos-2 (osteoblasts), HUVAC (aorta) or myocardium cell.

Example 25: Assays for SEAP Activity

As a reporter for the assays described in the examples disclosed herein, SEAP activity was determined with the Tropix Phospho-light kit (Cat. BP-400) according to the following general procedure. The TropixPhospho-light kit provides the dilution, assay and reaction buffers used below.

The dispenser was primed with 2.5x Dilution Buffer and 15 μl of 2.5x Dilution Buffer was dispensed into the Optiplate containing 35 μl of the solution containing the albumin fusion protein of the invention. The plates were sealed with plastic sealing film and incubated at 65°C for 30 minutes. Separate the Optiplate to avoid uneven heating.

The samples were allowed to cool to room temperature for 15 minutes. Empty the dispenser and prime with assay buffer. Add 50 μl of assay buffer and incubate at room temperature for 5 minutes. Empty the dispenser and prime with reaction buffer (see table below). Add 50 µl of reaction buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescence signal is time dependent and it takes about 10 min to read 5 plates on the luminometer, 5 plates were processed at a time and a second set was started 10 min later.

Read relative light units in a luminometer. Set H12 to blank and print the result. Increased chemiluminescence indicates reporter activity.

Table 6

Example 26: Assays to Identify Neuronal Activity

As cells undergo differentiation and proliferation, a set of genes is activated through many different signal transduction pathways. One of these genes, EGR1 (Early Growth Response Gene 1), is induced upon activation in a variety of tissues and cell types. The promoter of EGR1 is responsible for this induction. Using the EGR1 promoter linked to a reporter molecule, the ability of fusion proteins of the invention to activate cells can be assessed.

Specifically, neuronal activity was assessed in the PC12 cell line using the following protocol. PC12 cells (rat pheochromocytoma cells) are known to proliferate through the activation of various mitogens such as TPA (tetradecylphorbol acetate), NGF (nerve growth factor) and EGF (epidermal growth factor) and/or differentiation. EGR1 gene expression was activated during this treatment. Thus, activation of PC12 cells by albumin fusion proteins of the invention can be assessed by stably transfecting PC12 cells with a construct containing the EGR promoter linked to a SEAP reporter gene.

EGR/SEAP reporter constructs can be assembled by the following scheme. The EGR-1 promoter sequence (-633 to +1) (Sakamoto K et al., Oncogene 6:867-871, 1991) can be PCR amplified from human genomic DNA using the following primers:

First primer: 5'-GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ ID NO: 57)

Second primer: 5'-GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID NO: 58)

Using the GAS:SEAP/Neo vector generated in Example 24, the EGR1 amplification product can then be inserted into this vector. The GAS:SEAP/Neo vector was linearized using the restriction enzymes XhoI/HindIII and the GAS/SV40 stuff was removed. The EGR1 amplification product was restriction digested with the same enzymes. Ligate the vector and EGR1 promoter.

To prepare 96-well dishes for cell culture, add 2 ml of coating solution (type I collagen (Upstate Biotech Inc., catalog number 08-115) to each 10 cm dish diluted 1:30 in 30% ethanol (filter sterilized). )) or 50 μl per well of a 96-well plate, and air dry for 2 hours.

PC12 cells were routinely placed on a pre-coated 10 cm tissue culture dish in 10% horse serum (JRH BIOSCIENCES, catalog number 12449-78P) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 5% heated Routine culture in RPMI-1640 medium (Bio Whittaker) with inactivated fetal bovine serum (FBS). Divide it into quarters every 3 to 4 days. Cells were removed from the plate by scraping and resuspended by aspirating and blowing out more than 15 times.

The EGR/SEAP/Neo construct was transfected into PC12 using techniques known in the art. EGR-SEAP/PC12 stable cells were obtained by culturing cells in 300 μg/ml G418. Use medium without G418 for routine culture, except that cells should be re-cultured in 300 μg/ml G418 for several passages every 1 to 2 months.

To measure neuronal viability, 10 cm dishes of cells grown to approximately 70-80% confluency are screened, ie, old medium is removed. Cells were washed once with PBS (phosphate buffered saline). Cells were then starved overnight in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS and antibiotics).

The next morning, the medium was removed and the cells were washed with PBS. Scrape the cells from the plate and resuspend the cells well in 2 ml of low serum medium. Count the cells and add more low serum medium to achieve a final cell density of 5 x ¹⁰⁵ cells/ml.

Add 200 μl of cell suspension (equivalent to 1×10 ⁵ cells/well) to each well of a 96-well plate. A series of different concentrations of the albumin fusion protein of the present invention were added and incubated at 37° C. for 48 to 72 hours. A growth factor known to activate PC12 cells by EGR can be used as a positive control, such as 50 ng/μl neuronal growth factor (NGF). A greater than 50-fold induction of SEAP was typically observed in positive control wells. SEAP assays can be routinely performed using techniques known in the art and/or described in Example 25.

Example 27: Determination of T cell activity

The following protocol is used to assess T cell activity by identifying factors and determining whether albumin fusion proteins of the invention proliferate and/or differentiate T cells. T cell activity was assessed using the GAS/SEAP/Neo construct generated in Example 24. Thus, a fold increase in SEAP activity is indicative of the ability to activate the Jaks-STAT signaling pathway. The T cells used in this assay were Jurkat T-cells (ATCC No. TIB-152), although Molt-3 cells (ATCC No. CRL-1552) and Molt-4 cells (ATCC No. CRL-1582) could also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. To generate stable cell lines, approximately 2 million Jurkat cells were transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies) (transfection method described below). Transfected cells were seeded at a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml gentamicin were selected. Resistant colonies were expanded and then tested for their response to increasing concentrations of interferon gamma. Dose response of one selected clone is demonstrated.

Specifically, the protocol below will yield enough cells for 75 wells containing 200 μl of cells. Therefore, to generate enough cells for multiple 96-well plates, either scale up, or perform multiple times. Jurkat cells were maintained in RPMI + 10% serum with 1% penicillin-streptomycin. 2.5 ml OPTI-MEM (Life Technologies) and 10 μg plasmid DNA were mixed in a T25 flask. Add 2.5ml OPTI-MEM containing 50μl DMRIE-C and incubate at room temperature for 15-45 minutes.

During the incubation period, the cell concentration was counted, the required number of cells were spun down (10 ⁷ cells per transfection), and resuspended in OPTI-MEM to a final concentration of 10 ⁷ cells/ml. Then add 1 x ¹⁰⁷ cells in 1 ml OPTI-MEM to the T25 flask and incubate at 37 °C for 6 h. After incubation, 10ml RPMI+15% serum was added.

The Jurkat: GAS-SEAP stable reporter was maintained in RPMI + 10% serum, 1 mg/ml gentamicin and 1% penicillin-streptomycin. These cells are treated with varying concentrations of one or more fusion proteins of the invention.

On the day of treatment with the fusion protein, cells should be washed and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the amount of fusion protein and the number of different concentrations of fusion protein being screened. For a 96-well plate, approximately 10 million cells are required (for 10 plates, 100 million cells are required).

The wellware containing the fusion protein-treated Jurkat cells was placed in the incubator for 48 hours (note that this time can vary between 48-72 hours). Then transfer 35 μl of sample from each well into an opaque 96-well plate using a 12-channel pipette. Cover the opaque plates (with a sellophene cover) and store at -20°C until performing the SEAP assay according to Example 25. Plates containing remaining treated cells were placed at 4°C and used as a source of material to repeat assays on specific wells as needed.

100U/ml interferon gamma can be used as a positive clone, which is known to activate Jurkat T cells. More than 30-fold induction was typically observed in positive control wells.

It will be apparent to those skilled in the art that the above-described protocols can be used to generate transiently and stably transfected cells.

Example 28: Determination of T cell activity

NK-KB (nuclear factor KB) is produced by a variety of factors, including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-α and lymphotoxin-β through exposure to LPS or thrombin, and through certain A transcription factor activated by the expression of viral gene products. As a transcription factor, NF-KB regulation is involved in immune cell activation, apoptosis control (NF-KB has been shown to protect cells from apoptosis), B and T-cell development, antiviral and antimicrobial responses, and various stress responses. gene expression.

In the unexcited state, NF-KB and I-KB (Inhibitor KB) remain in the cytoplasm. However, upon stimulation, I-KB is phosphorylated and degraded, resulting in the shuttling of NF-KB into the nucleus, where it activates the transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and MHC class I.

Due to its essential role and ability to respond to multiple stimuli, reporter constructs utilizing the NF-KB promoter element were used to screen fusion proteins. Activators or inhibitors of NF-KB would be useful in the treatment, prevention and/or diagnosis of diseases. For example, inhibitors of NF-KB are useful in the treatment of diseases involving acute or chronic activation of NF-KB, such as rheumatoid arthritis.

To construct vectors containing the NF-KB promoter element, a PCR-based strategy was employed. The upstream primer contained 4 tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 59), an 18 bp sequence complementary to the 5' end of the SV40 early promoter sequence, and flanked by XhoI sites:

5'-GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCCTGCCATCTCAATTAG-3' (SEQ ID NO: 60)

The downstream sequence is complementary to the 3' end of the SV40 promoter and flanked by HindIII sites:

5'-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3' (SEQ ID NO: 55)

PCR amplification was performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The PCR fragment thus obtained was digested with XhoI and HindIII and subcloned into BLSK2-(Stratagene). Sequencing with T7 and T3 primers confirmed that the insert contained the following sequence:

5'-CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTT

TCCATCTGCCATCTCAAATTAGTCAGCAACCATAGTCCCGCCCCTAACTC

CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCA

TGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCC

TCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGC

TTTTGCAAAAAGCTT-3' (SEQ ID NO: 61)

Next, the present SV40 minimal promoter element in the pSEAP2-promoter plasmid (Clontech) was replaced by this NF-KB/SV40 fragment by XhoI and HindIII. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

To generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette was removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Specifically, after restriction digestion of pGFP-1 with Sal I and Not I, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene.

Once the NF-KB/SV40/SEAP/Neo vector was created, stable Jurkat T-cells were created and maintained according to the protocol described in Example 25. Similarly, Example 25 also describes the use of these stable Jurkat T-cells to assay fusion proteins. As a positive control, exogenous TNFα (0.1, 1, 10 ng) was added to wells H9, H10 and H11, where a 5-10 fold activation was typically observed.

Example 29: Assays to Identify Myeloid Viability

The following protocol is used to assess the myeloid activity of albumin fusion proteins of the invention by determining whether the fusion protein proliferates and/or differentiates myeloid cells. Bone marrow cell viability was assessed using the GAS/SEAP/Neo construct generated in Example 24. Thus, a fold increase in SEAP activity is indicative of the ability to activate the Jaks-STAT signaling pathway. The bone marrow cells used in this assay were U937, a promonocytic cell line, although TF-1, HL60 or KG1 could also be used.

For transient transfection of U937 cells with the GAS/SEAP/Neo construct generated in Example 24, the DEAE-Dextran method was used (Kharbanda et al., 1994, Cell Growth & Differentiation 5:259-265). First, 2 × ¹⁰⁷ U937 cells were collected and washed with PBS. U937 cells are usually cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin.

Then _, the _cells were suspended in ₁ ml of 20 _{mM Tris-} HCI (pH7.4) buffer. Incubate at 37°C for 45 minutes. The cells were washed with RPMI 1640 medium containing 10% FBS, then resuspended in 10 ml of complete medium, and incubated at 37°C for 36 hours.

GAS-SEAP/U937 stable cells were obtained by culturing cells in 400 μg/ml G418. Use medium without G418 for routine culture, except that cells should be re-cultured in 400 μg/ml G418 for several passages every 1 to 2 months.

To test these cells, 1 x ¹⁰⁸ cells (which is enough for 10 96-well plate assays) were collected and washed with PBS. Suspend the cells in 200 ml of the growth medium described above to a final density of 5 x ¹⁰⁵ cells/ml. Add 200 μl of cells (or 1×10 ⁵ cells/well) to each well of a 96-well plate.

Add different concentrations of fusion protein. Incubate at 37°C for 48 to 72 hours. 100 U/ml interferon gamma, which is known to activate U937 cells, can be used as a positive control. More than 30-fold induction was typically observed in positive control wells. Supernatants were subjected to SEAP assays according to methods known in the art and/or the protocol described in Example 25.

Example 30: Assays to Identify Small Molecule Concentration and Changes in Membrane Permeability

Binding of ligands to receptors is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, and to alter membrane potential. These changes can be measured in assays to identify fusion proteins that bind to receptors of specific cells. Although the following protocol describes an assay for calcium, this protocol can be easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe.

The following assay uses a Fluorescence Imaging Plate Reader ("FLIPR") to measure changes in fluorescent molecules (Molecular Probes) bound to small molecules. Obviously, any fluorescent molecule that detects small molecules can be used instead of the calcium fluorescent molecule used here, fluo-4 (Molecular Probes, Inc.; Cat. No. F-14202).

For adherent cells, cells are seeded in Co-star black 96-well plates with clear bottoms at 10,000-20,000 cells/well. Incubate the plates for 20 h in a CO _incubator . Adherent cells were washed twice with 200 μl HBSS (Hank's Balanced Salt Solution) in a Biotek washer, leaving 100 μl buffer after the final wash.

A 1 mg/ml fluo-4 stock solution was prepared in 10% pluronic acid DMSO. To load cells with fluo-4, 50 μl of 12 μg/ml fluo-4 was added to each well. Incubate the plate at 37 °C for 60 min in a CO _incubator . The plates were washed 4 times with HBSS in a Biotek washer, leaving 100 [mu]l of buffer.

For non-adherent cells, spin the cells down from the medium. Resuspend cells to 2-5 x ¹⁰⁶ cells/ml in HBSS in a 50 ml conical tube. Add 4 μl of 1 mg/ml fluo-4 in 10% pluronic acid DMSO per ml of cell suspension. The tubes were then placed in a 37°C water bath for 30-60 minutes. Cells were washed twice with HBSS, resuspended to 1×10 ⁶ cells/ml, and dispensed into microplates at 100 μl/well. The plates were centrifuged at 1000 rpm for 5 minutes. The plates were then washed once with 200 μl in Denley Cell Wash, followed by a pipetting step to a final volume of 100 μl. For cell-free based assays, each well contains a fluorescent molecule, such as fluo-4. A fusion protein of the invention is added to the wells, and the change in fluorescence is detected.

In order to measure the fluorescence of intracellular calcium, FLIPR set the following parameters: (1) system gain 300-800mW; (2) exposure time 0.4 seconds; (3) camera F/stop F/2; (4) excitation 488nm; (5) ) emission at 530 nm; and (6) adding 50 μl. An increase in emission at 530 nm is indicative of an extracellular signaling event induced by an albumin fusion protein of the invention or a molecule induced by an albumin fusion protein of the invention, which results in an increase in intracellular Ca ⁺⁺ concentration.

Example 31: Assays to Identify Tyrosine Kinase Activity

Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytoplasmic kinases. Within the receptor protein tyrosine kinase (RPTK) group are receptors for a variety of mitogenic and metabolic growth factors, including the PDGF, FGF, EGF, NGF, HGF and insulin receptor subfamilies. Additionally, there is a large family of RPTKs for which the corresponding ligands are unknown. Ligands for RPTKs mainly include small secreted proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTKs by ligands involves ligand-mediated dimerization of the receptors, leading to transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. Cytoplasmic tyrosine kinases include receptors associated with the src family of tyrosine kinases (e.g., src, yes, Ick, lyn, fyn) and receptor-free cytosolic protein tyrosine kinases such as the Jak family, which Members mediate signal transduction triggered by the cytokine receptor superfamily such as interleukins, interferons, GM-CSF and leptin.

Because of the wide range of factors known to promote tyrosine kinase activity, it was of interest to identify whether albumin fusion proteins of the invention or molecules induced by albumin fusion proteins of the invention are capable of activating tyrosine kinase signaling pathways of. Therefore, the following protocol was designed to identify such molecules capable of activating tyrosine signaling pathways.

Target cells (eg, primary keratinocytes) were seeded at a density of approximately 25,000 cells per well into 96-well Loprodyne Silent Screen plates purchased from Nalge Nunc (Naperville, IL). Plates were sterilized by two 30 minute rinses with 100% ethanol, rinsed with water, and dried overnight. Some plates were filled with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml ) (all available from Sigma Chemicals, St. Louis, MO) or 10% Matrigel (purchased from Becton Dickinson, Bedford, MA), or bovine serum was coated for 2 hours, rinsed with PBS, and stored at 4°C. To assay cells grown on these plates, 5,000 cells/well were seeded on growth medium and the number of cells was determined indirectly after 48 hours using alamarBlue as described by the manufacturer, Alamar Biosciences, Inc. (Sacramento, CA). Loprodyne Silent Screen plates were capped using Falcon plate lids #3071 from Becton Dickinson (Bedford, MA). Falcon Microtest III cell culture plates can also be used for some proliferation assays.

To prepare extracts, A431 cells were seeded onto nylon membranes of Loprodyne dishes (20,000/200 ml/well) and grown overnight in complete medium. Cells were quiesced by incubation in serum-free basal medium for 24 hours. After treating with EGF (60ng/ml) or different concentrations of the albumin fusion protein of the present invention for 5-20 minutes, remove the medium, and add 100ml of extraction buffer (20mM HEPES pH7.5, 0.15M NaCl, 1% Triton X-100, 0.1% SDS, 2mM Na ₃ VO ₄ , 2mM Na ₄ P ₂ O ₇ and Protease Inhibitor Cocktail (#1836170) obtained from Boeheringer Mannheim (Indianapolis, IN) and the plates were spun Shake at 4°C for 5 minutes on a shaker. The plates were then placed in a vacuum transfer apparatus and the extract was filtered through the bottom of each well with a 0.45 mm filter using a house vacuum. Extracts were collected into 96-well capture/assay dishes in the bottom of the vacuum apparatus and placed immediately on ice. To obtain clarified extracts by centrifugation, after 5 minutes of detergent dissolution, the contents of each well were removed and centrifuged at 16,000 xg for 15 minutes at 4°C.

The filtered extracts were tested for the level of tyrosine kinase activity. While many methods for detecting tyrosine kinase activity are known, only one of these methods is described herein.

Typically, the tyrosine kinase activity of an albumin fusion protein of the invention is assessed by measuring its ability to phosphorylate tyrosine residues on a specific substrate (biotinylated peptide). Biotinylated peptides useful for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for several tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction was set up by sequentially adding the following components. First, 10 μl of 5 μM biotinylated peptide was added, followed by 10 μl of ATP/Mg ₂₊ (5 mM ATP/50 mM MgCl ₂ ), then 10 μl of 5x assay buffer (40 mM imidazole hydrochloride pH 7.3, 40 mM β-glycerophosphate, 1 mM EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg/ml BSA), then 5 μl sodium vanadate (1 mM), then 5 μl water. The components were mixed gently and the reaction mixture was pre-incubated at 30°C for 2 minutes. Reactions were started by adding 10 [mu]l of control enzyme or filtered supernatant.

The tyrosine kinase assay reaction was then terminated by adding 10 μl of 120 mm EDTA and placing the reaction on ice.

Tyrosine kinase activity was determined by transferring 50 [mu]l aliquots of the reaction mixture to a microtiter plate (MTP) assembly and incubating at 37[deg.] C. for 20 minutes. This allows streptavidin-coated 96-well plates to be bound to biotinylated peptides. Wash the MTP assembly 4 times with 300 μl/well PBS. Then, 75 μl of anti-phosphotyrosine antibody (anti-P-Tyr-POD, 0.5u/ml) conjugated to horseradish peroxidase was added to each well, and incubated at 37°C for 1 hour. Wells were washed as above.

Then 100 μl of peroxidase substrate solution (Boehringer Mannheim) was added and incubated at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the samples at 405 nm was measured using an ELISA plate reader. The level of bound peroxidase activity was quantified using an ELISA plate reader and reflected the level of tyrosine kinase activity.

Example 32: Assays to identify phosphorylation activity

As a potential alternative to and/or in addition to the protein tyrosine kinase activity assays described in Example 31, assays that detect the activation (phosphorylation) of major intracellular signaling intermediates may also be used. For example, as described below, one particular assay detects tyrosine phosphorylation of Erk-1 and Erk-2 kinases. However, other molecules such as Raf, JNK, p38MAP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec and Janus and any other phospho-serine, phosphotyrosine or phosphothreonine Phosphorylation of amino acid molecules can be detected by replacing Erk-1 or Erk-2 in the assays described below with these molecules.

Specifically, assay plates were prepared by coating wells of 96-well ELISA plates with 0.1 ml protein G (1 μg/ml) for 2 hours at room temperature (RT). The plates were then rinsed with PBS and blocked with 3% BSA/PBS for 1 hour at room temperature. Protein G plates were then treated (1 hour at room temperature) with two commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (Santa Cruz Biotechnology). (To detect other molecules, this step can be easily modified by substituting the monoclonal antibodies that detect any of the above molecules) After rinsing 3-5 times with PBS, the plate was stored at 4°C until use.

A431 cells were seeded at 20,000/well in 96-well Loprodyne filter plates and cultured overnight in growth medium. Then the cells were starved and cultured in basal medium (DMEM) for 48 hours, and then treated with EGF (6 ng/well) or different concentrations of the fusion protein of the present invention for 5-20 minutes. Cells were then lysed and the extracts were filtered directly into assay plates.

After incubation with the extract for 1 hour at room temperature, the wells were rinsed again. As a positive control, a commercial preparation of MAP kinase (10 ng/well) was used in place of the A431 extract. The plates were then treated (1 hour at room temperature) with a commercial polyclonal (rabbit) antibody (1 μg/ml) that specifically recognizes phosphorylated epitopes of Erk-1 and Erk-2 kinases. This antibody was biotinylated by standard procedures. Bound polyclonal antibodies were then measured by continuous incubation with europium-streptavidin and europium fluorescence enhancing reagent in a Wallac DELFIA instrument (time-resolved fluorescence). An increase in fluorescent signal above background is indicative of phosphorylation of a molecule induced by a fusion protein of the invention or an albumin fusion protein of the invention.

Example 33: Phosphorylation Assay

To determine the phosphorylation activity of the albumin fusion proteins of the invention, the phosphorylation assay described in US Pat. No. 5,958,405 (herein incorporated by reference) was used. Briefly, phosphorylation activity can be measured by phosphorylating a protein substrate with gamma-labeled ³² P-ATP and measuring the incorporated radioactivity with a gamma radioisotope counter. The fusion protein of the invention is incubated with protein substrate, ³² P-ATP and kinase buffer. Substrate-incorporated ³² P was then separated from free ³² P-ATP by electrophoresis, and the incorporated ³² P was counted and compared to a negative control. Radioactive counts above the negative control indicate phosphorylation activity of the fusion protein.

Example 34: Detection of Phosphorylation Activity (Activation) of Albumin Fusion Proteins of the Invention in the Presence of Peptide Ligands

Methods known in the art or described herein can be used to determine the phosphorylation activity of albumin fusion proteins of the invention. A preferred method of determining phosphorylation activity is using the tyrosine phosphorylation assay described in US 5,817,471 (incorporated herein by reference).

Example 35: Assay to Stimulate Proliferation of Bone Marrow CD34+ Cells

This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of fusion proteins of the invention to stimulate proliferation of CD34+ cells.

It has previously been demonstrated that most mature precursors will only respond to a single signal. More immature precursors require at least two signals in response. Thus, to test the effect of fusion proteins of the invention on the hematopoietic activity of various progenitor cells, assays contain the indicated fusion proteins of the invention in the presence or absence of hematopoietic growth factors. Isolated cells were cultured for 5 days in the presence of stem cell factor (SCF) along with test samples. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, it only acts as a "survival" factor in this condition. However, SCF will cause a synergistic effect when combined with any factor that shows a stimulatory effect on these cells, such as IL-3. Therefore, if the fusion protein tested has a stimulatory effect in hematopoietic progenitor cells, this activity can be easily detected. Since normal BM cells have low levels of cycling cells, it is possible that any inhibitory effect of the indicated fusion protein could not be detected. Therefore, assays for the inhibitory effect on progenitor cells are preferably tested in cells first stimulated in vitro with SCF+IL+3 and then contacted with the compound to be assessed for inhibition of this induced proliferation.

Briefly, CD34+ cells were isolated using methods known in the art. Cells were thawed and resuspended in medium (QBSF 60 serum-free medium (500 ml) with 1% L-glutamine, Quality Biological, Inc., Gaithersburg, MD, Cat. No. 160-204-101). After several gentle centrifugation steps at 200xg, the cells were allowed to rest for 1 hour. Adjust the cell number to 2.5×10 ⁵ cells/ml. During this time, 100 µl of sterile water was added to the peripheral wells of the 96-well plate. Cytokines that can be tested in this assay with the albumin fusion proteins of the invention are 50 ng/ml rhSCF alone (R&D Systems, Minneapolis, MN, catalog number 255-SC) and in combination with rhSCF and 30 ng/ml rhIL-3 (R&D Systems , Minneapolis, MN, Catalog No. 203-ML) Associates. After 1 hour, 10 μl of prepared cytokines, different concentrations of albumin fusion proteins of the present invention, and 20 μl of diluted cells were added to the medium already present in the wells so that the final total volume was 100 μl. The plates were then placed in a 37 °C/5% _CO2 incubator for 5 days.

Assay Proliferation rate was determined by adding 0.5 μCi/well [3H]thymidine to each well in a volume of 10 μl 18 hours prior to harvest. Assays were terminated by harvesting cells from each 96-well plate onto filtermats using a Tomtec Harvester 96. After harvesting, the filter mats are dried, organized and placed in an OmniFilter Assembly consisting of an OmniFilter Plate and an OmniFilter Disk. 60 μl Microscint was added to each well and the plate was sealed with TopSeal-A imposed seal. Apply a barcode 15 sticker to the first plate for counting. The sealed plates were then loaded, radioactivity levels were determined by Packard Top Count, and printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.

The studies described in this example tested the activity of the indicated fusion proteins to promote proliferation of bone marrow CD34+ cells. Those skilled in the art can readily adapt the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (eg, gene therapy) and agonists and antagonists thereof. The ability of the albumin fusion proteins of the invention to promote the proliferation of bone marrow CD34+ cells indicates that the albumin fusion proteins and/or polynucleotides corresponding to the fusion proteins are effective for diagnosis and treatment of diseases affecting the immune system and hematopoiesis. Representative uses are described above in the "Immunological Competence" and "Infectious Diseases" sections, and elsewhere herein.

Example 36: Assay for Extracellular Matrix Enhanced Cellular Response (EMECR)

The purpose of the extracellular matrix-enhanced cellular response (EMECR) assay is to assess the ability of fusion proteins of the invention to act on hematopoietic stem cells in the context of extracellular matrix (ECM) induced signals.

Cells respond to regulatory factors in the context of receiving signals from the surrounding microenvironment. For example, fibroblasts and endothelial and epithelial stem cells cannot replicate in the absence of signals from the ECM. Hematopoietic stem cells can self-renew in bone marrow, but not in suspension culture in vitro. The ability of stem cells to self-renew in vitro depends on their interaction with stromal cells and the ECM protein fibronectin (fn). Cell adhesion to fn is mediated by the _α5.β1 and _α4.β1 integrin receptors, which are _expressed by human and _mouse hematopoietic stem cells. The factors that integrate with the ECM environment and are responsible for stimulating stem cell self-renewal have not been identified. The discovery of these factors will be of great interest in gene therapy and bone marrow transplantation applications.

Briefly, polystyrene tissue culture-untreated 96-well dishes were coated with fn fragments at a coating concentration of 0.2 μg/cm ² . Distribute mouse bone marrow cells (1,000 cells/well) in 0.2 ml serum-free medium. Cells cultured in the presence of IL-3 (5ng/ml) + SCF (50ng/ml) will serve as a positive control, under which stem cells are expected to undergo little self-renewal but significant differentiation. The albumin fusion protein of the invention was tested with a suitable negative control in the presence and absence of SCF (5.0 ng/ml), wherein the volume of the administered composition containing the albumin fusion protein of the invention was 10% of the total assay volume . Distributed cells were then grown by incubation for 7 days in a tissue culture incubator in a hypoxic environment (5% _CO2 , 7% _O2 and 88% _N2 ). The number of proliferating cells in the wells was then determined by measuring thymidine incorporation into the cellular DNA. Confirmation of a positive result in the assay requires phenotypic characterization of the cells, which can be performed by scaling up the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.

If a particular fusion protein of the invention is found to be a stimulator of hematopoietic progenitor cells, that fusion protein and polynucleotides corresponding to that fusion protein may be useful, for example, in the diagnosis and treatment of diseases affecting the immune system and hematopoiesis. Representative uses are described above in the "Immune Competence" and "Infectious Diseases" sections, and elsewhere herein. Fusion proteins may also be useful in the expansion of stem cells and committed progenitor cells of various blood cell lineages, and in the differentiation and/or proliferation of various cell types.

In addition, albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention can also be used to inhibit the proliferation and differentiation of hematopoietic cells, and thus can be used to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This anti-proliferative effect may allow administration of higher doses of chemotherapeutic agents and thus more effective chemotherapeutic treatment.

In addition, the fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention can also be used in the treatment and diagnosis of hematopoietic related disorders, such as anemia, pancytopenia, leukopenia, thrombocytopenia, or leukemia, because stromal cells in Important in the generation of cells of the hematopoietic lineage. Uses include in vitro culture of bone marrow cells, bone marrow transplantation, bone marrow remodeling, radiotherapy or chemotherapy for neoplasms.

Example 37: Proliferation of Human Dermal Fibroblasts and Aortic Smooth Muscle Cells

The albumin fusion protein of the present invention was added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC), and two co-assays were performed for each sample. The first assay examined the effect of the fusion protein on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Abnormal growth of fibroblasts or smooth muscle cells is part of several pathological processes, including fibrosis and restenosis. The second assay examined IL6 production by NHDFs and SMCs. IL6 production indicates functional activation. Activation of cells will increase the production of various cytokines and other factors that can lead to pro-inflammatory or immunomodulatory consequences. Assays were performed with and without co-TNFa stimulation to examine co-stimulatory or inhibitory activity.

Briefly, on day 1, 96-well black plates were set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl medium. NHDF medium contains: Clonetics FB basal medium, 1mg/ml hFGF, 5mg/ml insulin, 50mg/ml gentamicin, 2% FBS, while AoSMC medium contains Clonetics SM basal medium, 0.5g/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamicin, 50 μg/ml amphotericin B, 5% FBS. After incubation at 37°C for at least 4-5 hours, the medium was aspirated and replaced with growth arrest medium. The growth arrest medium of NHDF contains fibroblast basal medium, 50 mg/ml gentamicin, 2% FBS, while the growth arrest medium of AoSMC contains SM basal medium, 50 mg/ml gentamicin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37°C until day 2.

On day 2, serial dilutions of albumin fusion proteins of the invention and templates were designed such that they always included media controls and known protein controls. For both stimulation and inhibition experiments, proteins were diluted in growth arrest medium. For inhibition experiments, TNFa was added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1/3 volume of medium containing control or inventive albumin fusion protein and incubate at 37°C/5% _CO2 until day 5.

Transfer 60 [mu]l from each well to another labeled 96-well plate, cover with plate seal, and store at 4[deg.]C until day 6 (for IL6 ELISA). To the remaining 100 μl of the cell culture plate, Alamar Blue equivalent to 10% of the culture volume (10 μl) was aseptically added. Return the plate to the incubator for 3-4 hours. Excitation at 530nm and emission at 590nm were then measured using CytoFluor. This yielded growth promotion/inhibition data.

On day 5, IL6 ELISA was performed by coating 96-well plates with 50-100 μl/well anti-human IL6 monoclonal antibody diluted in PBS pH 7.4 and incubated overnight at room temperature.

On day 6, the plates were emptied into the sink and blotted dry on paper towels. Assay buffer containing PBS was prepared with 4% BSA. Plates were blocked with 200 μl/well Pierce Super Block blocking buffer in PBS for 1-2 hours, then washed with wash buffer (PBS, 0.05% Tween-20). The plates were blotted dry on paper towels. Then 50 μl/well of anti-human IL-6 monoclonal, biotinylated antibody diluted at 0.5 mg/ml was added. IL-6 stocks were diluted in culture medium (30, 10, 3, 1, 0.3, Ong/ml). Duplicate samples were added to the top row of plates. Cover the plate and incubate for 2 hours at room temperature on a shaker.

Plates were washed with wash buffer and blotted dry on paper towels. EU-labeled streptavidin was diluted 1:1000 in assay buffer and added 100 μl/well. Cover the plate and incubate at room temperature for 1 hour. The plates were washed again with wash buffer and blotted dry on paper towels.

Add 100 μl/well of enhancement solution. Shake for 5 minutes. Plates were read on a Wallac DELFIA fluorometer. The readings of the triplicate samples in each assay were tabulated and averaged.

Positive results in this assay indicate that AoSMC cells are proliferating and that the albumin fusion protein may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. Positive results also suggest many potential uses for fusion proteins and polynucleotides encoding albumin fusion proteins. For example, inflammation and immune response, wound healing and angiogenesis, as detailed throughout this specification. In particular, fusion proteins are useful in wound healing and skin regeneration, as well as promoting angiogenesis of both blood vessels and lymphatic vessels. The growth of blood vessels can be used in the treatment of, for example, cardiovascular disease. Furthermore, fusion proteins exhibiting antagonistic activity in this assay may be useful in the treatment of diseases, disorders and/or conditions involving angiogenesis by being effective as anti-angiogenic agents (eg, anti-angiogenesis). These diseases, disorders and/or conditions are known in the art and/or described herein, such as, for example, malignant tumors, solid tumors, benign tumors, such as hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas ; Atherosclerotic plaque; Angiogenic diseases of the eye such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolentic fibroplasia, redness, retinoblasts tumors, uveitis of the eye, and pterygium (abnormal blood vessel growth); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulation; hypertrophic scarring ( keloids); ununion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary artery collaterals; cerebral collaterals; arteriovenous malformations; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. In addition, albumin fusion proteins that are antagonists in this assay are useful in the treatment of anti-hyperproliferative diseases and/or anti-inflammation as known in the art and/or described herein.

Example 38: Cell Adhesion Molecule (CAM) Expression on Endothelial Cells

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between lymphocytes and cell surface adhesion molecules (CAMs) on the vascular endothelium. The adhesion process follows a multistep cascade in both normal and pathological conditions that involves intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression. The expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and extravasate into local tissues during an inflammatory response. Local concentrations of cytokines and growth factors are involved in the regulation of the expression of these CAMs.

Briefly, endothelial cells (e.g., human umbilical vein endothelial cells (HUVEC)) are grown to confluence in standard 96-well dishes, the growth medium is removed from the cells, and replaced with 100 μl of 199 medium (10% fetal bovine serum (FBS )). Test samples (containing albumin fusion proteins of the invention) and positive or negative controls were added to plates in triplicate (10 [mu]l volume). Plates were then incubated at 37°C for 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). The plates were aspirated to remove medium and 100 [mu]l of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) was added to each well. The plates were kept at 4°C for 30 minutes. The fixative was removed from the wells, the wells were washed once with PBS (+Ca, Mg) + 0.5% BSA and drained. Add 10 μl of diluted primary antibody to test and control wells. Anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-selectin-biotin were used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml antibody stock). Cells were incubated for 30 minutes at 37°C in a humidified environment. Wells were washed 3 times with PBS (+Ca, Mg)+0.5% BSA. 20 μl of diluted Extr avidin-alkaline phosphatase (1:5,000 dilution, referred to herein as the use diluent) was added to each well and incubated at 37°C for 30 minutes. Wells were washed three times with PBS (+Ca, Mg)+0.5% BSA. Dissolve 1 tablet of p-nitrophenol phosphate pNPP per 5 ml of glycine buffer (pH10.4). 100 μl of pNPP substrate in glycine buffer will be added to each assay well. Standard wells in triplicate were prepared from working dilutions of Extr avidin-alkaline phosphatase in glycine buffer: 1:5,000 (10 ⁰ ) > 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 . 5 μl of each dilution was added to triplicate wells, so that AP contents in each well were 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. Then add 100 μl of pNNP reagent to each standard well. The plates were incubated at 37°C for 4 hours. A volume of 50 μl of 3M NaOH was added to all wells. Plates were read on a plate reader at 405nm using the background subtraction option for blank wells containing glycine buffer only. In addition, the template was set to show the concentration of AP-conjugate [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng] in each standard well. Results will be displayed as the amount of bound AP-conjugate in each sample.

Example 39: Alamar Blue Endothelial Cell Proliferation Assay

This assay can be used to quantify protein-mediated inhibition of bFGF-induced proliferation of bovine lymphatic endothelial cells (LEC), bovine aortic endothelial cells (BAEC), or human microvascular myometrial cells (UTMEC). This assay incorporates a fluorescent growth indicator based on the detection of metabolic activity. A standard Alamar Blue proliferation assay was prepared in EGM-2MV supplemented with 10 ng/ml bFGF as a source of endothelial cell stimulation. With minor changes in growth medium and cell concentration, this assay can be applied to a variety of endothelial cells. Dilute appropriately the dilution of the protein batch to be tested. Serum-free medium without bEGF was used as a non-stimulation control, and angiostatin or TSP-1 was included as a known inhibition control.

Briefly, LECs, BAECs or UTMECs were seeded into 96-well dishes at a density of 5000 to 2000 cells/well in growth medium and left overnight at 37°C. After cells were incubated overnight, the growth medium was removed and replaced with GIBCO EC-SFM. Cells were treated in triplicate wells with appropriate dilutions of albumin fusion proteins of the invention or control protein samples (prepared in SFM) and bFGF was added to a concentration of 10 ng/ml. Once the cells were treated with the samples, the plates were returned to the 37°C incubator for 3 days. After 3 days, 10 ml of alamar blue stock solution (Biosource, catalog number DAL1100) was added to each well, and the plates were returned to the 37°C incubator for 4 hours. Plates were then read using a CytoFluor fluorescence reader at 530 nm excitation and 590 nm emission. Direct output is recorded in relative fluorescence units.

Alamar blue is a redox indicator, both fluorescence and color change reflect the chemical reduction of the growth medium caused by cell growth. Innate metabolic activity leads to chemical reduction in the immediate environment when cells are grown in culture. A reduction reaction involving growth causes the indicator to change from an oxidized (non-fluorescent blue) form to a reduced (fluorescent red) form (i.e., enhanced proliferation will produce a stronger signal, while inhibited proliferation will produce a weaker signal. signal, the total signal is proportional to the total number of cells and their metabolic activity). Background levels of activity were observed with starvation medium alone. This was compared to the output observed from positive control samples (bFGF in growth medium) and protein dilutions.

Example 40: Detection of Inhibition of Mixed Lymphocyte Reaction

This assay can be used to detect and evaluate the inhibition of mixed lymphocyte reaction (MLR) by fusion proteins of the invention. Inhibition of MLR may be due to direct effects on cell proliferation and viability, modulation of co-stimulatory molecules on interacting cells, modulation of adhesion between lymphocytes and accessory cells, modulation of cytokine production by accessory cells. Since the mononuclear fraction of peripheral blood used in this assay includes T, B, and natural killer lymphocytes as well as monocytes and dendritic cells, albumin fusion proteins that inhibit MLR can be directed against a variety of cells.

，密度1.0770g/ml，OrganonTeknika Corporation，West Chester，PA)通过密度梯度离心纯化来自人供体的PBMC。将来自两个供体的PBMS在补充有10％FCS和2mM谷氨酰胺的RPMI-1640(Life Technologies，Grand Island，NY)中调至2×10⁶细胞/ml。将来自第三供体的PBMC调至2×10⁵细胞/ml。将50μl来自每个供体的PBMC加到96孔圆底微量滴定平皿的孔中。将融合蛋白测试材料的稀释液(50μl)一式三份加到微量滴定孔中。加入测试样品(目的蛋白质)至最终1∶4稀释度；加入rhulL-2(R&D Systems，Minneapolis，MN，产品目录编号202-IL)至终浓度1μg/ml；加入抗CD4mAb(R&D Systems，克隆34930.11，产品目录编号MAB379)至终浓度10μg/ml。将细胞于37℃在5％CO₂中培养7-8天，在培养的最后16个小时向孔中加入1μC[³H]胸苷。收集细胞，并用Packard TopCount测定胸苷掺入。数据表述为三份测定的平均值和标准偏差。 Albumin fusion proteins of the invention that inhibit MLR will find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, Crowe Engler's disease, ulcerative colitis, arteriosclerosis, sclerosis, graft-versus-host disease, host-versus-graft disease, hepatitis, leukemia, and lymphoma. Briefly, lymphocyte isolation medium (

, density 1.0770 g/ml, OrganonTeknika Corporation, West Chester, PA) PBMCs from human donors were purified by density gradient centrifugation. PBMS from two donors were adjusted to 2 x ¹⁰⁶ cells/ml in RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor were adjusted to 2 x ¹⁰⁵ cells/ml. 50 [mu]l of PBMC from each donor was added to the wells of a 96-well round bottom microtiter plate. Dilutions (50 μl) of fusion protein test material were added to microtiter wells in triplicate. Add test sample (protein of interest) to a final 1:4 dilution; add rhulL-2 (R&D Systems, Minneapolis, MN, catalog number 202-IL) to a final concentration of 1 μg/ml; add anti-CD4 mAb (R&D Systems, clone 34930.11 , catalog number MAB379) to a final concentration of 10 μg/ml. Cells were cultured at 37°C in 5% CO ₂ for 7-8 days, and 1 μC of [ ³ H]thymidine was added to the wells during the last 16 hours of culture. Cells were harvested and thymidine incorporation was determined using a Packard TopCount. Data are expressed as mean and standard deviation of triplicate determinations.

Samples of the fusion protein of interest were screened in separate experiments and compared with a negative control treatment, anti-CD4 mAb, which inhibited lymphocyte proliferation, and a positive control treatment, IL-2, which enhanced lymphocyte proliferation (recombinant material or supernatant).

Example 41: Determination of protease activity

The following assays can be used to assess the protease activity of albumin fusion proteins of the invention.

Gelatin and casein zymography was performed essentially as described (Heusen et al., Anal. Biochem. 102:196-202, 1980; Wilson et al., Journal of Urology 149:653-658, 1993). Run samples on a 10% polyacrylamide/0.1% SDS gel containing 1% gelatin or casein, soak in 2.5% triton for 1 hour at room temperature, and soak in 0.1M glycine pH 8.3 at 37°C 5-16 hours. After staining in Amide Black, regions of proteolysis appear as clear areas on a blue-black background. Trypsin (Sigma T8642) was used as a positive control.

Protease activity can also be determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500). Reactions were set up in (25mM NaPO4, 1 mM EDTA and 1 mM BAEE), pH 7.5. Samples were added and the change in absorbance at 260 nm was monitored in time-driven mode on a Beckman DU-6 spectrophotometer. Trypsin was used as a positive control.

Other assays based on the release of soluble peptides from casein or hemoglobin by measuring absorbance at 280 nm or colorimetric titration using the Folin method are performed as described in Bergmeyer et al., Methods of Enzymatic Analysis 5, 1984). Other assays involve the dissolution of chromogenic substrates (Ward, Applied Science 251-317, 1983).

Example 42: Identification of Serine Protease Substrate Specificity

Methods known in the art or described herein can be used to determine the substrate specificity of albumin fusion proteins of the invention having serine protease activity. A preferred method of determining substrate specificity is the use of positional scanning synthetic combinatorial libraries as described in GB2324529 (herein incorporated in its entirety).

Example 43: Ligand Binding Assays

The following assays can be used to assess the ligand binding activity of albumin fusion proteins of the invention.

Ligand binding assays provide a straightforward method to confirm receptor pharmacology and are amenable to high-throughput formats. Ligands of purified albumin fusion proteins of the invention were radiolabeled with high specific activity (50-2000 Ci/mmol) for binding studies. Subsequent procedures to determine radiolabeling did not diminish the activity of the ligand on the fusion protein. Assay conditions for buffers, ions, pH, and other modifiers such as nucleotides are optimized to establish viable signal-to-noise ratios for both membrane and whole-cell polypeptide sources. For these assays, specific polypeptide binding is defined as the total associated radioactivity minus the radioactivity measured in the presence of excess unlabeled competing ligand. If possible, use more than one competing ligand to define residual nonspecific binding.

Example 44: Functional assays in Xenopus oocytes

Capped RNA transcripts of linearized plasmid templates encoding albumin fusion proteins of the invention were synthesized in vitro using RNA polymerase according to standard procedures. In vitro transcripts were suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes were removed from adult female toads, stage V defolliculated oocytes were obtained, and RNA transcripts (10 ng/oocyte) were injected in 50 nl bolus using a microinjection device. Two electrode voltage clamps were used to measure currents in individual Xenopus oocytes in response to exposure to fusion proteins and peptide agonists. Recordings were performed at room temperature in Ca2+-free Barth's medium. The Xenopus system can be used to screen for known ligands and tissue/cell extracts for activating ligands.

Embodiment 45: Microphysiometric Assay (Microphysiometric Assay)

Activation of a wide variety of second messenger systems results in the extrusion of small amounts of acid from the cell. The acid formed is primarily a result of increased metabolic activity required to drive intracellular signaling processes. Changes in pH in the medium surrounding the cells are small but detectable with a CYTOSENSOR microphysiological assay (Molecular Devices Ltd., Menlo Park, Calif.). CYTOSENSOR thus enables the detection of the ability of albumin fusion proteins of the invention to activate second messengers associated with intracellular signaling pathways for energy utilization.

Example 46: Extract/Cell Supernatant Screening

There are a variety of mammalian receptors for which there are as yet no associated activating ligands (agonists). Thus, active ligands for these receptors may not be included in the repertoire of ligands identified to date. Therefore, the albumin fusion protein of the present invention can also be functionally screened against tissue extracts (using calcium, cAMP, microphysiological function assays, oocyte electrophysiology, etc.) to identify the therapeutic properties of the albumin fusion protein of the present invention. Natural ligands for protein moieties and/or albumin protein moieties. Extracts producing a positive functional response can continue to be subfractionated until the active ligand is isolated and identified.

Example 47: ATP Binding Assay

The following assays can be used to assess the ATP binding activity of fusion proteins of the invention. the

The ATP binding activity of albumin fusion proteins of the invention can be tested using the ATP binding assay described in US Patent No. 5,858,719, which is incorporated herein by reference in its entirety. Briefly, ATP bound to albumin fusion proteins of the invention was measured by 8-azido-ATP labeled photoaffinity in a competition assay. Reaction mixtures containing 1 mg/ml ABC transporter were incubated with varying concentrations of ATP, or the nonhydrolyzable ATP analog adenine-5'-imidodiphosphate, at 4°C for 10 minutes. Add a mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, MO.) plus 8-azido-ATP ( ³² P-ATP) (5 mCi/μmol, ICN, Irvine, CA.) to 100 μM and place a 0.5ml aliquot in the well of a porcelain drip plate on ice. The plates were irradiated with a short wave 254 nm UV lamp at a distance of 2.5 cm from the plates for two one minute intervals with a one minute cooling interval in between. The reaction was terminated by adding dithiothreitol at a final concentration of 2 mM. The incubation solution was subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. The protein band corresponding to the albumin fusion protein of the present invention was excised and radioactivity was measured. Decreasing radioactivity with increasing ATP or adenine-5'-imidophosphate provides a measure of the fusion protein's affinity for ATP.

Example 48: Identification of Signal Transduction Proteins Interacting with Albumin Fusion Proteins of the Invention

The albumin fusion proteins of the present invention can be used as research tools for identifying, characterizing and purifying signal transduction pathway proteins or receptor proteins. Briefly, labeled fusion proteins of the invention can be used as reagents for the purification of molecules that interact with them. In one embodiment of affinity purification, an albumin fusion protein of the invention is covalently coupled to a chromatography column. Cell-free extracts derived from putative target cells, such as cancerous tissue, are passed through the column and molecules with suitable affinity bind to the albumin fusion protein. Protein complexes are recovered from the column, dissociated, and the recovered molecules are subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the corresponding gene from an appropriate cDNA library.

Example 49: IL-6 Bioassay

A variety of assays are known in the art for testing the proliferative effects of albumin fusion proteins of the invention. For example, one such assay is the IL-6 bioassay described by Marz et al. (Proc. Natl. Acad. Sci. U.S.A. 95:3251-56, 1998, which is incorporated herein by reference). After 68 hours of incubation at 37°C, in order to measure the number of surviving cells, tetrazolium salt thiazolyl blue (MTT) was added and incubated at 37°C for an additional 4 hours. B9 cells were lysed with SDS and the optical density was measured at 570nm. Controls containing IL-6 (positive) and no cytokine (negative) were added. (Briefly, IL-6-dependent B9 mouse cells were washed 3 times with IL-6-free medium, and distributed in 50 μl of a plate at a concentration of 5,000 cells per well, and 50 μl of the fusion protein of the present invention was added.) Enhanced proliferation in test samples (containing albumin fusion proteins of the invention) relative to negative controls indicates a proliferative effect mediated by the fusion protein.

Example 50: Support of Chick Embryo Neuronal Survival

In order to test whether the albumin fusion protein of the present invention supports the survival of sympathetic neuronal cells, the chicken embryo neuron survival assay of Senaldi et al. (Proc. Natl. Acad. Sci. USA96: 11458-63, 1998, which incorporated herein by reference). Briefly, motor and sympathetic neurons were isolated from chicken embryos and resuspended in L15 medium (containing 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville , MD.) and Dulbecco's modified Eagles' medium (containing 10% FCS, glucose, penicillin and 25mM Hepes buffer (pH7.2); Life Technologies, Rockville, MD.), and in the presence of different concentrations of purified The fusion protein of the present invention was incubated at 37° C. in 5% CO ₂ , and a negative control lacking any cytokines. After 3 days, neuronal survival was determined by assessing cell morphology and using Mosmann's colorimetric assay (Mosmann, T., J. Immunol. Methods 65:55-63, 1983). Enhanced neuronal cell survival compared to cytokine-deficient controls indicates the ability of albumin fusion proteins to enhance neuronal cell survival.

Example 51: Assays for Phosphatase Activity

The following assay can be used to assess the serine/threonine phosphatase (PTPase) activity of albumin fusion proteins of the invention.

To measure serine/threonine phosphatase (PTPase) activity, assays widely known to those skilled in the art can be utilized. For example, the serine/threonine phosphatase (PSPase) activity of the albumin fusion protein of the present invention can be measured using the PSPase Assay Kit of New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate of PSPase, is phosphorylated on serine and threonine residues by cAMP-dependent protein kinase in the presence of [ ^32P ]ATP. Protein serine/threonine phosphatase activity was then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.

Example 52: Interaction of Serine/Threonine Phosphatases with Other Proteins

Fusion proteins of the invention having serine/threonine phosphatase activity (e.g. assayed according to Example 51) can be used, for example, as research tools for the identification, characterization and purification of other interacting or receptor proteins, or other signal transducers. pathway proteins. Briefly, the tagged fusion protein of the invention can be used as a reagent for purifying molecules that interact with it. In one embodiment of affinity purification, an albumin fusion protein of the invention is covalently coupled to a chromatography column. Cell-free extracts from putative target cells, such as neural or hepatic cells, are passed through the column and molecules with suitable affinity bind to the fusion protein. The fusion protein-complex is recovered from the column, dissociated, and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the corresponding gene from an appropriate cDNA library.

Example 53: Determination of heparanase (heparanase) activity

Various assays are known in the art that can be used to determine the heparanase activity of albumin fusion proteins of the invention. In one example, the heparanase activity of an albumin fusion protein of the invention is assayed as described by Vlodavsky et al. (Vlodavsky et al., Nat. Med. 5:793-802, 1999). Briefly, cell lysates, conditioned media, intact cells (1×10 ⁶ cells per 35 mm dish), cell culture supernatants, or purified fusion proteins were incubated with ³⁵ S-labeled ESCs at pH 6.2-6.6 at 37°C or soluble ECM derived from Peak I proteoglycans for 18 hours. The incubation medium was centrifuged and the supernatant analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions were eluted with PBS and their radioactivity was measured. Degraded fragments of heparan sulfate side chains were eluted from Sepharose 6B at 0.5<K _av <0.8 (peak II). Each experiment was performed at least three times. The degradation fragment corresponding to "Peak II" indicates the activity of the albumin fusion protein of the invention in cleaving heparan sulfate as described by Vlodavsky et al.

Example 54: Immobilization of Biomolecules

This example provides methods for stabilizing albumin fusion proteins of the invention in non-host cell lipid bilayer constructs (see, e.g., Bieri et al., Nature Biotech 17:1105-1108, 1999, incorporated herein by reference in its entirety), It can be adapted to the study of the fusion protein of the present invention in various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation was used to confine the biotin tag to albumin fusion proteins of the invention, resulting in a uniform orientation after immobilization. A solution of 50 μM albumin fusion protein of the present invention in the washed membrane was incubated with 20 mM NaIO and 1.5 mg/ml ( ₄ mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide at room temperature for 1 hour (reaction volume, 150 μl). The samples were then first dialyzed (Pierce Slidealizer Cassett, 10 kDa cut-off; Pierce Chemical Co., Rockford, IL) at 4° C. for 5 hours, the buffer was changed every hour, and finally 500 ml of buffer R (0.15M NaCl, 1 mM MgCl ₂ , 10mM sodium phosphate pH7) dialyzed for 12 hours. Samples were diluted 1 :5 with buffer ROG50 (buffer R supplemented with 50 mM octylglucoside) just before adding to the cuvette.

Example 55: Assay for metalloprotease activity

Metalloproteases are peptidohydrolases that utilize metal ions such as Zn ²⁺ as a catalytic mechanism. The metalloprotease activity of the albumin fusion protein of the present invention can be determined according to methods known in the art. The following exemplary methods are provided:

Proteolysis of alpha-2-macroglobulin

In order to confirm the protease activity, the purified fusion protein of the present invention and the substrate α-2-macroglobulin (0.2 units/ml; Boehringer Mannheim, Germany) were mixed in 1x assay buffer (50mM HEPES pH7.5, 0.2M NaCl, 10mM CaCl ₂ , 25 μM ZnCl ₂ and 0.05% Brij-35), and incubated at 37° C. for 1-5 days. Trypsin was used as a positive control. Negative controls contained alpha-2-macroglobulin only in assay buffer. Samples were collected, boiled for 5 min in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol, and loaded onto an 8% SDS-polyacrylamide gel. After electrophoresis, proteins were visualized by silver staining. Proteolysis was evidenced by the appearance of lower molecular weight bands compared to negative controls.

Inhibition of proteolysis of α-2-macroglobulin by inhibitors of metalloproteases

Known inhibitors of metalloproteases (metal chelators (EDTA, EGTA and _HgCl2 ), peptide inhibitors of metalloproteases (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) can also be used to characterize the proteins of the invention. Proteolytic activity of protein fusion proteins. Three synthetic MMP inhibitors that can be used are: MMP Inhibitor I, [ _IC50 = 1.0 μM for MMP-1 and MMP-8; _IC50 = 30 μM for MMP-9; _IC50 = 150 μM for MMP-3]; MMP-3 (stromolysin-1) inhibitor I, [ _IC50 = 5 μM against MMP-3], and MMP-3 inhibitor II, [Ki = 130 nM against MMP-3]; inhibitors available from Calbiochem, The catalog numbers are 444250, 444218 and 444225 respectively. Briefly, different concentrations of small molecule MMP inhibitors were mixed with purified fusion protein of the present invention (50 μg/ml) in 22.9 μl 1× HEPES buffer (50 mM HEPES pH7.5, 0.2 M NaCl, 10 mM CaCl ₂ , 25 μM ZnCl ₂ and 0.05% Brij-35), and incubated at room temperature (24°C) for 2 hours, then added 7.1 μl of substrate α-2-macroglobulin (0.2 units/ml), and incubated at 37°C for 20 hours. The reaction was stopped by adding 4x sample buffer and immediately boiled for 5 minutes. After SDS-PAGE, protein bands were visualized by silver staining.

Synthetic fluorescent peptide substrate cleavage assay

The substrate specificity of fusion proteins of the invention with demonstrated metalloprotease activity can be determined using techniques known in the art, such as the use of synthetic fluorescent peptide substrates (available from BACHEMBioscience Inc). Substrates tested included M-1985, M-2225, M-2105, M-2110 and M-2255. The first four are MMP substrates, and the last is a substrate for tumor necrosis factor-α (TNF-α) converting enzyme (TACE). These substrates are preferably prepared in a 1:1 ratio of dimethylsulfoxide (DMSO) and water. Stock solutions are 50-500 μM. Fluorescence measurements were performed using a Perkin Elmer LS 50B Luminescence Spectrometer equipped with a constant temperature water bath. The excitation λ is 328nm and the emission λ is 393nm. Briefly, the assay was performed by incubating 176 μl of 1x HEPES buffer (0.2M NaCl, 10 mM CaCl ₂ , 0.05% Brij-35 and 50 mM HEPES pH 7.5) and 20 μl of substrate solution (50 μM) at 25° C. for 15 minutes and then incubating Add 20 μl of the purified fusion protein of the present invention to the measurement cuvette. The final concentration of substrate was 1 μM. The initial rate of hydrolysis was monitored for 30 minutes.

Example 56: Development of Diabetes in NOD Mice

Female NOD (non-obese diabetic) mice are characterized by displaying IDDM with a course similar to that found in humans, although the disease is more pronounced in females than in male NOD mice. Hereinafter, unless otherwise stated, the term "NOD mice" means female NOD mice. NOD mice have a progressive destruction of β-cells caused by chronic autoimmune disease. Thus, NOD mice begin life with normoglycemia, or normal blood glucose levels. By about 15-16 weeks of age, however, NOD mice began to become hyperglycemic, indicating destruction of most of their pancreatic beta cells and a corresponding inability of the pancreas to produce sufficient insulin. Thus, both the cause and progression of the disease are similar to those of human IDDM patients.

In vivo assays can assess the efficacy of immunization regimens in female NOD/LtJ mice (commercially available from The Jackson Laboratory, Bar Harbor, Me.). It is reported in the literature that 80% of female mice develop diabetes at the age of 24 weeks, and the onset of insulitis begins between 6 and 8 weeks of age. NOD mice are inbred and highly responsive to a variety of immune regulatory strategies. Adult NOD mice (6-8 weeks old) have an average weight of 20-25 g.

These mice can be untreated (control), treated with a therapeutic agent of the invention (eg, albumin fusion proteins of the invention, and fragments and variants thereof), alone or in combination with other therapeutic compounds as described above. The effect of these different therapies on the progression of diabetes can be measured as follows:

At 14 weeks of age, female NOD mice can be phenotyped based on glucose tolerance. Glucose tolerance can be measured by the intraperitoneal glucose tolerance test (IPGTT). Briefly, blood was taken from the paraorbital plexus at 0 and 60 minutes after intraperitoneal injection of glucose (1 g/kg body weight). Normal tolerance was defined as a plasma glucose of less than 144 mg% at minute 0, or less than 160 mg% at minute 60. Blood glucose levels were measured with a Glucometer Elite device.

Based on this phenotypic analysis, animals can be assigned to different experimental groups. Specifically, animals with elevated blood glucose levels can be assigned to an impaired glucose tolerance group. Mice were given food ad libitum and acidified water (pH 2.3).

Glucose tolerant and intolerant mice can be further subdivided into control groups, albumin fusion protein groups of the invention, and albumin fusion protein/therapeutic compound combination groups. Mice in the control group can receive daily intraperitoneal injections of vehicle 6 times per week. Mice in the albumin fusion group may receive daily intraperitoneal injections of therapeutic agents of the invention (eg, albumin fusion proteins of the invention, and fragments and variants thereof) in a vehicle, 6 times a week. Mice in the albumin fusion protein/therapeutic compound combination group can receive both the albumin fusion protein and the therapeutic compound combination as described above.

Urinary glucose levels in NOD mice can be measured biweekly with Labstix (Bayer Diagnostics, Hampshire, England). Body weight and fluid intake were also measured biweekly. Onset of diabetes was determined after the appearance of glycosuria in two consecutive measurements. After 10 weeks of treatment, an additional IPGTT can be performed and the animals sacrificed the next day.

After a 10-week course of treatment, 60% and 86% of control animals in the glucose-tolerant and intolerant groups developed diabetes, respectively (see US Patent No. 5,866,546, Gross et al.). Thus, without intervention, a high proportion of diabetes developed even in initially glucose-tolerant NOD mice.

Results can be confirmed by measuring blood glucose levels in NOD mice before and after treatment. Blood glucose levels in both glucose-tolerant and glucose-intolerant mice in all groups described were measured as described above.

In alternative embodiments, therapeutic agents of the invention (e.g., specific fusion proteins disclosed as SEQ ID NO: Y, and fragments and variants thereof) can be measured using spectroscopic analysis, and an appropriate amount of the protein is dosed prior to injection. Resuspend in 50 μl phosphate buffered saline (PBS) per dose. Two injections, one week apart, can be administered subcutaneously under the skin on the back of each mouse. Monitoring can be performed in two separate periods prior to immunization and can be performed weekly during treatment and continued thereafter. Urinary glucose can be tested weekly (Keto-Diastix.RTM.; Miles Inc., Kankakee, Ill.) and serum glucose can be checked in diabetic mice (ExacTech.RTM., MediSense, Inc., Waltham, Mass.). Diabetes is diagnosed when fasting blood glucose is higher than 2.5g/L.

Example 57: Histological examination of NOD mice

Histological examination of tissue samples from NOD mice can demonstrate the ability of compositions of the invention and/or combinations of compositions of the invention with other diabetes therapeutics to increase the relative concentration of beta cells in the pancreas. The experimental method is as follows:

Mice from Example 56 can be sacrificed at the end of the treatment period and tissue samples removed from the pancreas. Samples can be fixed in 10% formaldehyde in 0.9% saline and coated with wax. Two sets of 5 serial 5 μm sections can be cut at 150 μm cutting intervals for immunolabelling. Sections can be immunolabeled for insulin (1:1000 dilution of guinea pig anti-insulin antiserum, ICN Thames U.K.) and glucagon (1:2000 dilution of rabbit anti-glucagon antiserum) with peroxide-conjugated enzyme anti-guinea pig (Dako, High Wycombe, U.K.) or peroxidase-conjugated anti-rabbit serum (1:50 dilution, Dako) for detection.

The composition of the invention may or may not have as strong an effect on the visible mass of beta cells as it has on clinically demonstrated diabetes in glucose tolerant and intolerant animals.

Example 58: In vivo mouse model of NIDDM

Male C57BL/6J mice from the Jackson Laboratory (Bar Harbor, ME) were obtained at 3 weeks of age and fed either regular chow or enriched fat (35.5% wt/wt; Bioserv. Frenchtown, NJ) or fructose (60 % wt/wt; Harlan Teklad, Madison, Wl). The regular diet consisted of 4.5% wt/wt fat, 23% wt/wt protein, 31.9% wt/wt starch, 3.7% wt/wt fructose and 5.3% wt/wt fiber. The high fat (lard) diet consisted of 35.5% wt/wt fat, 20% wt/wt protein, 36.4% wt/wt starch, 0.0% wt/wt fructose and 0.1% wt/wt fiber. The high fructose food consisted of 5% wt/wt fat, 20% wt/wt protein, 0.0% wt/wt starch, 60% wt/wt fructose and 9.4% wt/wt fiber. Mice were housed in a controlled room with a 12-hour light (6:00 am to 6:00 pm)/dark cycle at a temperature of 22±3°C and a humidity of 50±20%, with no more than 5 per cage (Luo et al., 1998, Metabolism 47(6):663-8, "Nongenetic mouse models of non-insulin-dependent diabetes mellitus"; Larsen et al., Diabetes 50(11):2530-9, 2001, "Systemic administration of the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats"). After 3 weeks of administration of the corresponding food, the mice were injected intraperitoneally with 100 mg/kg body weight of streptozotocin, "STZ" (Sigma, St. Louis, MO) or vehicle (0.05 mol/L citric acid pH 4.5), And keep the same food for the next 4 weeks. Blood was drawn at 1, 2 and 4 weeks after STZ by clipping the tail end without fasting. Samples were used to measure non-fasting plasma glucose and insulin concentrations. Body weight and food intake were recorded weekly.

To directly measure the effect of a high-fat diet on the ability of insulin to promote glucose control, experiments can be initiated on three groups of mice at the end of the 7-week period described above, namely fat-fed mice, mice fed with vehicle-injected chow, and mice fed with vehicle-injected chow, and Mice were fed with STZ-injected fat. Mice were fasted for 4 hours before the experiment. In the first set of experiments, mice were anesthetized by inhalation of methoxyflurane (Pitman-Moor, Mundelein, IL). Regular insulin (Sigma) was injected intravenously ([IV] 0.1 U/kg body weight) through the tail vein and blood was drawn from different tail veins at 3, 6, 9, 12 and 15 minutes after injection. Plasma glucose concentrations can be determined for these samples and the half-life (t1/2) for glucose disappearance from plasma is calculated using WinNonlin (Scientific Consulting, Apex, NC), a pharmacokinetic/pharmacodynamic software program.

In a second set of experiments, mice were anesthetized with intraperitoneal pentobarbital sodium (Sigma). The abdominal cavity was opened, the major abdominal veins were exposed, and a 24 gauge IV catheter (Johnson-Johnson Medical, Arlington, TX) was inserted. The catheter is secured to the musculature near the abdominal vein, cut at the base where the syringe is connected, and a pre-filled PE50 plastic tubing is hung, which in turn is connected to a syringe containing perfusate. The abdominal cavity is then sutured. In this way, there will be no hindrance to the return of blood from the lower parts of the body. Mice were continuously infused with glucose (24.1 mg/kg/min) and insulin (10 mU/kg/min) at a perfusion volume of 10 μl/min. To measure plasma glucose and insulin concentrations, retro-orbital blood samples (70 μl each) can be collected at 90, 105, 120 and 135 minutes after the start of perfusion. The average of these 4 samples was used to estimate steady state plasma glucose (SSPG) and insulin (SSPI) concentrations for each animal.

Finally, the assays used to evaluate the albumin fusion proteins of the present application, therapeutic compositions, for their ability to lower plasma glucose when administered alone or in combination with any one or more of the therapeutic agents listed for the treatment of diabetes can be performed in the following two Groups were performed in the "NIDDM" mouse model injected with STZ: (1) fat-fed C57BL/6J, and (2) fructose-fed C57BL/6J. Plasma glucose concentrations of mice used in these studies ranged from 255 to 555 mg/dL. Mice were randomly assigned to be treated with the vehicle, or with the albumin fusion therapeutic agent of the present invention alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes. A total of three doses can be administered. Tail vein blood samples for measurement of plasma glucose concentrations may be collected prior to the first dose and 3 hours after the last dose.

Plasma glucose concentrations can be determined using the Glucose Diagnostic Kit from Sigma (Sigma No. 315), an enzymatic colorimetric assay. Plasma insulin levels can be determined using the mouse insulin RIA kit from Linco Research (#RI-13K; St. Charles, MO).

Example 59: In vitro H4IIe-SEAP reporter assay to determine involvement of insulin action

Various H4IIe reporter genes

H4IIe/rMEP-SEAP: Malic enzyme (rMEP) isolated from rat contains the PPAR-γ element in the insulin pathway. This reporter construct was stably transfected into the hepatic H4IIe cell line.

H4IIe/SREBP-SEAP: Sterol regulatory element-binding protein (SREBP-1c) is a transcription factor that acts on the promoters of several insulin-responsive genes, such as fatty acid synthase (FAS), and is expressed in fibroblasts, lipid Regulates expression of key genes involved in fatty acid metabolism in cells and hepatocytes. SREBP-1c, also known as adipocyte determination and differentiation factor 1 (ADD-1), is thought to be a major mediator of insulin-induced gene expression in adipocytes. Its activity is regulated by insulin, sterol and glucose levels. This reporter construct was stably transfected into the hepatic H4IIe cell line.

H4IIe/FAS-SEAP: Fatty acid synthase reporter construct contains a minimal SREBP-responsive FAS promoter. This reporter construct was stably transfected into the hepatic H4IIe cell line.

H4IIe/PEPCK-SEAP: The phosphoenolpyruvate carboxykinase (PEPCK) promoter is the major hormonal regulatory site of transcription of the PEPCK gene that regulates PECK activity. PEPCK catalyzes a critical and rate-limiting step in hepatic gluconeogenesis and therefore must be carefully controlled to maintain blood glucose levels within normal limits. This reporter construct was stably transfected into the hepatic H4IIe cell line.

These reporter constructs can also be stably transfected into 3T3-L1 fibroblasts and L6 myoblasts. These stable cell lines were then differentiated into 3T3-L1 adipocytes and L6 myotubes as previously described in Example 13. The differentiated cell lines can then be used in the SEAP assay described below.

Growth and Assay Media

Growth medium containing 10% fetal bovine serum (FBS), 10% calf serum, 1% NEAA, 1x penicillin/streptomycin, and 0.75mg/ml G418 (for H4IIe/rFAS-SEAP and H4IIe/SREBP-SEAP ) or 0.50mg/ml G418 (for H4IIe/rMEP-SEAP). For H4IIe/PEPCK-SEAP, growth medium consisted of 10% FBS, 1% penicillin/streptomycin, 15 mM HEPES buffered saline, and 0.50 mg/ml G418.

For H4IIe/rFAS-SEAP, H4IIe/SREBP-SEAP, H4IIe/rMEP-SEAP reporter genes, the assay medium consisted of low glucose DMEM medium (Life Technologies), 1% NEAA, 1x penicillin/streptomycin. Assay medium for the H4IIe/PEPCK-SEAP reporter gene consisted of 0.1% FBS, 1% penicillin/streptomycin, and 15 mM HEPES buffered saline.

method

96-well plates were seeded at 75,000 cells/well in 100 [mu]l/well growth medium until cells in logarithmic growth phase became attached. Cells were starved for 48 hours by replacing growth medium with 200 μl/well assay medium. (For H4IIe/PEPCK-SEAP cells, 100 μl/well of assay medium containing 0.5 μM dexamethasone was added and incubated for about 20 hours). The assay medium was then replaced with 100 μl/well of fresh assay medium, and the wells were added to the wells obtained from transfected cell lines expressing therapeutic agents of the invention (such as albumin fusion proteins of the invention, and fragments and variants thereof). A 50 μl aliquot of the cell supernatant. Supernatants from empty vector-transfected cell lines were used as negative controls. 10 nM and/or 100 nM insulin was added to the wells as a positive control. After 48 hours of incubation, conditioned medium was harvested and SEAP activity was measured (Phospha-Light System protocol, Tropix #BP2500). Briefly, samples were diluted 1:4 in dilution buffer and incubated at 65°C for 30 minutes to inactivate endogenous non-placental forms of SEAP. A 50 μl aliquot of the diluted sample was mixed with 50 μl SEAP assay buffer containing a cocktail of inhibitors active against non-placental SEAP isoenzymes and incubated for an additional 5 minutes. A 50 [mu]l aliquot of CSPD chemiluminescent substrate diluted 1:20 in Emerald Luminescence Enhancer was added to the mixture and incubated for 15-20 minutes. Plates were read in a Dynex plate luminometer.

Example 60: Transgenic animals

Albumin fusion proteins of the invention can also be expressed in transgenic animals. Animals of any species, including but not limited to mice, rats, rabbits, hamsters, guinea pigs, pigs, miniature pigs, goats, sheep, cattle, and non-human primates such as baboons, monkeys, and orangutans can be used to generate transgenic animals . In specific embodiments, fusion proteins of the invention are expressed in humans as part of a gene therapy regimen using techniques described herein or otherwise known in the art.

Any technique known in the art can be used to introduce polynucleotides encoding albumin fusion proteins of the invention into animals to generate founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698, 1994; Carver et al., Biotechnology (NY) 11:1263-1270, 1993; Wright et al. , Biotechnology (NY) 9:830-834,1991; and Hoppe et al., U.S. Pat. No. 4,873,191,1989); retrovirus-mediated gene transfer to the germline (VanderPutten et al., Proc.Natl.Acad.Sci .USA 82:6148-6152, 1985), blastocyst or embryo; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321, 1989); electroporation of cells or embryos (Lo, 1983, Mol .Cell.Biol.3:1803-1814,1983); Use the introduction of the polynucleotide of the present invention of gene gun (seeing such as Ulmer et al., Science 259:1745,1993); Nucleic acid construct is introduced into embryonic pluripotent stem cell and Transfer of stem cells back to blastocysts; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989); et al. For a review of these techniques see Gordon, "Transgenic Animals", Intl. Rev. Cytol. 115:171-229, 1989), which is incorporated herein by reference in its entirety.

Transgenic clones containing a polynucleotide encoding an albumin fusion protein of the invention can be generated using any technique known in the art, for example, nuclear transfer of nuclei from cultured embryos, fetuses, or adult cells induced to quiescence into enucleated In oocytes (Campell et al., Nature 380:64-66, 1996; Wilmut et al., Nature 385:810-813, 1997).

The invention provides transgenic animals that carry polynucleotides encoding albumin fusion proteins of the invention in all of their cells, as well as animals that carry these nucleotides in some but not all of their cells, ie, chimeric animals or chimeras. The transgene may be integrated as a single transgene or as multiple copies, eg, concatemers, such as head-to-head tandems or head-to-tail tandems. Transgenes can also be selectively introduced into and activated in specific cell types according to eg Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992). The regulatory sequences required for such cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. Gene targeting is preferred when integration of the polynucleotide encoding the fusion protein of the invention into the chromosomal location of the endogenous gene corresponding to the Therapeutic protein portion or the albumin portion of the fusion protein of the invention is desired. Briefly, when using such a technique, a vector containing some nucleotide sequence homologous to an endogenous gene is designed to integrate into the nucleotide sequence of an endogenous gene by homologous recombination with a chromosomal sequence and destroy its functionality. Transgenes can also be selectively introduced into specific cell types, thereby inactivating endogenous genes only in that cell type, as taught eg by Gu et al. (Gu et al., Science 265: 103-106, 1994). The regulatory sequences required for such cell type specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art.

Once transgenic animals have been produced, expression of the recombinant gene can be assayed using standard reductions. Primary screening is accomplished by analyzing animal tissues by Southern blot analysis or PCR techniques to confirm that integration of the polynucleotide encoding the fusion protein of the invention has occurred. The following techniques can also be used to assess mRNA expression levels of polynucleotides encoding fusion proteins of the present invention in tissues of transgenic animals, including but not limited to Northern blot analysis, in situ hybridization analysis, and reverse transcription PCR of tissue samples obtained from animals (rt-PCR). Samples of tissues expressing the fusion protein can also be evaluated by immunocytochemistry or immunohistochemistry using antibodies specific for the fusion protein.

Once founder animals have been produced, they can be bred, inbred, outbred, or crossed to create a population of particular animals. Examples of such mating strategies include, but are not limited to: outcrossing of founder animals with more than one integration site to create separate lines; Compound transgenic animals that express the transgene at higher levels as a result of the effect; crossing of heterozygous transgenic animals to generate animals homozygous at a given integration site, thereby increasing expression and eliminating the need to screen animals by DNA analysis; isolation of homozygous lines to produce compound heterozygous or homozygous lines; and mating to place the transgene (ie, the polynucleotide encoding the albumin fusion protein of the invention) in a unique context suitable for the experimental model of interest. Uses of the transgenic animals of the present invention include, but are not limited to, useful in specifying the biological functions of the fusion proteins of the present invention and therapeutic proteins and/or albumin components of the fusion proteins of the present invention, studying conditions associated with abnormal expression, and/or or diseases, and animal model systems for screening compounds effective in ameliorating these conditions and/or disorders. Example 61: Therapeutic method using gene therapy - ex vivo

One approach to gene therapy involves transplanting fibroblasts capable of expressing albumin fusion proteins of the invention into a patient. Typically, fibroblasts are obtained from a subject by skin biopsy. The tissue thus obtained was placed in tissue culture medium and divided into small pieces. Place small pieces of tissue on the wet surface of tissue culture flasks, placing approximately ten pieces in each flask. The flask was turned upside down, sealed, and left overnight at room temperature. After 24 hours at room temperature, the flask is inverted with the tissue pieces still fixed at the bottom of the flask, and fresh medium (eg, Ham's F12 medium containing 10% FBS, penicillin, and streptomycin) is added. The flask was then incubated at 37°C for approximately one week.

At this point, fresh medium was added and replaced every few days thereafter. After an additional 2 weeks of culture, a monolayer of fibroblasts appeared. Cell monolayers were trypsinized and scaled up to larger flasks.

pMV-7 flanked by the long terminal repeats of Moloney murine sarcoma virus (Kirschmeier, P.T. et al., DNA 7:219-25, 1988) was digested with EcoRI and HindIII and then treated with calf intestinal phosphatase. Linear vectors were fractionated on agarose gels and purified using glass beads.

A polynucleotide encoding an albumin fusion protein of the invention can be generated using techniques known in the art, amplified using PCR primers corresponding to its 5' and 3' end sequences, optionally with suitable restriction sites and start/stop codon. Preferably, the 5' primer contains an EcoRI site and the 3' primer contains a HindIII site. Equal amounts of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragments were added together in the presence of T4 DNA ligase. The mixture thus obtained is maintained under conditions suitable for ligation of the two fragments. The ligation mixture was then used to transform bacteria HB 101 and subsequently plated on agar containing kanamycin to confirm correct insertion of the gene of interest in the vector.

Amphotropic pA317 or GP+aml2 packaging cells were grown to confluent density in tissue culture in Dulbecco's Modified Eagles' Medium (DMEM) containing 10% calf serum (CS), penicillin and streptomycin . The MSV vector containing the gene is then added to the medium, and the packaging cells are transduced with the vector. The packaging cells now produce infectious virus particles that contain the genes (packaging cells are now called producer cells).

Fresh medium was added to the transduced producer cells and subsequently harvested from confluent 10 cm dishes of producer cells. Spent medium containing infectious viral particles is filtered through a millipore filter to remove detached producer cells, and this medium is then used to infect fibroblasts. The medium was removed from the sub-confluent plate of fibroblasts and replaced with medium from the producer cells. Remove this medium and replace with fresh medium. If the titer of the virus is high, virtually all fibroblasts will be infected without selection. If the titer is very low, a retroviral vector with a selectable marker such as neo or his must be used. Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine whether the albumin fusion protein is produced.

The engineered fibroblasts were then grown to confluence, alone or on cytodex3 microcarrier beads, and transplanted onto the host.

Example 62: Methods of treatment using gene therapy - in vivo

Another aspect of the invention is the use of in vivo gene therapy to treat disorders, diseases and conditions. Gene therapy methods involve introducing into animals naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences encoding albumin fusion proteins of the invention. A polynucleotide encoding an albumin fusion protein of the invention may be operably linked (ie, linked) to a promoter or any other genetic element necessary for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see for example WO90/11092, WO98/11779; U.S. Application Nos. 5693622, 5705151, 5580859; Tabata et al., Cardiovasc. 479,1997; Chao et al., Pharmacol.Res.35(6):517-522,1997; Wolff, Neuromuscul.Disord.7(5):314-318,1997; Schwartz et al., Gene Ther.3(5 ): 405-411, 1996; Tsurumi et al., Circulation 94(12): 3281-3290, 1996 (incorporated herein by reference).

The polynucleotide constructs can be delivered by any method that delivers an injectable substance into the cells of an animal, such as injection into the interstitial space of tissue (heart, muscle, skin, lung, liver, intestine, etc.). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term "naked" polynucleotide, DNA or RNA refers to sequences free from any delivery vehicle that assists, facilitates or accelerates entry into cells, including viral sequences, viral particles, liposome formulations, lipofection or precipitation reagents, and the like. However, polynucleotides encoding albumin fusion proteins of the invention may also be delivered in liposomal formulations which can be prepared by methods well known to those skilled in the art (such as Felgner P.L. et al., 1995, Ann.NY Acad.Sci.772 : 126-139 and taught by Abdallah B. et al., 1995, Biol. Cell 85(1): 1-7).

Polynucleotide vector constructs for use in gene therapy methods are preferably constructs that neither integrate into the host genome nor contain sequences that allow replication. Any strong promoter known to those skilled in the art can be used to drive expression of the DNA. A major advantage of introducing naked nucleic acid sequences into target cells, unlike other gene therapy techniques, is the transient nature of polynucleotide synthesis in cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for the production of desired polypeptides for a period of up to 6 months.

The polynucleotide constructs can be delivered to the interstitial tissue of animals, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, Intestines, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue. The interstitial spaces of tissue include the intercellular fluid, the mucopolysaccharide layer between the reticular fibers of organ tissues, the elastic fibers in the walls of vessels or chambers, the collagen fibers of fibrous tissues, or the same matrix incorporated into connective tissue or bone spaces of muscle cells . The space occupied by circulating plasma and lymphatic fluid of the lymphatic ducts is also similar. Delivery to the interstitial space of muscle tissue is preferred for reasons discussed below. They can be conveniently delivered by injection to tissues containing these cells. They are preferably delivered to and expressed in persistent, non-dividing differentiated cells, although delivery and expression may be accomplished in undifferentiated or less fully differentiated cells such as, for example, blood stem cells or skin fibroblasts. Muscle cells are particularly competent in their ability to uptake and express polynucleotides in vivo.

For naked polynucleotide injections, effective doses of DNA or RNA will range from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably, the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, those of ordinary skill in the art will appreciate that this dosage will vary with the tissue site of injection. Appropriate and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art, and may depend on the condition being treated and the route of administration. The preferred route of administration is by parenteral injection into the interstitial space of the tissue. However, other parenteral routes may also be used, such as aerosol inhalation, particularly suitable for delivery to the lung or bronchial tissue, larynx or nasal mucosa. In addition, naked polynucleotide constructs can be delivered to an artery during angioplasty through the catheter used in the procedure.

The drug-response effect of intramuscular injection of polynucleotides in vivo was determined as follows. Suitable template DNA for production of mRNA encoding a polypeptide of the invention is prepared according to conventional recombinant DNA methods. Template DNA, which may be circular or linear, is used either as naked DNA or complexed with liposomes. The quadriceps muscles of the mice were then injected with varying amounts of template DNA.

Five to six week old female and male Balb/C mice were anesthetized by intraperitoneal injection of 0.3 ml 2.5% Avertin. A 1.5 cm incision is made on the front thigh to directly expose the quadriceps muscle. Template DNA was injected in 0.1 ml vector with a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal muscle insertion site to the knee and approximately 0.2 cm deep. For future positioning, sutures were placed over the injection site and the skin was closed with stainless steel clips.

After an appropriate incubation period (eg, 7 days), muscle extracts are prepared by dissecting the whole quadriceps muscle. Every five 15 μm transverse sections of individual quadriceps muscles were histologically stained for protein expression. The time course of fusion protein expression can be done in a similar fashion, except that the quadriceps muscles of different mice are collected at different times. The persistence of DNA in muscle after injection can be determined by Southem blot analysis of total cellular DNA and HIRT supernatants prepared from injected and control mice. The results of the above experiments in mice can be used to extrapolate appropriate dosages and other parameters of treatment using naked DNA in humans and other animals.

Example 63: Biological effects of the fusion protein of the present invention

Astrocyte and Neuronal Assays

The albumin fusion protein of the present invention can be tested for its activity in promoting the survival of cortical neuron cells, neurite outgrowth, or phenotypic differentiation and the ability to induce glial fibrillary acidic protein immunopositive cells and astrocyte proliferation . The choice of cortical cells for the bioassay was based on the ubiquitous expression of FGF-1 and FGF-2 in cortical structures and the previously reported enhanced survival of cortical neurons due to FGF-2 treatment. For example, a thymidine incorporation assay can be used to elucidate the activity of albumin fusion proteins of the invention on these cells.

Furthermore, previous reports describing the biological effects of FGF-2 (the basal FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuronal survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension", Proc. Nntl. Acad. Sci. USA 83:3012-3016, 1986, the assay is incorporated herein by reference in its entirety). However, reports from experiments on PC-12 cells suggest that the two responses are not necessarily synonymous, but may depend not only on which FGF is tested but also on which receptor is expressed on the target cell. Using primary cortical neuron culture models, the ability of albumin fusion proteins of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2, for example, using a thymidine incorporation assay.

Fibroblast and endothelial cell assays

Human lung fibroblasts were obtained from Clonetics (San Diego, CA) and maintained in growth medium obtained from Clonetics. Skin microvascular endothelial cells were obtained from Cell Applications (San Diego, CA). For proliferation assays, human lung fibroblasts and skin microvascular endothelial cells can be cultured in 96-well dishes at 5,000 cells/well in growth medium for 1 day. Cells were then incubated for 1 day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells were incubated with the test fusion protein of the protein of the invention for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, CA) was added to each well to a final concentration of 10%. Cells were incubated for 4 hours. Cell viability was measured by reading in a CytoFluor fluorescence reader. For the PGE2 assay, human lung fibroblasts were cultured in 96-well dishes at 5,000 cells/well for 1 day. After the medium was replaced with 0.1% BSA basal medium, the cells were incubated with FGF-2 or the fusion protein of the present invention for 24 hours with or without IL-1α. Supernatants were collected and measured for _PGE2 using an EIA kit (Cayman, Ann Arbor, MI). For the IL-6 assay, human lung fibroblasts were cultured in 96-well dishes at 5,000 cells/well for 1 day. After changing the medium to 0.1% BSA basal medium, cells were incubated for 24 hours with FGF-2 or with or without albumin fusion protein of the invention and/or IL-1α. Supernatants were collected and assayed for IL-6 using an ELISA kit (Endogen, Cambridge, MA).

Human lung fibroblasts were cultured with FGF-2 or the albumin fusion protein of the invention in basal medium for 3 days before adding Alamar Blue to assess the effect on fibroblast growth. FGF-2 should show a stimulating effect at 10-2500 ng/ml, which can be used to compare the stimulating effect with the fusion protein of the present invention.

Cell proliferation based on [3H]thymidine incorporation

The following [3H]thymidine incorporation assay can be used to measure the proliferative effect of therapeutic proteins, such as growth factor proteins, on cells such as fibroblasts, epithelial cells or immature muscle cells.

Subconfluent cultures were arrested at the G1 phase by incubation in serum-free medium for 18 hours. Therapeutic proteins were then added for 24 hours and the cultures were labeled with [3H]thymidine for the final 4 hours at a final concentration of 0.33 μΜ (25 Ci/mmol, Amersham, Arlington Heights, IL). Incorporated [3H]thymidine was precipitated with ice-cold 10% trichloroacetic acid for 24 hours. Subsequently, cells were rinsed sequentially with ice-cold 10% trichloroacetic acid followed by ice-cold water. After dissolution in 0.5M NaOH, the lysate and PBS rinse (500ml) were combined and the amount of radioactivity was measured.

Parkinson's model

Loss of motor function in Parkinson's disease is attributable to striatal dopamine deficiency due to degeneration of nigrostriatal dopaminergic projection neurons. A well-characterized animal model of Parkinson's disease involves systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenylpyridine (MPP ⁺ ) and released. Subsequently, MPP ⁺ is actively accumulated in dopaminergic neurons via the high-affinity reuptake transporter of dopamine. MPP ⁺ then concentrates and selectively inhibits nicotinamide adenine diphosphate:ubiquinone oxidoreductase (complex 1) through an electrochemical gradient in the mitochondria, thereby interfering with electron transport and ultimately generating oxygen free radicals.

FGF-2 (basal FGF) has been demonstrated in a tissue culture paradigm to have trophic activity against substantia nigra dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administration of FGF-2 in the striatum as a gel foam implant results in near complete protection of substantia nigra dopaminergic neurons from toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the FGF-2 data, the albumin fusion protein of the invention can be evaluated to determine whether it has a similar effect to FGF-2 in enhancing the survival of dopaminergic neurons in vitro, and can also be tested in vivo to protect dopaminergic neurons in the striatum. Effects of dopaminergic neurons on protection from damage associated with MPTP processing. The potential effect of the albumin fusion proteins of the invention was first tested in vitro in a dopaminergic neuronal cell culture paradigm. To prepare cultures, the midbrain floor plate was dissected from gestational day 14 Wistar rat embryos. Tissues were dissociated with trypsin and seeded onto polyorthinine-laminin-coated glass coverslips at 200,000 cells/ ^cm2 . Cells were maintained in Dulbecco's modified Eagle's medium and F12 medium with hormone supplements (N1). After 8 days the cultures were fixed in vitro with paraformaldehyde and processed for immunohistochemical staining for tyrosine hydroxylase, a specific marker of dopaminergic neurons. Preparation of dissociated cell cultures from embryonic rats. Medium was changed every three days and factors were added at that time.

Since dopaminergic neurons were isolated from animals at 14 days of gestation, a developmental time past the stage of proliferation of dopaminergic precursor cells, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent Increase in the number of surviving dopaminergic neurons in vitro. Therefore, if a therapeutic protein of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the fusion protein may be involved in Parkinson's disease.

Example 64: Combination therapy with pancreatic β-cell transplantation

Transplantation is a common form of treatment for autoimmune diseases, especially when the target self tissue has been severely damaged. For example, without limitation, pancreas transplantation and islet cell transplantation are common treatment options for IDDM (see, e.g., Stewart et al., Journal of Clinical Endocrinology & Metabolism 86(3):984-988, 2001; Brunicardi, Transplant.Proc.28:2138- 40, 1996; Kendall and Robertson, Diabetes Metab. 22: 157-163, 1996; Hamano et al., Kobe J. Med. Sci. 42: 93-104, 1996; Larsen and Stratta, Diabetes Metab. 22: 139-146 , 1996; and Kinkhabwala et al., Am. J. Surg. 171:516-520, 1996). As with any transplantation procedure, transplantation therapy in patients with autoimmune disease includes management to minimize the risk of host rejection of the transplanted tissue. However, autoimmune disease involves the additional, independent risk that a pre-existing host's own immune response involving the destruction of the original self tissue will have the same damaging effect on the transplanted tissue. Accordingly, the invention encompasses methods and compositions for the treatment of autoimmune pancreatic disease using albumin fusion proteins of the invention in combination with immunomodulators/immunosuppressants in individuals undergoing transplant therapy for the autoimmune disease.

According to the present invention, the albumin fusion-based compositions and formulations described above are administered to prevent and treat damage to transplanted organs, tissues or cells caused by the host individual's initial autoimmune response against the original self-tissue. Administration can be performed pre- and post-implantation in 2 to 4 doses, each one week apart.

The following immunomodulators/immunosuppressants, including but not limited to AI-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal antibody), Linoramide (Roquinimex), NBI-6024 (altered peptide ligand), TM-27, VX-740 (HMR-3480), caspase 8 protease inhibitor, thalidomide, hOKT3gammal (Ala-ala) (anti-CD3 monoclonal antibody), oral interferon-α, oral lactobacilli, and LymphoStat-B ^™ , can be used together with the albumin fusion therapeutic agent of the present invention in islet cells and pancreas transplantation.

Example 65: Identification and cloning of VH and VL domains

试剂(Life Technologies，Rockville.MD)溶解细胞，并用五分之一体积的氯仿抽提。加入氯仿后，使溶液于室温保温10分钟，并于4℃在台式离心机中在14,000rpm离心15分钟。收集上清液，并用等体积的异丙醇沉淀RNA。通过在台式离心机中于4℃以14,000rpm离心15分钟来沉降沉淀的RNA。离心后，丢弃上清液并用75％乙醇清洗。清洗后，将RNA再次于4℃以800rpm离心5分钟。丢弃上清液并使沉淀物空气干燥。将RNA溶于DEPC水中并加热至60℃达10分钟。可通过光密度测量法来测定RNA的数量。 One method of identifying and cloning VH and VL domains from cell lines expressing a particular antibody is by PCR with VH and VL specific primers on cDNA prepared from antibody expressing cell lines. Briefly, RNA was isolated from cell lines and used as template in RT-PCR designed to amplify the VH and VL domains of antibodies expressed by EBV cell lines. Can use

Reagent (Life Technologies, Rockville, MD) lysed cells and extracted with one-fifth volume of chloroform. After the addition of chloroform, the solution was incubated at room temperature for 10 minutes and centrifuged at 14,000 rpm in a tabletop centrifuge at 4°C for 15 minutes. The supernatant was collected and the RNA was precipitated with an equal volume of isopropanol. Precipitated RNA was pelleted by centrifugation at 14,000 rpm for 15 minutes at 4°C in a tabletop centrifuge. After centrifugation, discard the supernatant and wash with 75% ethanol. After washing, the RNA was centrifuged again at 800 rpm for 5 minutes at 4°C. Discard the supernatant and allow the pellet to air dry. RNA was dissolved in DEPC water and heated to 60°C for 10 minutes. The amount of RNA can be determined by densitometry.

cDNA can be synthesized from 1.5-2.5 micrograms of RNA using reverse transcriptase and random hexamer primers according to methods well known in the art. The cDNA was then used as a template for PCR amplification of the VH and VL domains. Primers used to amplify VH and VL genes are shown in Table 7. Typically, a PCR reaction uses a single 5' primer and a single 3' primer. Sometimes, when the amount of RNA template available is limited, or for greater efficiency, sets of 5' and/or 3' primers can be used. For example, all five VH-5' primers and all JH 3' primers are sometimes used in a single PCR reaction. PCR reactions were performed in a volume of 50 microliters containing 1X PCR buffer, 2 mM of each dNTP, 0.7 units of high-fidelity Taq polymerase, 5' primer mix, 3' primer mix, and 7.5 microliters of cDNA. 5' and 3' primer mixes for both VH and VL can be prepared by combining 22 pmole and 28 pmole of each individual primer, respectively. PCR conditions were: 96°C for 5 minutes; followed by 25 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute; followed by an extension cycle of 72°C for 10 minutes. After the reaction was complete, the sample tubes were stored at 4°C.

Table 7: Primer sequences used to amplify VH and VL domains

PCR samples were then electrophoresed on a 1.3% agarose gel. DNA bands of the expected size (approximately 506 bp for the VH domain and 344 bp for the VL domain) can be excised from the gel and purified using methods well known in the art. Purified PCR products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, CA). PCR products of individual clones can be isolated after transfection of E. coli and blue/white color selection. The cloned PCR products can then be sequenced by methods generally known in the art.

PCR bands containing VH and VL domains can also be used to create full-length Ig expression vectors. The VH and VL domains can be cloned into vectors containing the nucleotide sequence of the heavy chain (e.g. human IgG1 or human IgG4) or light chain (human kappa or human lambda) constant region such that upon transfection into an appropriate host cell Afterwards, complete heavy or light chain molecules can be expressed from these vectors. Furthermore, when both cloned heavy and light chains are expressed in a single cell line (from one or both vectors), they can be assembled into complete functional antibody molecules that are secreted into the cell culture medium. Methods for generating expression vectors encoding intact antibody molecules using polynucleotides encoding VH and VL antibody domains are well known in the art.

Example 66: Preparation of HA-cytokine or HA-growth factor fusion proteins such as NGF, BDNFa, BDNFb and BDNFc

cDNA for a cytokine or growth factor of interest such as NGF can be isolated by a variety of methods, including from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences of all these proteins are known and available. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. Cloning the NGF (or other cytokine) cDNA into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, and then excising the complete expression cassette from it and inserting it into the plasmid pSAC35, so that it can be expressed in yeast Albumin fusion protein. Albumin fusion proteins secreted by yeast can then be collected and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 67: Preparation of HA-IFN fusion proteins such as IFNa

The cDNA for an interferon of interest, such as IFNa, can be isolated by a variety of methods including, but not limited to, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences of interferons such as IFNa are known and available, eg, in US Patents 5,326,859 and 4,588,585, EP32134, and public databases such as GenBank. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. IFNa (or other interferon) cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, and then the complete expression cassette is excised from it and inserted into plasmid pSAC35, so that it can be expressed in yeast Albumin fusion protein. Albumin fusion proteins secreted by yeast can then be collected and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Maximum protein recovery from tubes

The albumin fusion proteins of the invention are highly stable even when they are packaged at low concentrations. Furthermore, despite the low protein concentration, good recovery of the fusion protein was observed even when the aqueous solution was free of other proteins added to minimize binding to the tube wall. The recovery of the HA-IFN solution stored in tubes was compared to the stock solution. 6 or 30 μg/ml HA-IFN solutions were placed in tubes and stored at 4°C. After 48 or 72 hours, an initial volume equivalent to 10 ng of sample was withdrawn and measured with an IFN sandwich ELISA. Compare the estimated value to the high concentration stock solution. As demonstrated, there was essentially no loss of sample in these tubes, indicating that the addition of exogenous substances such as albumin was not necessary to prevent loss of sample through the tube wall.

In Vivo Stability and Bioavailability of HA-α-IFN Fusion

To determine the in vivo stability and bioavailability of the HA-[alpha]-IFN fusion molecule, monkeys were administered the purified fusion molecule (from yeast). Pharmaceutical compositions formulated from HA-[alpha]-IFN fusions result in increased serum half-life and bioavailability. Accordingly, pharmaceutical compositions can be formulated that contain lower doses of alpha-interferon activity compared to native alpha-interferon molecules.

Pharmaceutical compositions containing HA-α-IFN fusions are useful for treating or preventing disease in patients suffering from any disease or condition that can be modulated by administration of α-IFN. Such diseases include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, genital and anal warts, chronic hepatitis B, chronic non-A non-B hepatitis, especially hepatitis C, hepatitis D, chronic myelogenous leukemia, renal Cell carcinoma, bladder cancer, ovarian and cervical cancer, skin cancer, recurrent respiratory papillomatosis, non-Hodgkin's and cutaneous T-cell lymphoma, melanoma, multiple myeloma, AIDS, Multiple sclerosis, glioblastoma, etc. (see Interferon Alpha, in "AHFS Drug Information", 1997).

Accordingly, the present invention includes pharmaceutical compositions comprising HA-α-IFN fusion proteins, polypeptides or peptides formulated in correct dosage for human administration. The present invention also includes methods of treating a patient in need of such treatment comprising at least one step of administering a pharmaceutical composition comprising at least one HA-alpha-IFN fusion protein, polypeptide or peptide.

Bifunctional HA-α-IFN fusion

The HA-α-IFN expression vector can be modified to contain an insert for expression of a bifunctional HA-α-IFN fusion protein. For example, the cDNA of the second protein of interest can be inserted in the same reading frame downstream of the "rHA-IFN" sequence after removing the double stop codon or moving downstream of the coding sequence.

In one version of the bifunctional HA-α-IFN fusion protein, an antibody or fragment against a B-lymphocyte stimulator protein (GenBank Accession No. 4455139) or polypeptide can be fused to one end of the HA component of the fusion molecule. This bifunctional protein is useful for modulating any immune response generated by the α-IFN component of the fusion.

Example 68: Preparation of HA-hormone fusion protein

cDNA for the hormone of interest can be isolated by a variety of methods including, but not limited to, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences of all these proteins are known and available, for example, in public databases such as GenBank. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. The albumin fusion protein can be expressed in yeast by cloning the hormone cDNA into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette is excised and inserted into plasmid pSAC35. Albumin fusion proteins secreted by yeast can then be collected and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then subsequently excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 69: Preparation of HA-Soluble Receptor or HA-Binding Protein Fusion Proteins

cDNA for a soluble receptor or binding protein of interest can be isolated by a variety of methods including, but not limited to, from a cDNA library, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods . The nucleotide sequences of all these proteins are known and available eg in GenBank. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. The receptor cDNA was cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette was excised and inserted into plasmid pSAC35, allowing expression of the albumin fusion protein in yeast. Albumin fusion proteins secreted by yeast can then be collected and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfection of mammalian cell lines.

Example 70: Preparation of HA-growth factor

The cDNA for the growth factor of interest can be isolated by a variety of methods including, but not limited to, from a cDNA library, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods (see GenBank no. NP_000609). The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. Albumin fusion proteins can be expressed in yeast by cloning the growth factor cDNA into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette is excised and inserted into plasmid pSAC35. The albumin fusion protein secreted by the yeast was then collected and purified from the culture medium and tested for its biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 71: Preparation of HA-single-chain antibody fusion protein

ScFvs are produced by several methods including, but not limited to: selection from phage libraries, cloning the variable region of a specific antibody by cloning the antibody's cDNA and using the flanking constant regions as primers to clone the variable region, or by synthesis with any An oligonucleotide corresponding to the variable region of a particular antibody. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. Albumin fusion proteins can be expressed in yeast by cloning cellular cDNA into vectors such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette is excised and inserted into plasmid pSAC35.

_In the fusion molecule of the present invention, VH and VL can be connected by one or a combination _of the following methods: a peptide linker between the C-terminus of _VH and the N-terminus of _VL ; Kex2p protease cleavage site so that the two can cleave after secretion and subsequently self-associate; and a cystine residue positioned such that _VH and _VL can form a disulfide bond between them to link them together. An alternative is to place the _VH at the N-terminus of the HA or HA domain fragment and the _VL at the C-terminus of the HA or HA domain fragment.

Albumin fusion proteins secreted by yeast can then be harvested and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines. Antibodies prepared in this manner can be purified from culture medium and tested for binding to antigen using standard immunochemical methods.

Example 72: Preparation of HA-cell adhesion molecule fusion protein

cDNA for a cell adhesion molecule of interest can be isolated by a variety of methods including, but not limited to, from a cDNA library, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences of known cell adhesion molecules are known and available eg in GenBank. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. Cell adhesion molecule cDNA is cloned into vectors such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette is excised and inserted into plasmid pSAC35, allowing expression of albumin fusions in yeast protein. The albumin fusion protein secreted by the yeast was then collected and purified from the culture medium and tested for its biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 73: Preparation of inhibitors and peptides as HA fusion proteins such as HA-antivirals, HA-antibiotics, HA-enzyme inhibitors and HA-antiallergic proteins

cDNA for a peptide of interest, such as an antibiotic peptide, can be isolated by a variety of methods including, but not limited to, from a cDNA library, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. Albumin fusion proteins can be expressed in yeast by cloning the peptide cDNA into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, from which the complete expression cassette is excised and inserted into plasmid pSAC35. The albumin fusion protein secreted by the yeast was then collected and purified from the culture medium and tested for its biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 74: Preparation of targeted HA fusion protein

The cDNA for the protein of interest can be isolated from a cDNA library using standard molecular biology methods or can be prepared synthetically using several overlapping oligonucleotides. Appropriate nucleotides in the cDNA can be engineered to create convenient restriction sites and allow attachment of the protein cDNA to the albumin cDNA. Likewise, a targeting protein or peptide cDNA, such as a single chain antibody or peptide, such as a nuclear localization signal, capable of directing the protein within the cell, can be fused to the other end of the albumin. The protein of interest and targeting peptide are cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, resulting in a fusion to the albumin cDNA. In this manner, other proteins are fused to both the N- and C-termini of albumin. The fusion cDNA is then excised from pPPC0005 and inserted into a plasmid such as pSAC35, allowing expression of the albumin fusion protein in yeast. All of the above procedures can be performed using standard methods of molecular biology. Albumin fusion proteins secreted by yeast can be collected and purified from the culture medium and tested for their biological activity and their targeting activity using appropriate biochemical and biological assays.

Example 75: Preparation of HA-enzyme fusions

The cDNA for the enzyme of interest can be isolated by a variety of methods including, but not limited to, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be trimmed at the 5' and 3' ends to create restriction sites that allow oligonucleotide linkers to be used to clone the cDNA into a vector containing the HA cDNA. This can be at the N- or C-terminus, with or without a spacer sequence. The enzyme cDNA was cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:HSA, and then the complete expression cassette was excised from it and inserted into plasmid pSAC35, so that the albumin fusion protein could be expressed in yeast. Albumin fusion proteins secreted by yeast can then be collected and purified from the culture medium and tested for biological activity. For expression in mammalian cell lines, a similar procedure was followed, except that the expression cassette used employed a mammalian promoter, leader and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for transfecting mammalian cell lines.

Example 76: Construct ID 2053, Production of IFNb-HSA

Construct ID 2053, pEE12.1: IFNb.HSA contained in the NSO expression vector pEE12.1 DNA encoding an IFNb-albumin fusion protein with full-length IFNb protein including native IFNb fused to the amino terminus of the mature form of HSA leading sequence.

Cloning of IFNb cDNA

The polynucleotide encoding IFNb was PCR amplified using primers IFNb-1 and IFNb-2 described below, cut with Bam HI/Cla I, and ligated into Bam HI/Cla I cut pC4:HSA to generate construct 2011. The Eco RI/Eco RI fragment of construct ID #2011 was subcloned into the Eco RI site of pEE12.1, resulting in construct ID #2053, which contained DNA encoding an albumin fusion protein containing a leader sequence and The mature form of IFNb, followed by the mature HSA protein.

Two oligonucleotides suitable for PCR amplification of polynucleotides encoding full-length IFNb were synthesized, IFNb-1 and IFNb-2:

IFNb-1: 5′-GCGC GGATCC GAATTACCAACAAGTGT

CTCCTCCAAATTGCTCTCCTGTTGTGCTTTCCCACTACAG

CTCTTTCCATGAGCTACAACTTGCTTGG-3' (SEQ ID NO:

107)

IFNb-2: 5′-GCGCGC ATCGAT GAGCAACCTCACTCTTGTGTGCATCG

TTTCGGAGGTAACCTGT-3' (SEQ ID NO: 108)

The IFNb-1 primer incorporates a Bam HI cloning site (shown underlined), an Eco RI cloning site, and a Kozak sequence (shown in italics), followed by 80 nucleotides encoding the first 27 amino acids of the full-length form of IFNb. In IFNb-2, the Cla I site (shown underlined) and the following DNA are the reverse complements of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1), while the last 18 nuclear The nucleotides are the reverse complement of DNA encoding the last 6 amino acid residues of IFNb (see Example 2). PCR amplicons were generated with these primers, purified, digested with Bam HI and Cla I restriction enzymes, and cloned into the Bam HI and Cla I sites of the pC4:HSA vector. After sequence confirmation, the Eco RI fragment containing the IFNb-albumin fusion protein expression cassette was subcloned into Eco RI digested pEE12.1.

In addition, analysis of the N-terminus of expressed albumin fusion proteins by amino acid sequencing confirmed the presence of the expected IFNb sequence (see below).

The IFNb-albumin fusion protein of the invention preferably comprises the mature form of HSA, Asp-25 to Leu-609, fused to the N- or C-terminus of the mature form of IFNb, Met-22 to Asn-187. In one embodiment of the invention, the IFNb-albumin fusion protein of the invention further comprises a signal sequence for directing the nascent fusion polypeptide in the secretory pathway of the host for expression. In another preferred embodiment, the signal peptide encoded by the signal sequence is cleaved and the mature IFNb-albumin fusion protein is directly secreted into the culture medium. The IFNb-albumin fusion protein of the present invention may comprise a heterologous signal sequence, including but not limited to MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants including but not limited to A chimeric HSA/MAF leader sequence, or other heterologous signal sequence known in the art. In a preferred embodiment, the IFNb-albumin fusion protein of the invention comprises native IFNb. In another preferred embodiment, the IFNb-albumin fusion protein of the invention further comprises an N-terminal methionine residue. The invention also encompasses polynucleotides encoding these polypeptides, including fragments and/or variants.

Expression and purification of construct ID 2053

Expression in murine myeloma NSO cell line

Construct ID #2053, pEE12.1:IFNb-HSA was electroporated into NSO cells by methods known in the art (see Example 6).

Purification from NSO cell supernatant

Purification of IFNb-HSA from NSO cell supernatants may include Q-Sepharose anion exchange chromatography at pH 7.4 with a gradient of NaCl from 0 to 1 M in 20 mM Tris-HCl, followed by 5 to 40 mM lemon at pH 6.5. Poros PI 50 anion exchange chromatography with a sodium nitrate gradient and 6DV diafiltration into 10 mM citrate pH 6.5 and 140 mM NaCl as the final buffer composition. N-terminal sequencing should yield the sequence MSYNLL, which is the amino terminus of the mature form of IFNb. The protein has a molecular weight of approximately 88.5 kDa.

For larger scale purifications, for example, 50 L of NSO cell supernatant can be concentrated to approximately 8-10 L. The concentrated sample was then passed through a Q-Sepharose anion exchange column (10 x 19 cm, 1.5 L), pH 7.5, eluting with a profile consisting of 50 mM NaOAc pH 6.0 and 150 mM NaCl. The eluted samples can then be inactivated with 0.75% Triton-X 100 for 60 minutes at room temperature. SDR-reverse phase chromatography (10 cm x 10 cm, 0.8 L) at pH 6.0 using 50 mM NaOAc and 150 mM NaCl can then be employed, or the sample can be passed through an SP-Sepharose column at pH 4.8 using 50 mM NaOAc pH 6.0 and 150 mM Distribution elution of NaCl. Subsequent filtration through DV 50 to remove any viral content. This can be followed by Phenyl-650M chromatography at pH 6.0 (20 cm x 12 cm, 3.8 L), eluting with a profile consisting of 350 mM (NH4) _2SO4 and 50 mM NaOAc or 50 mM NaOAc pH 6.0. Diafiltration of 6-8DV can be buffer exchanged to 10mM _Na2HPO4 + _58mM Sucrose + 120mM NaCl pH7.2 or 10mM Citrate pH6.5 and 140mMNaCl or 25mM Na2HPO4, _100mM NaCl pH7.2 Expected final Prepare buffer.

IFNb activity can be determined using the in vitro ISRE-SEAP assay

All type I interferon proteins signal through a common receptor complex and a similar Jak/STAT signaling pathway, the latter culminating in the activation of interferon "IFN" responsive genes through the interferon sequence response element "ISRE". A convenient assay for type I IFN activity is a promoter-reporter based assay system containing multiple copies of an ISRE element fused to a downstream reporter gene. Generates a stable HEK293 cell line containing a stably integrated copy of the ISRE-SEAP reporter gene that is extremely sensitive to type I IFN and exhibits linearity over 5 log concentrations.

Screening method for stable clones of IFNb-HSA NSO

Construct 2053 was electroporated into NSO cells as described in Example 6. NSO cells transfected with construct ID #2053 were screened for activity by testing conditioned growth media in the ISRE-SEAP assay. ISRE-SEAP/293F reporter cells were dispensed into poly-D-lysine-coated 96-well dishes at 3×10 ⁴ cells/well one day before treatment. Reporter cells were treated with various dilutions (including but not limited to 1:500 and 1:5000) of conditioned supernatants or purified preparations of IFNb-albumin fusion protein encoded by construct ID 2053 or rhIFNb as a control. The reporter cells were then incubated for 24 hours before 40 [mu]l was removed for use in the SEAP reporter gene chemiluminescence assay (Roche Cat. No. 1779842). Recombinant human interferon beta, "rhIFNb" (Biogen), was used as a positive control.

result

A purified preparation of NSO-expressed IFNb-HSA had a greater EC50 of 9.3 x 10 ^-9 g/ml than rhIFNb (Biogen) with an EC50 of 1.8 x 10 ^-10 g/ml (see Example 4).

In vivo induction of OAS by interferon

method

The OAS enzyme, 2'-5'-oligoadenylate synthase, is transcriptionally activated by interferon in response to antiviral infection. The effect of the interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of two OAS mRNAs, p41 and p69. 0.5 ml volumes can be obtained from 4 animals per group at 7 different time points per animal, Day 0, Day 1, Day 2, Day 4, Day 8, Day 10, and Day 14 of whole blood. Different groups may include injection of vehicle control, 30 μg/kg and/or 300 μg/kg IFN-albumin fusion protein intravenously and/or subcutaneously on day 1, and 40 μg/kg subcutaneously on days 1, 3 and 5 Interferon α (Schering-Plough) was used as a positive control. The levels of p41 and p69 mRNA transcripts can be determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS mRNA levels can be quantified relative to the 18S ribosomal RNA endogenous control.

In vivo induction of OAS by an interferon beta-albumin fusion encoded by construct ID 2053

method

The activity of the HSA-IFNb fusion protein encoded by construct 2053 can be determined by an in vivo OAS assay, as previously described above under the subheading "In vivo induction of OAS by interferon".

Example 77: Indications for IFNb-albumin fusion protein

IFN beta-albumin fusion proteins (including but not limited to those encoded by construct 2053) are useful in the treatment, prevention, amelioration and/or detection of multiple sclerosis. Other indications include, but are not limited to, viral infections, including severe acute respiratory syndrome (SARS) and other coronavirus infections; filoviruses, including but not limited to Ebola virus and Marburg virus; arenaviruses, including but not limited to Pichende virus, Lassa virus, Junin virus, Machupo virus, Guanarito virus; and lymphocytic choriomeningitis virus (LCMV); bunya virus, including but not limited to Ponta Toru virus, Crimea - Congo hemorrhagic fever virus, sandfly fever virus, Rift Valley fever virus, La Crosse virus, and Hantaan virus; flaviviruses, including but not limited to yellow fever, banzie virus, West Nile virus , dengue virus, Japanese encephalitis virus, tick-borne encephalitis, Omsk hemorrhagic fever, and Kosanur forest disease virus; togaviruses, including but not limited to Venezuelan, Eastern, and Western equine encephalitis viruses, Ross River Viruses, and rubella viruses; orthopoxviruses, including but not limited to vaccinia, vaccinia, smallpox, and monkeypox; herpesviruses; influenza A/B; respiratory syncytial virus (RAV); parainfluenza; measles; rhinoviruses; adenoviruses ; Semliki Forest virus; viral hemorrhagic fever; baculoviruses; paramyxoviruses, including but not limited to Nipah virus and Hendra virus; Other viral factors (i.e. A, B, and C class factors; see, e.g., Moran, Emerg. Med. Clin. North. Am. 20(2): 311-30, 2002 and Darling et al., Emerg. Med. Clin. North Am. 20(2):273-309, 2002).

Example 78: Construct ID 2249, Production of IFNa2-HSA

Construct ID 2249, pSAC35:IFNa2.HSA contained DNA encoding an IFNa2-albumin fusion protein with a chimeric HSA leader sequence followed by the mature form of the IFNa2 protein, C1-E165, in the Saccharomyces cerevisiae expression vector pSAC35, Fused to the amino terminus of the mature form of HSA.

Cloning of IFNa2 cDNA

The polynucleotide encoding IFNa2 was PCR amplified using the primers IFNa2-1 and IFNa2-2 described below. the

The PCR amplicon was cut with Sal I/Cla I and ligated into pScCHSA cut with Xho I/Cla I. Construct ID #2249 encodes an albumin fusion protein containing a chimeric HSA leader sequence, the mature form of IFNa2, followed by the mature HSA protein.

Two oligonucleotides, IFNa2-1 and IFNa2-2, were synthesized suitable for PCR amplification of polynucleotides encoding the mature form of IFNa2:

IFNa2-1: 5′-CGCGCGC GTCGAC AAAAGATGTGATCTGCCTCAAACC

CACA-3' (SEQ ID NO: 109)

IFNa2-2: 5′-GCGCGC ATCGAT GAGCAACCTCACTCTTGTGTGCATCT

TCCTTACTTCTTAAACTTTCT-3' (SEQ ID NO: 110)

The IFNa2-1 primer incorporates the Sal I cloning site (shown underlined), the nucleotides encoding the last three amino acid residues of the chimeric HSA leader sequence, and the 22 cores encoding the first seven amino acid residues of the mature form of IFNa2 Nucleotides (shown in bold). In IFNa2-2, the ClaI site (shown in underline) and the following DNA is the reverse complementary sequence of the DNA encoding the first 10 amino acids of the mature HSA protein, while the last 22 nucleotides (shown in bold) are Reverse complement of DNA encoding the last 7 amino acid residues of IFNa2 (see Example 2). These primers were used to generate a PCR amplicon of IFNa2-HSA, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pScCHSA vector. After sequence confirmation, the expression cassette encoding this IFNa2-albumin fusion protein was subcloned into Not I digested pSAC35.

Furthermore, analysis of the N-terminus of expressed albumin fusion proteins by amino acid sequencing could confirm the presence of the expected IFNa2 sequence (see below).

Other IFNa2-albumin fusion proteins employing different leader sequences were constructed by methods known in the art (see Example 2). Examples of various leader sequences include, but are not limited to, invertase "INV" (constructs 2343 and 2410) and mating alpha factor "MAF" (construct 2366). These IFNa2-albumin fusion proteins can be subcloned into mammalian expression vectors such as pC4 (construct 2382) and pEE12.1 as previously described (see Example 5). An IFNa2-albumin fusion protein with the therapeutic moiety fused to the C-terminus of HSA can also be constructed (construct 2381).

The IFNa2-albumin fusion protein of the present invention preferably comprises the mature form of HSA, Asp-25 to Leu-609, fused to the N- or C-terminus of the mature form of IFNa2, Cys-1 to Glu-165. In one embodiment of the invention, the IFNa2-albumin fusion protein of the invention further comprises a signal sequence directing the initial fusion polypeptide in the secretory pathway of the host for expression. In another preferred embodiment, the signal peptide encoded by the signal sequence is cleaved and the mature IFNa2-albumin fusion protein is directly secreted into the culture medium. The IFNa2-albumin fusion protein of the present invention may comprise a heterologous signal sequence, including but not limited to MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants including but not limited to A chimeric HSA/MAF leader sequence, or other heterologous signal sequence known in the art. In a preferred embodiment, the IFNa2-albumin fusion protein of the invention comprises native IFNa2. In another preferred embodiment, the IFNa2-albumin fusion protein of the invention further comprises an N-terminal methionine residue. The invention also encompasses polynucleotides encoding these polypeptides, including fragments and/or variants.

Expression and purification of construct ID 2249

Expression in Saccharomyces cerevisiae

Construct 2249 was transformed into S. cerevisiae strain BXP10 by methods known in the art (see Example 3). Cells in the stationary phase can be harvested after 72 hours of growth. The supernatant was collected by centrifuging the cells at 3000g for 10 minutes. Expression levels were examined using anti-HSA serum (Kent Laboratories) or as primary antibody by immunoblotting. An IFNa2-albumin fusion protein with a molecular weight of approximately 88.5 kDa is available.

Purification from Saccharomyces cerevisiae cell supernatant

Cell supernatants containing the IFNa2-albumin fusion protein expressed in S. cerevisiae cells by construct ID #2249 can be performed on a small scale using a Dyax peptide affinity column (see Example 4), or on a large scale by following 5 steps Purification: Diafiltration, Anion Exchange Chromatography with DEAE-Sepharose Fast Flow, Hydrophobic Interaction Chromatography (HIC) with Butyl 650S, Cation Exchange with SP-Sepharose Fast Flow or Blue-Sepharose Chromatography, and high-efficiency chromatography with a Q-Sepharose high-efficiency column (see Example 4). IFNa2-albumin fusion protein can be washed from DEAE-Sepharose Fast Flow column with 100-250mM NaCl, from SP-Sepharose Fast Flow column with 150-250mM NaCl, and from Q-Sepharose high-efficiency column at 5-7.5mS/cm take off. The N-terminal sequence should yield the sequence CDLPQ (SEQ ID NO: 98) identical to the mature form of IFNa2.

IFNa2 activity can be determined using the in vitro ISRE-SEAP assay

method

The IFNa2-albumin fusion protein encoded by construct ID #2249 can be tested for activity in the ISRE-SEAP assay as previously described in Example 76. Briefly, conditioned yeast supernatants were tested at a 1:1000 dilution for their ability to direct ISRE signaling on the ISRE-SEAP/293F reporter cell line. ISRE-SEAP/293F reporter cells were dispensed into poly-D-lysine-coated 96-well dishes at 3×10 ⁴ cells/well one day before treatment. The reporter cells were then incubated for 18 to 24 hours before 40 μl was withdrawn for use in the SEAP reporter gene chemiluminescence assay (Roche Cat. No. 1779842). Recombinant human interferon beta, "rhIFNb" (Biogen), was used as a positive.

result

Purified preparations of IFNa2-HSA exhibited relative relative concentration in the ISRE-SEAP assay over the concentration range 10 ⁻¹ to 10 ¹ ng/ml (see Figure 5) or 10 ⁻¹⁰ to 10 ⁻⁸ ng/ml (see Figure 6). linear increase.

In vivo induction of OAS by an interferon alpha fusion encoded by construct ID 2249

method

The OAS enzyme, 2'-5'-oligoadenylate synthase, is transcriptionally activated by interferon in response to antiviral infection. The effect of the interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of two OAS mRNAs, p41 and p69. A volume of 0.5 ml was obtained from 4 animals per group at 7 different time points per animal, Day 0, Day 1, Day 2, Day 4, Day 8, Day 10, and Day 14. Whole blood. The different groups included vehicle control, iv injection of 30 μg/kg HSA-IFN on day 1, 30 μg/kg HSA-IFN subcutaneously on day 1, 300 μg/kg HSA-IFN subcutaneously on day 1, and day 1, 40 μg/kg interferon α (Schering-Plough) was subcutaneously injected on days 3 and 5 as a positive control. The levels of p41 and p69 mRNA transcripts were determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS mRNA levels were quantified relative to the 18S ribosomal RNA endogenous control. Similar experiments were performed with the albumin fusion encoded by construct 2249.

result

Significant increases in mRNA transcript levels of both OAS, p41 and p69, were observed in monkeys treated with HSA-interferon, in contrast to monkeys treated with IFNa (see Figure 7 for p41 data). The effect lasts for nearly 10 days.

Example 79: Indications for IFNa2-albumin fusion protein

IFNα-albumin fusion proteins (including but not limited to those encoded by constructs 2249, 2343, 2410, 2366, 2382 and 2381) are useful for treating, preventing, ameliorating and/or detecting multiple sclerosis. Other indications include but are not limited to viral infections, including severe acute respiratory syndrome (SARS) and other coronavirus infections; filoviruses, including but not limited to Ebola virus and Marburg virus ); Arenaviruses, including but not limited to Pichende virus, Lassa virus, Junin virus, Machupo virus, Guanarito virus and Lymphocytic choriomeningitis virus (LCMV); Bunyaviruses, including but not limited to Puntatoro virus, Crimean-Congo hemorrhagic fever virus hemorrhagic fever virus), sandfly fever virus, Rift Valley fever virus, La Crosse virus, and hantavirus; Flaviviruses, including but Not limited to yellow fever, Banzi vires, West Nile virus, dengue virus, Japanese encephalitis virus, tick-borne encephalitis, Omsk Hemorrhagic fever, and Kosanu Kyasanur Forest Disease virus; Togaviruses, including but not limited to Venezuelan, Oriental, and Western equine encephalitis viruses, Ross River virus, and rubella virus; Orthopoxviruses, Including but not limited to vaccinia, vaccinia, smallpox, and monkeypox; herpes virus; influenza A/B; respiratory syncytial virus (RAV); parainfluenza; measles; rhinovirus; adenovirus; Semliki Forest virus virus); viral hemorrhagic fevers; baculoviruses; paramyxoviruses, including but not limited to Nipah virus and Hendra virus; and Other viral agents (i.e., class A, B, and C agents; see, e.g., Moran, Emerg. Med. Clin. North. Am. 20(2): 311-30, 2002 and Darling et al., Emerg. Med. Clin. North Am. 20(2):273-309, 2002).

Preferably, the IFNa-albumin fusion protein or the IFN hybrid fusion protein is administered in combination with a CCR5 antagonist, and then combined with ribavirin (ribavirin), IL-2, IL-12, pentafuside (pentafuside) At least one anti-HIV drug therapy alone or in combination, such as HAART, for the preparation of HIV-1 infection, HCV, or HIV-1 and HCV co-infection in treatment-naïve and treatment-experienced adult and pediatric patients drug.

Example 80: Construct #3691, Production of BNP-HSA

Construct ID#3691, pC4:SPCON.BNP1-32/HSA contains DNA encoding a BNP-albumin fusion protein in the mammalian expression vector pC4, which has a consensus leader sequence, secrecon, followed by a fusion to the amino terminus of the mature form of HSA processed, active BNP peptide (amino acids 1-32).

Cloning of BNP cDNA for construct 3691

A polynucleotide encoding BNP was PCR amplified using primers BNP-1 and BNP-2 described below, cut with Bam HI/Cla I, and ligated into Bam HI/Cla I cut pC4:HSA to generate construct ID# 3691. Construct ID #3691 encodes an albumin fusion protein containing a consensus leader sequence (SEQ ID NO: 111) and a processed active form of BNP, followed by the mature HSA protein (see SEQ ID for construct 3691 in Table 2 NO: 321).

Synthesis of two nucleotides suitable for PCR amplification of polynucleotides encoding active, processed forms of BNP, BNP-1 and BNP-2:

BNP-1: 5′-GAGCGC GGATCC AAGCTTCCGCCATC

AGCCCCAAGCTGGTGCAAGG  -3′

AGCCCCAAGCTGGTGCAAGG-3′

(SEQ ID NO: 463)

BNP-2: 5′-AGTCCC ATCGAT GAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3' (SEQ ID NO: 464)

BNP-1 incorporates the Bam HI cloning site (underlined), the polynucleotide encoding the consensus leader sequence (SEQ ID NO: 111) (italics), and the polynucleotide encoding the first 7 amino acid sequences of BNP (bold) . In BNP-2, the underlined sequence is the Cla I site, and the polynucleotide following it contains the reverse complement of DNA encoding the last 6 amino acids of BNP (in bold) and the first 10 amino acids of the mature HSA protein. The BNP protein was PCR amplified using these two primers. Annealing and extension temperatures and times must be determined empirically for each specific primer and template pair.

Purify the PCR product (e.g. using the Wizard PCR Preps DNA Purification System; Promega) and digest with Bam HI and Cla I. After further purification of the Bam HI-Cla I fragment by gel electrophoresis, the product was cloned into Bam HI/Cla I digested pC4:HSA, resulting in construct ID #3691. Verify the sequence of the expression construct.

Expression and purification of construct ID 3691

Expression in 293F cells

Construct ID #3691, pC4:SPCON.BNP1-32/HSA was transfected into 293F cells by methods known in the art (see Example 6).

Purified from 293F cell supernatant

2 liters of supernatant were collected 3 days after transfection. Recombinant protein was captured with 5ml Blue Sepharose CL-6B column (Amersham Biosciences, Piscataway, NJ, USA) and eluted with 2M NaCl. Material was bound to a HiPrep 16/10 Phenyl FF (high sub) column and eluted with 20 mM MES pH 6.7. BNP-HSA was further purified by hydroxyapatite column chromatography at pH 6.8 with a gradient of sodium phosphate buffer (0-20 mS/cm in 200 ml). The final product was converted into PBS pH 7.2 by HiPrep 26/10 desalting column (Amersham Biosciences).

The activity of BNP-HSA can be determined using an in vitro NPR-A/cGMP assay

Natriuretic peptide receptor-A (NPR-A) is the signaling receptor for BNP and is thus responsible for most of the biological effects of BNP. The biological activity of BNP is mediated by the NPR-A guanylate cyclase domain that converts GTP to cGMP upon activation. A convenient assay for BNP activity is to measure BNP stimulation in the 293F cell line stably overexpressing NPR-A. cGMP production in cells after exposure to BNP can be measured by cGMP ELISA.

Screening method of NPR-A 293F stable clone

The open reading frame of human NPR-A was constructed into the pcDNA3.1 expression vector (Invitrogen). 293F cells were stably transfected with plasmid DNA by Lipofectamine method and selected with 0.8 μg/ml G418. 293F/NPR-A stable clones were screened for the best response to recombinant BNP.

Measurement of cGMP activation

Activation of cGMP by BNP was performed in 293F/NPR-A cells and measured using the CatchPoint cGMP Fluorometric Assay Kit (Molecular Devices, Sunnyvale, CA, USA). Briefly, 293F/NPR-A cells cultured at 50,000 cells/well in a 96-well plate were treated with 80 μl of prestimulation buffer (containing 10 mM glucose pH 7.4, 15 nM sodium bicarbonate, and 0.75 mM 3-isobutyl- 1-methylxanthine in Krebs-Ringe bicarbonate buffer) for washing. 40 μl of BNP-HSA or recombinant BNP in pre-stimulation buffer was added to the cells for 10 minutes at 37°C. Cells were lysed by shaking with 40 μl of lysis buffer for 10 min. The amount of cGMP in the lysate was determined following the manufacturer's instructions.

result

The dose-response relationship of BNP-HSA and recombinant BNP was determined (see Figure 8). The maximal activities of construct ID#3691 and recombinant BNP were similar (1.63±0.016 and 1.80±0.016 pm, respectively), with EC50 values of 28.4±1.2 and 0.46±1.1 nM, respectively.

BNP-HSA lowers blood pressure in vivo

method

BNP lowers blood pressure both through direct vasodilation and through inhibition of the renin/angiotensin/endothelin/aldosterone system. The ability of BNP-HSA to lower arterial blood pressure was tested in 3-month-old male spontaneously hypertensive rats purchased from Taconic (Germantown, NY, USA). Spontaneously hypertensive rats are genetically hypertensive, with onset of hypertension after 3 months of age. BNP-HSA or recombinant BNP reconstituted in 0.3cc PBS was administered to each rat. The drug was delivered by tail vein injection. Systolic and diastolic blood pressures were recorded by the cuff-tail method using an XBP-1000 system (Kent Scientific, Torrington, CT, USA). For each blood pressure data point, 4-5 consecutive readings were taken and averaged. Calculate mean arterial pressure (MAP) using 1/3 systolic blood pressure + 2/3 diastolic blood pressure. For dose-response determinations, blood pressure was measured 20 hours after administration of pC4:SPCON.BNP1-32/HSA at doses of 0.5, 2, 6 and 18 nmol/kg.

result

Typical systolic blood pressure in spontaneously hypertensive rats was 180-200 mmHg before administration. A single bolus of 6 nmol/kg BNP-HSA delivered via tail vein injection lowered both systolic and diastolic blood pressure, achieving a mean arterial pressure (MAP) reduction of more than 30 mmHg. The reduced blood pressure was stable and persisted for 1 day, and then gradually returned to baseline over several days (see Figure 9). In contrast, a single 6 nmol/kg bolus of recombinant BNP produced only a very transient decrease in MAP of about 15 mmHg due to its transient clearance.

In addition, the dose-response 20 hours after bolus injection of BNP-HSA was determined in 4 spontaneously hypertensive rats. 0.5nmol/kg BNP-HSA has an average MAP reduction of 7mmHg, while 6nmol/kg BNP-HSA has an average MAP reduction of 30mmHg, and the high-dose BNP-HSA of 18nmol/kg can only slightly lower blood pressure than 6nmol/kg.

Induction of plasma cGMP by BNP-HSA in vivo

method

Intracellular cGMP activation of BNP results in its release from cells into circulation. Plasma cGMP levels are associated with BNP-induced cardiovascular and renal physiology. Plasma cGMP has been used as a biomarker of BNP action in vivo. To test induction of plasma cGMP by BNP-HSA in vivo, 11-12 week old male C57/BL6 mice were delivered recombinant BNP or BNP-HSA at a dose of 6 nmol/kg via a single bolus injection via the tail vein. Plasma was prepared from tail blood at time points of 5, 10, 20, 40 and 80 minutes in the recombinant BNP dosed group and an additional 640, 1440, 2880 and 5760 minutes in the BNP-HSA group. Plasma samples from mice treated with PBS were collected at time point zero as vehicle controls. Determine cGMP levels using the CatchPoint cGMP Fluorometric Assay Kit following the manufacturer's instructions.

result

Following a single intravenous bolus of 6 nmol/kg recombinant BNP or BNP-HSA, peak plasma cGMP levels over baseline were increased 3.9-fold or 5.6-fold, respectively (see Figure 10). Furthermore, the one-phase exponential decay half-life of cGMP after treatment with recombinant BNP was 16 minutes (10 to 42 minutes, 95% CI), while the one-phase exponential decay half-life of cGMP after administration of BNP-HSA The exponential decay half-life was 1538 minutes (1017 to 3153 minutes, 95% CI).

In vivo pharmacokinetic analysis of BNP-albumin fusion encoded by construct ID 3691

method

11-12 week old male C57/BL6 mice (obtained from Ace Animals, Boyertown, PA, USA) weighed 25.1 ± 0.12 g at the time of the study. All animals were dosed in a volume of 10 ml/kg body weight. Pre-dosed animals were injected with PBS. Recombinant BNP was injected intraperitoneally into the tail or subcutaneously into the mid-scapular region.

Pharmacokinetic analyzes were performed on the following groups:

Table 8

Blood samples were collected from the inferior vena cava, placed in EDTA-coated microcontainers, and stored on ice. The samples were centrifuged at 14,000 rpm (16,000 xg) for 10 minutes at room temperature in a microcentrifuge. Plasma was transferred to cluster tubes and stored at -80°C.

BNP-HSA concentrations in plasma samples were determined using the BNP EIA kit (Phoenix Pharmaceutical, Belmont, CA, USA). Standard curves were generated on the same plates as the test samples at the same time. The detection limit of recombinant BNP was 0.11 ng/ml. This assay detects recombinant BNP and does not cross-react with mouse BNP.

Analysis was performed by non-compartmental methods (WinNonlin; version 4.1; Pharsight Corp., Mountain View, CA, USA). The mean plasma concentration at each time was used in the analysis. AUC _0-t was calculated using the linear ascending/log descending trapezoidal method. Extrapolation to infinite AUC _0-∞ was performed by dividing the last observed concentration by the terminal elimination rate constant. The data in these analyzes were uniformly weighted.

result

Mean baseline concentrations of BNP-HSA in plasma detected in predose samples were approximately 0.081-0.095 μg/ml. Following a single intravenous or subcutaneous injection, BNP-HSA has a terminal elimination half-life of 11.2 (intravenous delivery) or 19.3 hours (subcutaneous delivery) compared with 3.1 minutes for recombinant BNP in mice. Noncompartmental analysis of BNP-HSA revealed that BNP-HSA has the following characteristics:

Table 9

Five points in the final phase of the intravenous curve and 4 points in the final phase of the subcutaneous curve were chosen for the calculation of the final half-life. The AUC thus obtained for this final phase was approximately 10% of the total AUC for the intravenous and subcutaneous curves, respectively. This compares to only 2% and 4% of the total AUC for the intravenous and subcutaneous curves, respectively, when the last 3 points were selected for the calculation of the final half-life.

Example 81: Construct ID#3618, Production of BNP(2X)-HSA

Construct ID #3618, pC4: SPCON.BNP1-32(2x)/HSA contains DNA encoding a BNP-albumin fusion protein in the mammalian expression vector pC4, which has a consensus leader sequence, secrecon, followed by a fusion in the HSA mature Forms two processed, active BNP peptides amino-terminal (amino acids 1-32).

Cloning of BNP cDNA for construct 3618

A polynucleotide encoding a dual BNP module was first PCR amplified from the processed active form of BNP (amino acids 1-32) using the four primers BNP-1, BNP-2, BNP-3 and BNP-4 described below to generate Two fragments A and B. After amplification, the two purified fragments (A and B) were mixed in equimolar amounts and used as PCR templates to amplify using primers BNP-5 and BNP-6 described below. Then use Bam

The BNP (2x) insert was digested with HI and ClaI and ligated into the pC4:HSA vector predigested with BamHI and ClaI, resulting in construct ID #3618. Construct ID #3618 encodes an albumin fusion protein containing the consensus leader sequence, secrecon, (SEQ ID NO: 111), and two tandem copies of the processed active form BNP, followed by the mature HSA protein (see Table 2 SEQ ID NO: 483 of construct 3618).

First synthesize four oligonucleotides suitable for PCR amplification of polynucleotides encoding two fragments of the BNP protein:

BNP-15′ AGCCCCAAGATGGTGCAAGGGTCTGGCTGCTTTGGGAG

GAAGATGGACCGGATCAGCTCCTCCAGTGGCTGGGCTGC

AAAGTGCTGAGGCGGCAT-3' (SEQ ID NO: 486)

BNP-25′-CCTTGCACCATCTTGGGGCTATGCCGCCTCAGCACTTT

GC-3' (SEQ ID NO: 487)

BNP-35′-GCAAAGTGCTGAGGCGGCATAGCCCCAAGATGGTGCA

AGG-3' (SEQ ID NO: 488)

BNP-45′-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3' (SEQ ID NO: 489)

Two BNP protein fragments (A and B, respectively) were PCR amplified using primer sets BNP-I/BNP-2 and BNP-3/BNP-4. Annealing and extension temperatures and times must be determined empirically for each specific primer and template pair. Fragments A and B were purified (e.g. using the Wizard PCR Preps DNA Purification System; Promega Corp), mixed in equimolar amounts, and used as templates for PCR amplification using the other two oligonucleotides BNP-5 and BNP- 6. PCR amplification:

BNP-55′-GAGCGC GGATCC AAGCTTCCGCCATC

AGCCCCAAGCTGGTGCAAGG-3′(SEQ ID

AGCCCCAAGCTGGTGCAAGG-3' (SEQ ID

NO: 463)

BNP-65′-AGTCCC ATCGAT GAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3' (SEQ ID NO: 464)

BNP-5 incorporates the Bam HI cloning site (underlined), the polynucleotide encoding the consensus leader sequence (SEQ ID NO: 111) (italics), and the polynucleotide encoding the first 7 amino acid sequences of BNP (bold ). In BNP-6, the underlined sequence is the Cla I site, and the polynucleotide following it contains the reverse complement of DNA encoding the last 6 amino acids of BNP (in bold) and the first 10 amino acids of the mature HSA protein. Using these two primers, two tandem copies of the consensus leader sequence and the active BNP peptide were PCR amplified. Annealing and extension temperatures and times must be determined empirically for each specific primer and template pair.

Purify the PCR product (e.g. using the Wizard PCR Preps DNA Purification System; Promega) and digest with Bam HI and Cla I. After further purification of the BamHI-ClaI fragment by gel electrophoresis, the product was cloned into BamHI/ClaI digested pC4:HSA, resulting in construct ID #3618. Verify the sequence of the expression construct.

Expression and purification of construct ID 3618

Expression in 293F cells

Construct ID #3618, pC4:SPCON.BNP1-32(2x)/HSA was transfected into 293F cells by methods known in the art (see Example 6).

Purified from 293F cell supernatant

pC4:SPCON.BNP1-32(2x)/HSA encoded by construct ID #3618 was purified as previously described in Example 80 above under the subheading "Purification from 203F Cell Supernatant".

The activity of BNP(2X)-HSA can be determined using an in vitro NPR-A/cGMP assay

The activity of BNP(2X)-HSA encoded by construct ID #3618 can be determined as previously described in Example 80 under the subheadings "The activity of BNP-HSA can be determined using an in vitro NPR-A/cGMP assay" and "NPR-A 293F Screening method for stable clones" was determined in vitro by the NPR-A/cGMP assay.

result

The dose-response relationship of BNP(2X)-HSA and recombinant BNP was determined (see Figure 8). The maximal activities of BNP(2X)-HSA and recombinant BNP encoded by construct ID#3618 were similar (1.68±0.021 and 1.80±0.016 pm, respectively), with EC50 values of 9.8±1.1 and 0.46±1.1 nM, respectively.

The entire disclosure of each document (including patents, patent applications, patent publications, journal articles, abstracts, experimental guidelines, books, or other publications) cited in this application and available through GenBank, GeneSeq or CAS mentioned in this application The information obtained from the Registry-specific identifiers is incorporated herein by reference in its entirety.

In addition, the specification and sequence listing of each of the following U.S. applications are hereby incorporated by reference in their entirety: U.S. Application No. 60/542,274, filed February 4, 2005; U.S. Application No. 60/549,901, filed March 5, 2004; US Application No. 60/556,906, filed March 29, 2004; and US Application No. 60/636,603, filed December 17, 2004.

Instructions for Preserving Biological Materials

A. The following description refers to the deposited biological material mentioned in Table 3 on page 44 of the specification.

B. Identification of deposits

Name of depository unit: American Type Culture Collection

Address of depository unit: 10801 University Boulevard

Manassas, Virginia 20110-2209

United States of America

Canada: The applicant requests that, until a Canadian patent is granted on the basis of the application or the application is rejected, or abandoned without reinstatement, or withdrawn, the Commissioner of Patents can only authorize the provision of the patents mentioned in this application to an independent expert appointed by the Commissioner. Samples of biological material are deposited, therefore the applicant must notify the International Bureau by a written statement before the technical preparations for publication of the international application are completed.

Norway: The applicant hereby requests that until the application has been made available to the public (through the Norwegian Patent Office) or, in the absence of publication, has finally been decided by the Norwegian Patent Office, samples should only be issued to experts in the field. Pursuant to Sections 22 and 33(3) of the Norwegian Patent Act, a claim to this effect shall be made by the applicant to the Norwegian Patent Office no later than the date of publication to the public. Any request by a third party for samples should indicate the experts to be used if the applicant has made such a request. The expert can be anyone on the list of recognized experts drawn up by the Norwegian Patent Office or anyone approved by the applicant in the individual case.

Australia: The applicant hereby notifies that samples of micro-organisms shall be issued only to persons who are skilled practitioners having no interest in the invention before the grant of a patent, or before the termination, refusal or withdrawal of the application (Australian Patents Regulations Regulation 3.25(3)).

Finland: The applicant hereby requests that samples shall only be issued to this domain experts.

United Kingdom: The applicant requires that microbiological samples can only be provided to experts. The application must be submitted by the applicant to the International Bureau before the application is technically ready for international publication in order to be effective.

Denmark: The applicant requires that until the application has been published internationally (by the Danish Patent Office), or a final decision has been taken by the Danish Patent Office without publication to the public, biological samples can only be issued to experts in the field. Pursuant to Sections 22 and 33(3) of the Danish Patents Act, the requirement is not valid until it has been submitted by the applicant to the Danish Patent Office no later than the date of publication to the public. If the applicant has submitted such a request, any request by a third party to provide samples is subject to the use of experts. Said expert can be any expert on the list of experts recognized by the Danish Patent Office, or anyone recognized by the applicant on a case-by-case basis.

Sweden: The applicant requires that until the application has been published internationally (by the Swedish Patent Office), or a final decision has been taken by the Swedish Patent Office without publication to the public, biological samples can only be released to experts in the field. The request must be submitted to the International Bureau (preferably in form PCT/RO/134 in Annex Z to Volume I of the PCT Applicant's Guide) before the expiry of 16 months from the priority date to become effective. If the applicant has submitted such a request, any request by a third party for a sample is subject to the use of experts. Said expert can be any expert on the list of experts recognized by the Danish Patent Office, or anyone recognized by the applicant on a case-by-case basis.

The Netherlands: The applicant requests that, until the date of grant of the Dutch patent or until the date of the refusal or withdrawal or abandonment of the application, the micro-organisms may only be issued to specialists in accordance with patent law 31F(1). This requirement must be submitted to the Netherlands Industrial Property Office by the applicant before the date on which the application is made available to the public in accordance with Section 22C or Section 25 of the Dutch Patent Act, whichever comes first.

Claims (35)

1. the albumin fusion proteins be made up of two connect orientation and the GLP-1 merged in heredity (7-36 (A8G)) polypeptide shown in the SEQ ID NO:387 merged in heredity with the N-terminal of human serum albumin via C-terminal and merge in heredity via the C-terminal of the HSA/kex2 targeting sequencing modified shown in N-terminal with SEQ ID NO:112, wherein said human serum albumin extends the serum plasma half-life of not merging GLP-1 polypeptide, and wherein said fusion rotein has GLP-1 activity.

2. the albumin fusion proteins of claim 1, it is generated by the host cell comprising the construction ID3610 expressing described albumin fusion proteins.

3. the albumin fusion proteins of claim 1, it comprises SEQ ID NO:1.

4. the albumin fusion proteins of claim 2, it comprises SEQ ID NO:1.

5. the albumin fusion proteins of any one of claim 1-4, it is nonglycosylated.

6. the albumin fusion proteins of any one of claim 1-4, it expresses in yeast.

7. the albumin fusion proteins of claim 6, wherein said yeast is saccharomyces cerevisiae, Kluyveromyces lactis or Pichia pastoris.

8. the albumin fusion proteins of claim 6, wherein said yeast is glycosylation defect.

9. the albumin fusion proteins of claim 7, wherein said yeast is glycosylation defect.

10. the albumin fusion proteins of claim 6, wherein said yeast is glycosylation and Deficient In Extracellular Proteases.

11. the albumin fusion proteins of claim 7, wherein said yeast is glycosylation and Deficient In Extracellular Proteases.

The albumin fusion proteins of 12. any one of claim 1-4, it is by mammalian cell expression.

13. the albumin fusion proteins of claim 12, wherein said mammalian cell is Chinese hamster ovary celI, COS cell or NS0 cell.

The albumin fusion proteins of 14. claim 1, it is as shown in SEQ ID NO:319.

The albumin fusion proteins of 15. any one of claim 1-4, wherein said targeting sequencing is removed.

The albumin fusion proteins of 16. claim 5, wherein said targeting sequencing is removed.

The albumin fusion proteins of 17. claim 6, wherein said targeting sequencing is removed.

The albumin fusion proteins of 18. claim 7, wherein said targeting sequencing is removed.

The albumin fusion proteins of 19. claim 8, wherein said targeting sequencing is removed.

The albumin fusion proteins of 20. claim 9, wherein said targeting sequencing is removed.

The albumin fusion proteins of 21. claim 10, wherein said targeting sequencing is removed.

The albumin fusion proteins of 22. claim 11, wherein said targeting sequencing is removed.

The albumin fusion proteins of 23. claim 12, wherein said targeting sequencing is removed.

The albumin fusion proteins of 24. claim 13, wherein said targeting sequencing is removed.

The albumin fusion proteins of 25. claim 14, wherein said targeting sequencing is removed.

The polynucleotide of the albumin fusion proteins of 26. any one of coding claim 1-4.

27. carriers comprising the polynucleotide of claim 26.

28. host cells comprising the carrier of claim 27.

The host cell of 29. claim 28, it is yeast cells or mammalian cell.

The host cell of 30. claim 29, wherein said yeast is saccharomyces cerevisiae, Kluyveromyces lactis or Pichia pastoris.

The host cell of 31. claim 29, wherein said yeast is glycosylation defect.

The host cell of 32. claim 30, wherein said yeast is glycosylation defect.

The host cell of 33. any one of claim 29-32, wherein said yeast is glycosylation and Deficient In Extracellular Proteases.

34. the host cell of claim 29, wherein said mammalian cell is Chinese hamster ovary celI, COS cell or NS0 cell.

35. comprise the albumin fusion proteins of any one of claim 1-25 and the compositions of pharmaceutical acceptable carrier.

Images