HK1141303B - Immunoglobulin fusion proteins - Google Patents

Description

Immunoglobulin fusion proteins

Technical Field

The present invention relates to hybrid human Fc and immunoglobulin fusion proteins in which the hybrid human Fc is linked to a biologically active molecule. In particular, it relates to hybrid human Fc derived from a combination of human immunoglobulin g (IgG) subclasses or a combination of human IgD and IgG; and fusion proteins in which such Fc is coupled to a biologically active molecule via a covalent bond.

Background

Biologically active molecules can have great therapeutic advantages. However, they may have disadvantages as therapeutic agents because of their lower in vivo stability. They have a short circulating half-life or serum half-life because they are digested by various enzymes in vivo. Thus, it is always desirable to increase the circulating half-life of a biologically active molecule.

It is known that: increasing the size of the protein, its half-life can be increased by preventing removal of the protein via the kidney (Knauf et al, J.biol.chem.1988.263: 15064-15070). For example, it has been reported that protein stability can be increased by coupling an active protein to human albumin (Kinstler et al, pharm. Res.1995.12: 1883-1888). However, since the conjugation of active proteins to human albumin only slightly increases its residence time, the development of effective pharmaceutical preparations containing active proteins conjugated to human albumin is not an effective approach.

Other reported methods are to modulate glycosylation of proteins. Increasing glycosylation on and introduction of sialic acid into proteins can prevent degradation of proteins in the liver. However, an increase in glycosylation of a protein also results in a decrease in protein activity.

To stabilize the protein and prevent clearance through the kidney, the protein is linked to polyethylene glycol (PEG). Covalent attachment to PEG has been widely used to deliver drugs with extended half-lives (Delgado et al, 1992.9: 249-304). However, it has been reported that attachment of PEG to a cytokine or hormone results in a decrease in receptor binding affinity due to steric hindrance caused by the attachment.

Recently, fusion proteins prepared using immunoglobulin (Ig) have been studied and developed. Ig is the major component of blood. Human Ig (humanIg, hIg) includes different species, such as IgG, IgM, IgA, IgD and IgE (Roitt et al, "Immunology" 1989, Gowermedical publishing, London, U.K.; New York, N.Y.). Human IgGs can be further divided into various subtypes known as human IgG1(hIgG1), human IgG2(hIgG2), human IgG3(hIgG3), and human IgG4(hIgG 4).

Immunoglobulins consist of four polypeptide chains, two heavy chains and two light chains, which form tetramers via disulfide bonds. Each chain is composed of a variable region and a constant region. The constant region of the heavy chain is further divided into three or four regions (CH1, CH2, CH3 and CH4) based on the isotype (isotypes). Based on the Ig isotype, the Fc portion of the heavy chain constant region includes hinge, CH2, CH3, and/or CH4 domains.

Regarding serum half-life, IgG1, IgG2, and IgG4 have long half-lives of 21 days, while other immunoglobulins have relatively short half-lives of less than one week. Chimeric proteins fused to the Fc portion of IgG show increased stability and increased serum half-life (Capon et al, Nature1989.337: 525-531). The biologically active protein has been fused at the N-terminus of the CH1 region, the N-terminus of the Fc region, or the C-terminus of the IgGCH3 region.

In the beginning, IgG fusion proteins were generated using extracellular domains of cell surface receptors such as CD4(Capon et al, Nature 1989.337: 525-531), TNFR (Mohler et al, J.Immunoglogy1993.151: 1548-1561), CTLA4(Linsley et al, J.exp.Med.1991.173: 721-730), CD86(Morton et al, J.Immunoglogy1996.156: 1047-1054). Meanwhile, there are several cytokines and growth hormones that have been fused to IgG domains. However, unlike fusion with the extracellular domain of a cell surface receptor, fusion with soluble proteins to IgG results in a reduction in biological activity compared to non-fused cytokines or growth factors. Chimeric proteins exist as dimers, which result in steric hindrance due to interactions with their target molecule-like receptors, due to the presence of two active proteins in close proximity to each other. Therefore, this problem should be overcome to produce an efficient fusion protein.

Other limitations of Fc fusion technology are the presence of unwanted immune responses. The Fc domain of an immunoglobulin has both effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This effector function is generally achieved through the interaction of the Fc region of Ig with FcRs on effector cells or through complement binding. Thus, blocking of effector functions of Fc should be performed to reduce undesired reactions such as cell killing, cytokine release or inflammation.

Disclosure of the invention

Overall, there is a need for improved Fc fusion proteins with minimal loss of biological activity and minimal risk of undesired immune responses.

Technical scheme

The present invention provides hybrid Fc (hybrid Fc) derived from a combination of human IgG subclasses or a combination of human IgD and IgG. The hybrid Fc is effective to increase the serum half-life of the biologically active molecule when the hybrid Fc is linked to the biologically active molecule, and to increase the expression level of the polypeptide when the nucleotide encoding the Fc-polypeptide fusion protein is expressed.

The invention also provides hybrid Fc fusion polypeptides in which a hybrid Fc is linked to a biologically active molecule. Such fusion proteins are sometimes referred to as "bioactive molecule-Fc fusion proteins" or simply "fusion proteins". The fusion protein may have a linker between the Fc and the biologically active molecule. The Fc can be coupled at its N-terminus to the C-terminus of the bioactive molecule.

Fusion proteins can be produced by: constructing nucleotides encoding and capable of expressing the fusion protein; expressing it in a host cell; and harvesting the fusion protein. Alternatively, the fusion protein may be produced by expressing the Fc-encoding nucleotide and linking it to the biologically active molecule in a conventional manner.

A polypeptide according to an embodiment of the present invention may be represented by the following formula:

N′-(Z1)_p-Y-Z2-Z3-Z4-C′

wherein N 'is the N-terminus and C' is the C-terminus of the polypeptide;

z1 represents an amino acid sequence which is comprised in seq id no:11 or at least the C-terminal part of an amino acid residue at position 90 to 98 of seq id no:14 at least a portion of amino acid residues at positions 90-98;

y represents an amino acid sequence comprised in seq id no:11 or at least the C-terminal part of the amino acid residues in positions 99 to 113 of seq id no:14 at least a portion of amino acid residues 99 to 162;

z2 represents an amino acid sequence which is comprised in seq id no: 12 or at least the N-terminal part of the amino acid residues at positions 111 to 147 of seq id no:14 at least a portion of the amino acid residues at positions 163 to 199;

z3 represents an amino acid sequence which is comprised in seq id no: 118 of 11, SEQ ID NO: 114 of 12, SEQ ID NO: 165 of 24 and 270 or SEQ ID NO:13 at least the C-terminal portion of amino acid residues at positions 115 to 220;

z4 represents an amino acid sequence which is comprised in seq id no: 224 of 11 and 330, SEQ ID NO: 220 of 12 and 326, SEQ ID NO: 271 of 24 and 377 or SEQ ID NO:13 at least the N-terminal portion of the amino acid residue at position 221-327 and

p is an integer of 0 or 1,

wherein the total number of amino acid residues Z2 and Z3 is between 80 and 140, inclusive.

Z1 may be an amino acid sequence comprising a sequence derived from seq id no:11, 5 to 9 consecutive amino acid residues C-terminal to the amino acid residue at position 90-98 of seq id no: 5 to 9 consecutive amino acid residues on the C-terminal side of the amino acid residue at the 90 to 98 positions of 14. In some embodiments, Z1 may be the 5, 6, 7, 8, or 9C-terminal amino acid residues of the IgG1CH1 domain (SEQ ID NO: 11) or the IgDCH1 domain (SEQ ID NO: 14).

In another embodiment, Z1 is an amino acid sequence comprising seq id no:11 or seq id no:14, amino acid residues at positions 90-98. Z1 may be a peptide consisting of seq id no:11 or position 90-98 of seq id no:14, positions 5 to 9 of positions 90-98. Z1 may also be represented by seq id no:11 or seq id no:14 from 90 to 98 amino acid residues.

Y may be an amino acid sequence comprising a sequence from seq id no:11, or 5 or more, or 10 or more consecutive amino acid residues from the C-terminal side of the amino acid residues at positions 99 to 113 of seq id no: 10 or more continuous amino acid residues C-terminal to the amino acid residues at positions 99 to 162 of 14. In certain embodiments, Y may be an amino acid sequence comprising seq id no:11, seq id no: amino acid residues 158 to 162 of 14, seq id no:14, amino acid residues at positions 153 to 162 of seq id no:14, amino acid residues at positions 143 to 162 of seq id no: amino acid residues at positions 133 to 162 of 14 or seq id no:14 at amino acid residues 99 to 162.

Z2 may be an amino acid sequence comprising a sequence derived from seq id no: 12(hIgG2) from 4 to 37 or 6 to 30 consecutive amino acid residues on the N-terminal side of the amino acid residues at positions 111 to 147 of seq id no:14 (hIgD) from 6 to 30 consecutive amino acid residues N-terminal to the amino acid residues in positions 163 to 199. In certain embodiments, Z2 may be the 6N-terminal amino acid residues of the human IgG2CH2 domain or the 8N-terminal amino acid residues of the human IgDCH2 domain.

The total number of amino acid residues of Z2 and Z3 may be between 80 and 140. In one embodiment, the total number of amino acid residues in Z2 and Z3 can be between 90 and 120, inclusive. In another embodiment, the total number of amino acid residues in Z2 and Z3 can be between 105 and 115, inclusive. In one embodiment, the total number of amino acid residues of Z2 and Z3 is 108. In a further embodiment, the total number of amino acid residues of Z2 and Z3 is 109.

Z4 may be an amino acid sequence comprising a sequence derived from seq id no: 224-330 of 11(hIgG1), SEQ ID NO: 12(hIgG2) 220-326, SEQ ID NO: 271-377 of 24(hIgG3) or SEQ ID NO:13 (hIgG4) at positions 221 to 327 of 90 or more, or 100 or more consecutive amino acid residues. Z4 can be seq id no: 224 of 11 and 330, SEQ ID NO: 220 of 12 and 326, SEQ ID NO: 271 of 24 and 377 or SEQ ID NO:13 at amino acid residues 221 to 327.

According to an embodiment, Z3-Z4 is an amino acid sequence selected from: (i) consists of SEQ ID NO:11 and the C-terminal portion of amino acid residues at positions 118 to 223 of seq id no:11, (ii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 224 to 330 of seq id no: 12 and the C-terminal portion of amino acid residues at positions 114 to 219 of seq id no: 12, (ii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 220 to 326 of seq id no: 24 and the C-terminal portion of amino acid residues at positions 165 to 270 of seq id no: (iii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 271 to 377 of position 24, and (iv) a sequence consisting of seq id no:13 and the C-terminal portion of amino acid residues at positions 115 to 220 of seq id no:13 from position 221 to 327 of amino acid residue.

According to one embodiment of the invention, the total number of amino acid residues of the polypeptide is from 154 to 288.

In one embodiment, Y may be an amino acid sequence comprising seq id no:11, p may be 0 or 1; z2 may be an amino acid sequence comprising seq id no: 12 at least a portion of the amino acid residue at position 111-147; and Z3 can be an amino acid sequence comprising seq id no: 118 of 11, SEQ ID NO: 114 of 12, SEQ ID NO: 165 of 24 and 270 or SEQ ID NO:13 at least a portion of amino acid residues at positions 115 to 220. In this embodiment, when p is 1, Z1 can be an amino acid sequence comprising seq id no:11, at least a portion of amino acid residues at positions 90 to 98.

In a further embodiment, Z3 can be seq id no: 118 of 11, SEQ ID NO: 114 of 12, SEQ ID NO: 165 of 24 and 270 or SEQ ID NO:13 from 73 to 106 consecutive amino acid residues at the 115-position 220, and the total number of amino acid residues of Z2 and Z3 may be 110. Z2 can be seq id no: 12, and Z3 can be the amino acid residue at position 111-116 of seq id no: 120 of 11, SEQ ID NO: 116 of 12, SEQ ID NO: 167-: 13 from position 118 to 220.

In another embodiment, Y may be an amino acid sequence comprising seq id no:14, p may be 1 or 0 (zero); z2 may be an amino acid sequence comprising seq id no: at least a portion of the amino acid residue at position 163-199 of 14; and Z3 can be an amino acid sequence comprising seq id no:13 at least a portion of the amino acid residues at positions 121 to 220. In this embodiment, when p is 1, Z1 can have an amino acid sequence comprising the amino acid sequence of seq id no:14 at least a portion of amino acid residues at positions 90 to 98.

In further embodiments, Y can be seq id no:14 at positions 99-162, 20 consecutive amino acid residues or more, 30 consecutive amino acid residues or more, 40 consecutive amino acid residues or more, 50 consecutive amino acid residues or more, or 60 consecutive amino acid residues or more. Z2 can be seq id no:14, Z3 may comprise amino acid residues at positions 163 to 170 of seq id no: 124 of 11, SEQ ID NO: 12, 120-219, SEQ ID NO: 171 and 270 of 24 or SEQ ID NO: 71-100 consecutive amino acid residues on the C-terminal side of the amino acid residues at positions 121-220 of 13. The total number of amino acid residues of Z2 and Z3 may be 108.

In one embodiment, the polypeptide may be encoded by a sequence selected from seq id nos: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 26 and seq id no: 27 is encoded by a nucleotide sequence of seq id no. The polypeptide is selected from SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 28 and seq id no: 29.

In one embodiment, the polypeptide is fused at its N-terminus to a biologically active molecule that exhibits an increased circulatory half-life compared to the circulatory half-life of the native form (native form) of the biologically active molecule. The biologically active molecule may be a polypeptide, protein or non-peptide (apeptide). The biologically active molecule may be a polypeptide, peptide or protein drug. The bioactive molecule can be a soluble protein such as, but not limited to: hormones, cytokines, growth factors, co-stimulatory molecules, hormone receptors, cytokine receptors, growth factor receptors, or short peptides. The biologically active molecule can be EPO or a variant/fragment thereof, p40 or a variant/fragment thereof (e.g., a p40 variant containing an Asn303Gln substitution), G-CSF or a variant/fragment thereof, TNF receptor, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10 receptor, TGF-beta, TGF-beta receptor, IL-17 receptor, factor VII, CXCL-11, FSH, human growth hormone, bone morphogenetic protein-1 (BMP-1), CTLA4, PD-1, GLP-1, betacellulin (betacellulin), OPG, RNAK, interferon alpha (interferon-alpha), interferon beta (interferon-beta) or variants/fragments thereof. The biologically active molecule may be a secreted protein, which may be in a mature form.

In one embodiment, there is provided a method of producing a polypeptide according to claim 1, wherein the method comprises the steps of: (i) introducing a DNA molecule encoding the polypeptide into a mammalian host cell, (ii) growing the cell in its culture medium under conditions in which the polypeptide can be expressed; and (iii) harvesting the expressed polypeptide. The mammalian host cell may be a CHO, COS or BHK cell.

In another embodiment, the following method is provided: (i) reducing the symptoms of, preventing or treating an autoimmune disease, (ii) inhibiting transplant rejection, or (iii) treating or preventing endotoxin induced shock, comprising administering a therapeutically effective amount of a polypeptide as described above, wherein the polypeptide is fused to a biologically active molecule.

In one embodiment, an isolated nucleic acid molecule is provided that encodes a polypeptide according to an embodiment of the invention. The polypeptide may have an amino acid sequence selected from the group consisting of: SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 28 and seq id no: 29. the nucleic acid molecule may have a nucleotide sequence shown in the following sequence: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 26 or seq id no: 27. the nucleic acid molecule may further comprise a signal sequence or a leader sequence.

According to embodiments of the invention, expression vectors comprising the nucleic acid molecules and host cells containing the vectors are provided. Examples of expression vectors include, but are not limited to, pAD11EPO-hFc-1, pAD11G-CSF-hFc-1, pAD11p40N303Q-hFc-1, pAD11EPO-hFc-6, pAD11G-CSF-hFc-6, pAD11p40N303Q-hFc-6, pAD11EPO-hFc-5, pAD11G-CSF-hFc-5, pAD11p40N303Q-hFc-5, and pAD11 TNFR-hFc-5.

In one embodiment, a method of delivering a biologically active molecule to a mammal is provided, comprising the step of administering a nucleic acid molecule to a mammal in need thereof.

In another embodiment, the polypeptide comprises an Fc domain consisting of a hinge region, a CH2 domain, and a CH3 domain in the N-terminal to C-terminal direction, wherein the hinge region comprises at least a portion of amino acid residues of a human IgD hinge region or a human IgG1 hinge region; the CH2 domain includes at least a portion of the amino acid residues of the human IgG4CH2 domain, wherein 4-37 consecutive amino acid residues at the N-terminus of the human IgG4CH2 domain are replaced with at least a portion of the amino acid residues of the N-terminal portion of the human IgG2CH2 domain or the N-terminal portion of the human IgDCH2 domain, and the CH3 domain includes at least a portion of the amino acid residues of the human IgG4CH3 domain.

The hinge region may comprise at least a portion of the amino acid residues of the hinge region of human IgG1, the CH2 domain comprising at least a portion of the amino acid residues of the human IgG4CH2 domain, wherein 4 to 37 consecutive amino acid residues at the N-terminus of the human IgG4CH2 domain are substituted with at least a portion of the amino acid residues of the N-terminal region of the human IgG2CH2 domain.

The hinge region may comprise at least a portion of the amino acid residues of a hinge region of human IgD, said CH2 domain comprising at least a portion of the amino acid residues of a human IgG4CH2 domain, wherein 4-37 consecutive amino acid residues at the N-terminus of the human IgG4CH2 domain are replaced with at least a portion of the amino acid residues of an N-terminal region of a human IgG2CH2 domain.

The polypeptide may further comprise a CH1 domain, wherein the CH1 domain comprises at least a portion of the amino acid residues of a human IgG1CH1 domain, and wherein the CH1 domain is coupled to the N-terminus of the hinge region. The polypeptide may further comprise a CH1 domain, wherein the CH1 domain comprises at least a portion of the amino acid residues of the human IgDCH1 domain, and wherein the CH1 domain is coupled to the N-terminus of the hinge region. The polypeptide may further comprise a second polypeptide coupled to the N-terminus of the hinge region, wherein the second polypeptide is a biologically active non-immunoglobulin polypeptide. The polypeptide may further comprise a biologically active molecule coupled to the N-terminus of said CH1 domain or to the C-terminus of said CH4 domain via a linker, wherein said biologically active molecule is not an immunoglobulin polypeptide. The polypeptide and the biologically active molecule may be linked to each other via a linker. The linker molecule is an albumin linker or a synthetic linker. The albumin linker comprises seq id no:25 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333 or 306 to 338. The synthetic linker may be a peptide of 10 to 20 amino acid residues consisting of Gly and Ser residues. In one embodiment, this Gly-Ser linker is GGGGSGGGGSGGGSG (SEQ ID NO: 32).

The invention also includes antibody molecules comprising a recombinant Fc region, as described above.

Brief Description of Drawings

FIG. 1 shows a schematic representation of hybrid Fcs (hybridFcs, hFcs) which can be used as carrier proteins for biologically active molecules denoted by "X".

FIG. 2 shows a schematic representation of hFcs wherein the amino acid positions derived from IgG1(SEQ ID NO: 11), IgG2(SEQ ID NO: 12), IgG4(SEQ ID NO: 13) and IgD (SEQ ID NO: 14) are described in detail. The nomenclature of amino acid positions in the polypeptides of the present application follows the same convention, unless otherwise indicated.

FIG. 3 shows a schematic representation of hFcs in which each hFc is coupled at the C-terminus to a biologically active molecule denoted by "X" via an albumin linker denoted by "AL".

FIG. 4 shows a schematic representation of hFcs coupled to a linker, wherein the amino acid positions of the albumin linker derived from human albumin (SEQ ID NO: 25) are described in detail.

FIG. 5 shows the results of hydrophobicity distribution (hydrophityplot) of hFc-6.

FIG. 6(a) shows an assay using a specific ELISA(Rituximab)、hIgG1、(etanercept), EPO-hFc-5, G-CSF-hFc-5, p40N 303Q-hFc-5; FIG. 6(b) shows analysis using specific ELISA(Rituximab)、hIgG1、(etanercept), EPO-hFc-5, G-CSF-hFc-5, p40N 303Q-hFc-5.

FIG. 7(a) shows EPO-IgG1Fc, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6 and EPO-hFc-78, respectively, compared to the biological activity of EPO in the human F36E cell lineBiological activity results of (derbepoetinalfa); FIG. 7(b) shows in a mouse hematopoietic cell line (NFS-60)Results of in vitro bioactivity of (pegfilgrastim) and G-CSF-hFc-5; FIG. 7(c) shows the in vitro bioactive junctions of p40 and p40N303Q-hFc-5 in human PBMCsFruit; FIG. 7(d) shows in murine L929 cells(etanercept) and TNFR-hFc-5; and FIG. 7(e) shows the results of in vitro bioactivity of thFc-1-AL (0) -IFN- β and thFc-1-AL (3) -IFN- β in human WISH cells.

FIG. 8(a) shows macaque administration via SC route (left panel) and IV route (right panel)(derbepoetinalfa), EPO-hFc-1 or EPO-hFc-5; FIG. 8(b) shows administration to Sprague Dawley rats by subcutaneous injection route (left panel) and intravenous injection route (right panel)(filgrastim) and the pharmacokinetic results for G-CSF-hFc-1; FIG. 8(c) shows the subcutaneous injection route applied to rhesus monkeys(etanercept) pharmacokinetic results; FIG. 8(d) shows TNFR-hFc-5 and TNFR-hFc-5 administered to Sprague Dawley rats by subcutaneous injection(etanercept) results of pharmacokinetics.

FIG. 9(a) shows macaque administration via subcutaneous injection (top left panel) and intravenous injection (bottom right panel)(derbepoetinalfa) and EPO-hFc-5, FIG. 9(b) shows administration to Sprague Dawley rats via subcutaneous (upper panel) and intravenous (lower panel) routesBiologically active knots of (filgrastim) and G-CSF-hFc-1 in vivoAnd (5) fruit.

Best mode for carrying out the invention

The present invention provides a hybrid human immunoglobulin Fc fragment comprising, from N-terminus to C-terminus, a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region is at least a partial amino acid sequence of a human IgD hinge region or a human IgG1 hinge region; the CH2 domain is the human IgG4CH2 domain with a portion of the N-terminal region of the CH2 domain replaced with amino acid residues 4-37 of the N-terminal region of the human IgG2CH2 or human IgDCH2 domain. When used to produce an Fc fusion protein linked to a biologically active molecule, such as a biologically active polypeptide, such a hybrid Fc fragment minimizes non-specific immune reactions of the Fc fusion protein, extends the serum half-life of the biologically active molecule of the biologically active polypeptide, and optimizes the activity of the biologically active molecule of the biologically active polypeptide.

In the Fc fusion protein according to one embodiment of the present invention, the combination of the N-terminus of the IgDCH2 domain with the remainder of the IgG4CH2 domain is designed such that the region of the fusion protein produced at the recombination of two different Ig subunits is hydrophobic. The hydrophobic region of the resulting fusion protein should be located within the folded protein, which minimizes undesired non-specific immune reactions.

As used herein, the term "Fc fragment" or "Fc" refers to a protein that contains the heavy chain constant region 1(CH1), heavy chain constant region 2(CH2), and heavy chain constant region 3(CH3) of an immunoglobulin and does not contain the variable regions of the heavy and light chains of the immunoglobulin and light chain constant region 1(CL 1). It may further comprise a hinge region at the heavy chain constant region. Hybrid Fc or hybrid Fc fragment is sometimes referred to herein as "hFc".

In addition, the Fc fragment of the present invention may be in the form of having a natural sugar chain, an increased sugar chain as compared to a natural sugar chain, a decreased sugar chain as compared to a natural sugar chain, or may be in the form of deglycosylation. The addition, reduction or removal of the immunoglobulin Fc sugar chain can be achieved by methods common in the art, such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment causes a sharp decrease in binding affinity to the C1q portion of the first complement component C1 and a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and thus does not induce an unnecessary immune response in vivo. In this regard, in some cases, deglycosylated or non-glycosylated (aglycosylated) forms of immunoglobulin Fc fragments as drug carriers may be more suitable for the purposes of the present invention.

As used herein, the term "deglycosylation" refers to the enzymatic removal of the sugar moiety from the Fc fragment, and the term "aglycosylation" means the production of the Fc fragment in an unglycosylated form by prokaryotes, preferably e.

As used herein, the term "hybridize (heterozygous)" means that the sequences encoding two or more immunoglobulin Fc fragments of different origin are present as single-chain immunoglobulin Fc fragments.

In one embodiment, a hybrid human Fc comprises, in the N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region is at least a partial amino acid sequence of a human IgD hinge region or a human IgG1 hinge region; and the CH2 domain is a human IgG4CH2 domain which has a portion of its N-terminal region replaced with 4-37 amino acid residues from the N-terminal region of the human IgG2CH2 or human IgDCH2 domain. The hybrid human Fc can be linked at its N-terminus to the C-terminus of a biologically active molecule via a covalent bond.

In another embodiment, the biologically active molecule-hybrid Fc fusion polypeptide can be represented by the formula:

N′-X-(Z1)_p-Y-Z2-Z3-Z4-C', or

N′-(Z1)_p-Y-Z2-Z3-Z4- (linker)_q-X-C′

Wherein N 'is the N-terminus and C' is the C-terminus of the polypeptide; z1 represents an amino acid sequence comprising seq id no:11, or at least the C-terminal portion of the amino acid residues at positions 90 to 98 of seq id no:14 at least a portion of amino acid residues at positions 90-98; y represents an amino acid sequence comprising seq id no:11, or at least the C-terminal part of the amino acid residues in positions 99 to 113 of seq id no:14 at least a portion of amino acid residues 99 to 162; z2 represents an amino acid sequence comprising seq id no: 12, or at least the N-terminal portion of the amino acid residues at positions 111 to 147 of seq id no: at least the N-terminal portion of amino acid residues 163 to 199 of 14; z3 represents an amino acid sequence comprising seq id no: 118 of 11, SEQ ID NO: 114 of 12, SEQ ID NO: 165 of 24 and 270 or SEQ ID NO:13 at least the C-terminal portion of amino acid residues at positions 115 to 220; z4 represents an amino acid sequence comprising seq id no:13 at least the N-terminal portion of the amino acid residue at position 221-327 and p and q are each integers of 0 or 1, wherein the total number of amino acid residues of Z2 and Z3 can be between 80 and 140, both end values are included, the linker is a linker molecule, and X is a biologically active molecule of interest.

In one embodiment, Z3-Z4 is an amino acid sequence selected from the group consisting of: (i) consists of SEQ ID NO:11 and the C-terminal portion of amino acid residues at positions 118 to 223 of seq id no:11, (ii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 224 to 330 of seq id no: 12 and the C-terminal portion of amino acid residues at positions 114 to 219 of seq id no: 12, (ii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 220 to 326 of seq id no: 24 and the C-terminal portion of amino acid residues at positions 165 to 270 of seq id no: (iii) a contiguous amino acid sequence consisting of the N-terminal portion of the amino acid residues at positions 271 to 377 of position 24, and (iv) a sequence consisting of seq id no:13 and the C-terminal portion of amino acid residues at positions 115 to 220 of seq id no:13 from position 221 to 327 of amino acid residue.

According to one embodiment of the invention, the total number of amino acid residues of said polypeptide is from 154 to 288.

When administered to a subject, the formula N' -X- (Z1)_p-Y-Z2-Z3-Z4-C 'and N' - (Z1)_p-Y-Z2-Z3-Z4- (linker)_q-X-C' increases the circulating half-life of the biologically active molecule X.

The linker may be derived from human albumin (CAA00606SEQ ID NO: 25). The linker may include seq id no:25 from 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333 or 306 to 338. Alternatively, the linker may be a synthetic linker. The synthetic linker may be a peptide consisting of a total of 10-20 Gly and Ser amino acid residues. In one embodiment, the Gly-Ser linker is GGGGSGGGGSGGGSG (SEQ ID NO: 32).

Z1 may include at least a portion of the CH1 domain of human IgG1(SEQ ID NO: 11) or IgD (SEQ ID NO: 14). Z1 may comprise 5 to 9 or 7 to 9 consecutive amino acid residues in the C-terminal region of the IgG1CH1 domain (positions 90-98 of SEQ ID NO: 11) or the C-terminal region of the IgDCH1 domain (positions 90-98 of SEQ ID NO: 14). In some embodiments, Z1 may be the 5, 6, 7, 8, or 9C-terminal amino acid residues of the IgG1CH1 domain or the IgDCH1 domain.

In some embodiments, Z1 is an amino acid sequence comprising seq id no:11 or the amino acid residue at position 90 to 98 of seq id no:14, amino acid residues at positions 90 to 98. Z1 may be a peptide consisting of seq id no:11 or position 90 to 98 of seq id no:14, 5 to 9 amino acid residues at positions 90-98. Z1 may also be represented by seq id no:11 or seq id no:14 from 90 to 98 amino acid residues.

Y may comprise at least a portion of the hinge region of human IgG1 or IgD. Y may comprise 5 or more, or 10 or more, consecutive amino acid residues at the C-terminus of the IgG1 hinge region (amino acids 99 to 113 of SEQ ID NO: 11) or of the IgD hinge region (amino acids 99 to 162 of SEQ ID NO: 14). In certain embodiments, Y may be an amino acid sequence comprising seq id no:11, seq id no: amino acid residues 158 to 162 of 14, seq id no:14, amino acid residues at positions 153 to 162 of seq id no:14, amino acid residues at positions 143 to 162 of seq id no: amino acid residues at positions 133 to 162 of 14 or seq id no:14 at amino acid residues 99 to 162.

Z2 may comprise 4 to 37, 6 to 30, 6 to 12, 6 to 8, 8 or 6 consecutive amino acid residues from the N-terminus of the human IgG2CH2 domain (amino acid residues from positions 111 to 147 of SEQ ID NO: 12) or from the N-terminus of the IgDCH2 domain (amino acid residues from positions 163 to 199 of SEQ ID NO: 14). In certain embodiments, Z2 can be the 6N-terminal amino acid residues of the human IgG2CH2 domain (amino acid residues at positions 111-116 of SEQ ID NO: 12) or the 8N-terminal amino acid residues of the human IgDCH2 domain (amino acid residues at positions 163-170 of SEQ ID NO: 14).

The total number of amino acid residues of Z2 and Z3 may be between 90 and 120 inclusive; or between 105 and 115, inclusive.

Z4 may be an amino acid sequence comprising 90 or more, or 100 or more consecutive amino acid residues of the IgG4CH3 domain (amino acid residues in positions 224-330 of SEQ ID NO:11, 220-326 of SEQ ID NO: 12, 271-377 of SEQ ID NO: 24 or 221 to 327 of SEQ ID NO: 13). Z4 may be an amino acid residue that is greater than 98% or 95% of the human IgG1, IgG2, IgG3 or IgG4CH3 domain. In an exemplary embodiment, Z4 is an amino acid sequence that includes the entire amino acid sequence of the human IgGCH3 domain. For example, Z4 is the amino acid sequence of the human IgG4CH3 domain corresponding to amino acid residue 341-447 of human IgG4, as numbered according to EUIndex, Kabat (which corresponds to the amino acid residue at position 221-327 of SEQ ID NO: 13).

In one embodiment, Y may be an amino acid sequence comprising at least a portion of the C-terminal amino acid residues of the hinge region of human IgG1 (positions 99 to 113 of SEQ ID NO: 11), p may be 1 or 0; z2 may be an amino acid sequence comprising at least a portion of the N-terminal region of human IgG2CH2 (amino acid residues at positions 111-147 of SEQ ID NO: 12); and Z3 may be an amino acid sequence which includes at least a portion of the C-terminal region of any one of the human IgG subclasses (amino acid residues at positions 118-223 of SEQ ID NO:11, 114-219 of SEQ ID NO: 12, 165-270 of SEQ ID NO: 24 or 115-220 of SEQ ID NO: 13). In this embodiment, when p is 1, Z1 may be an amino acid sequence comprising at least a portion of the C-terminal region of the human IgG1CH1 domain (amino acid residues 90 to 98 of SEQ ID NO: 11). For example, Z1 can be seq id no:11, amino acid residues 90 to 98.

In further embodiments, Z3 may be 73 to 106 consecutive amino acid residues from the C-terminal region of the human IgG4CH2 domain (at positions 115-220 of SEQ ID NO: 13), the human IgG1CH2 domain (at position 118-223 of SEQ ID NO: 11), the human IgG2CH2 domain (at position 114-219 of SEQ ID NO: 12), the human IgG3CH2 domain (at position 165-270 of SEQ ID NO: 24), and the total number of amino acid residues of Z2 and Z3 may be 110. For example, Z2 can be a residue as set forth in seq id no: 12, and Z3 can be the amino acid sequence at amino acid residue position 111-116 of seq id no:13 at amino acid residues 117 to 220.

In another embodiment, Y may be an amino acid sequence comprising at least a portion of the C-terminal region of the human IgD hinge region (amino acid residues 99 to 162 of SEQ ID NO: 14), and p may be 1 or 0 (zero); z2 may be an amino acid sequence comprising at least a portion of the N-terminal region of the human IgDCH2 domain (amino acid residues at positions 163 to 199 of SEQ ID NO: 14) and Z3 may be an amino acid sequence comprising at least a portion of the C-terminal region of the human IgG4CH2 domain (amino acid residues at positions 121 to 220 of SEQ ID NO: 13). For example, Y can be the same as in seq id no:14 at amino acid residue position 158 to 162, 133 to 162, or 99 to 162, Z2 may be at the amino acid residue position of seq id no: amino acid residues 163 to 170 of 14 and Z3 may be the amino acid residues in seq id no:13 at position 121-220.

In this embodiment, when p is 1, Z1 can be the amino acid sequence comprising the C terminal region of the human IgDCH1 domain (amino acid residues at positions 90 to 98 of SEQ ID NO: 14). For example, Z1 can be seq id no:14 from position 90 to 98.

In this embodiment, Y may be 20 consecutive amino acid residues or more, 30 consecutive amino acid residues or more, 40 consecutive amino acid residues or more, 50 consecutive amino acid residues or more, or 60 consecutive amino acid residues or more of the C-terminal side of the human IgD hinge region (amino acid residues at positions 99-162 of seq id no: 14). Z3 may include seq id no:13 from 71 to 100 consecutive amino acid residues C-terminal to the amino acid residue at position 121-220. The total number of amino acid residues of Z2 and Z3 may be 108.

Table 1 shows the amino acid sequences of human IgG1, IgG2, IgG3, and IgD fragments useful in constructing hFcs according to embodiments of the present invention.

TABLE 1

hFc domain	Range of acceptable Ig fragments	Sequence of the longest fragment within an acceptable range in the N-terminal to C-terminal direction	SEQ ID NO：	Position in SEQ ID	Position in EU index
						CH1(Z1)	5-9C-terminal amino acid residues of IgG1CH1	SNTKVDKRV **	11	90-98	207-215
CH1(Z1)	5-9C-terminal amino acid residues of IgD CH1	ASKSKKEIF	14	90-98	Is not obtained
						Hinge (Y)	5-15C-terminal amino acid residues of the hinge region of IgG1	EPKSCDKTHTCPPCP	11	99-113	216-230
Hinge (Y)	5-64C-terminal amino acid residues of the IgD hinge region	RWPESPKAQASSVPTAQPQAE GSLAKATTAPATTRNTGRGGE EKKKEKEKEEQEERETKTPECP	14	99-162	Is not obtained

N-terminal side of CH2 (Z2)	4-37N-terminal amino acid residues of IgG2CH2	APPVAGPSVFLFPPKPKDTLMI SRTPEVTWVVVDVSH	12	111-147	231-267
						CH2, N-terminal side (Z2)	4-37N-terminal amino acid residues of the IgD CH2 domain	SHTQPLGVYLLTPAVQDLWLR DKATFTCFVVGSDLKD	14	163-199	Is not obtained
C-terminal side of CH2 (Z3) + CH3(Z4)	71-106C-terminal amino acid residues of IgG4CH2 + 80-107N-terminal amino acid residues of the IgG4CH3 domain	LGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAK +GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLS LGK	13	115-220+22 1-327	235-340+341-44 7
						CH2, C-terminal side (Z3) + CH3(Z4)	71-106C-terminal amino acid residues of IgG3CH2 + 80-107N-terminal amino acid residues of the IgG3CH 3 domain	LGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFKW YVDGVEVHNAKTKPREEQYN STFRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKTK +GQPREPQVYTLPPSREEMTK NQVSLTCPVKGFYPSDIAVEW ESSGQPENNYNTTPPMLDSDG SFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKS LSLSPGK	24	165-270+27 1-377	235-340+341-44 7
CH2, C-terminal side (Z3) + CH3(Z4)	71-106C-terminal amino acid residues of IgG2CH2 + 80-107N-terminal amino acid residues of the IgG2CH 3 domain	VAGPSVFLFPPKPKDTLMISRT PEVTWVVVDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQF NSTFCVVSVLTVVHQDWLNG KEYKCKVSNKGLPAPIEKTISK TK+GQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSL SPGK	12	114-219+22 0-326	234-340+341-44 7
						CH2, C-terminal side (Z3) + CH3(Z4)	IgG1CH2 of 71-106C-terminal amino acid residues + 80-107N-terminal amino acid residues	LGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKA K+GQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEW	11	18-223+22 4-330	235-340+341-44 7

ESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLS PGK

^*The EU index is described in "sequencesof proteins of immune 1-logical interest, 5th edition, unitedstates deparatmentof health and human services.

^**The underlined region in each amino acid sequence represents the shortest fragment of the range of acceptable amino acid residues.

In one embodiment, the invention provides a hybrid Fc which is one of hFc-1, hFc-2, hFc-3, hFc-4, hFc-5 or hFc-6 as shown in FIGS. 1 and 2 or thFc-1 or thFc-2 as shown in FIGS. 3 and 4. Although fig. 1 and 3 depict double-stranded Fcs, the present invention encompasses single-stranded hybrid Fc molecules. The amino acid sequences of hFc-1 to hFc-6 are shown in SEQ ID NOs: 18-23, and the Fc-1 and thFc-2 amino acid sequences are shown in seq id nos: 28 and seq id no: 29 (b). The invention also includes polynucleotide molecules encoding hybrid Fc. They include, but are not limited to, the amino acid sequences as shown in seq id no: 1(hFc-1), seq id no: 2(hFc-2), seq id no: 3(hFc-3), seq id no: 4(hFc-4), seq id no: 5(hFc-5), SEQ ID NO: 6(hFc-6), SEQ ID NO: 26(thFc-1) and SEQ ID NO: 27 (thFc-2).

Human immunoglobulin amino acid sequences are known in the art and are stored in publicly accessible storage. For example, the amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, human IgG4 constant region, and human IgD constant region were obtained from CAA75032, CAC20455, CAC20456, AAH25985, and P01880, respectively. These sequences are (reproducedas) seq id nos: 11. 12, 24, 13 and 14.

The biologically active molecule X may be a soluble protein. It may include, but is not limited to, hormones, cytokines, growth factors, co-stimulatory molecules, hormone receptors, cytokine receptors, growth factor receptors, or short peptides. For example, X may be EPO, p40, G-CSF, TNF receptor, or variants/fragments thereof. X may be GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10 receptor, TGF- β receptor, IL-17 receptor, factor VII, CXCL-11, FSH, human growth hormone, bone morphogenetic protein-1, CTLA4, PD-1, GLP-1, betacellulin, OPG, RNAK, interferon alpha, interferon beta or variants/fragments thereof. It may also include, but is not limited to, the Fab region of an antibody. The biologically active molecule may also be a secreted protein. In one embodiment, the biologically active molecule does not belong to the immunoglobulin family.

The term "variant" refers to a polynucleotide or nucleic acid that differs from a reference nucleic acid or polypeptide, but retains its essential properties. In general, variants are very similar overall and are identical in many regions to a reference nucleic acid or polypeptide. Also, the term "variant" refers to a biologically active portion of a biologically active molecular drug that retains at least one functional and/or therapeutic property thereof as otherwise described herein or otherwise known in the art. In general, variants are very similar overall and are identical in many regions to the amino acid sequence of the biologically active polypeptide of interest.

The invention also provides proteins, including proteins that are homologous to, for example, seq id nos: 18-23 and 28-29, or alternatively, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. The invention also provides fragments of these polypeptides. Further, the polypeptides encompassed by the invention are polypeptides encoded by polynucleotides that hybridize to a DNA-binding filter under stringent hybridization conditions (e.g., hybridization to a DNA-binding filter in 6 x sodium chloride/sodium citrate (SSC) at about 45 ℃, followed by one or more washes in 0.2 x SSC, 0.1% SDS at about 50-65 ℃), highly stringent conditions (e.g., hybridization to a DNA-binding filter in 6 x sodium chloride/sodium citrate (SSC) at about 45 ℃, followed by one or more washes in 0.2 x SSC, 0.1% SDS at about 68 ℃) or other stringent conditions known to those of ordinary skill in the art (see, e.g., Ausubel, f.m. et al, eds., 1989 currentprocollution molecular, greenbelt, genshui, inc, john wiley, soyork, NewYork, page 6.3.16.3.6, and 2.10.3), nucleic acid encoding a polynucleotide molecule that hybridizes to a nucleic acid of the invention (inc). The invention also includes polynucleotides encoding these polypeptides.

For polypeptides having an amino acid sequence that is at least, e.g., 95% "identical" to the query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query amino acid sequence, except that the subject polypeptide sequence may include up to 5 amino acid changes in every 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having at least 95% identity to the query amino acid sequence, up to 5% of the amino acid residues in the target sequence may be inserted, deleted or substituted with another amino acid. These changes to the reference sequence can occur at a position at the amino-or carboxy-terminus of the reference amino acid sequence or anywhere between these two terminal positions, interspersed either individually among residues in the reference sequence or among residues in one or more adjacent groups within the reference sequence.

Indeed, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the amino acid sequence of the albumin fusion protein of the invention, or a fragment thereof, can generally be determined using known computer programs. A preferred method of determining the best overall match between a query sequence (a sequence of the invention) and a target sequence, also known as sequence global alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al (Comp.App.Biosci.6: 237245 (1990)). In sequence alignment, the query and target sequences are either both nucleotide sequences or both amino acid sequences. The results of the global alignment of sequences are expressed as percent identity. Preferred parameters used in the FASTDB amino acid ratio are: the matrix PAM0, k-tuple 2, mismatch penalty 1, ligation penalty 20, random set length 0, cut score 1, window size sequence length, gap penalty 5, gap size penalty 0.05, window size 500 or the length of the target amino acid sequence, whichever is shorter.

The variant typically HAs at least 75% (preferably at least about 80%, 90%, 95%, or 99%) sequence identity to a standard HA or therapeutic protein that is the same length as the variant. Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (BasiclLocalAlignmentSearchTool) analysis using the algorithms employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, Proc. Natl. Acad. Sci. USA 87: 22642268(1990) and Altschul, J.Mol. Evol. 36: 290300(1993), all incorporated by reference) designed to look for sequence similarity.

Polynucleotide variants of the invention may contain changes in coding regions, non-coding regions, or both. Particularly preferred are polynucleotide variants that contain changes that produce silent substitutions, additions or deletions without altering the properties or activity of the encoded polypeptide. Nucleotide variants resulting from silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which 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 are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants may arise for a variety of reasons, for example to optimize codon expression for a particular host (changing codons in human mRNA to codons preferred by a bacterial host such as yeast or e.

To construct various Fc fusion proteins such as EPO-Fc fusion construct, G-CSF-Fc fusion construct or human p40-Fc fusion construct, the amino acid sequences of human EPO, human G-CSF, human p40 and human TNF receptor can be obtained from NP000790(SEQ ID NO: 15), CAA27291(SEQ ID NO: 16), AAG32620(SEQ ID NO: 17) and NP001057(SEQ ID NO: 31), respectively. In one embodiment, a modified human p40 wherein the amino acid residue Asn at position 303 is replaced with a gin is linked to the polypeptide.

According to another aspect of the present invention, there is provided a whole antibody comprising an engineered Fc region. As used herein, the term "antibody" includes whole antibodies and antibody fragments comprising at least two of CH1, hinge region, CH2, or CH 3. Fully monoclonal antibodies are preferred. The heavy chain variable region of an antibody is selected for its binding specificity and may be of any type, such as, for example, non-human, humanized or fully human (fullyhuman). When the heavy chain variable region of an antibody is non-human (such as, for example, murine) and is recombinantly combined with an engineered Fc region according to the present disclosure, the resulting recombinant antibody is referred to as a chimeric antibody. When the heavy chain variable region of an antibody is humanized and recombinantly combined with an engineered Fc region according to the present disclosure, the resulting recombinant antibody is referred to as a humanized antibody. When the antibody heavy chain variable region is human and is recombinantly combined with an engineered Fc region according to the present disclosure, the resulting recombinant antibody is referred to as a fully human antibody. For example, the heavy chain variable region is humanized and includes human framework regions and non-human (in this case murine) Complementarity Determining Regions (CDRs). It is to be understood that the framework regions may be derived from one source or more than one source and the CDRs may be derived from one source or more than one source. Methods for humanizing antibodies are known to those skilled in the art and are known in the art.

The light chain of an antibody may be human, non-human or humanized. In the embodiment shown in fig. 1, the light chain is humanized and comprises human framework regions, non-human (in this case murine) CDRs and human constant regions. It is to be understood that the framework regions may be derived from one source or more than one source and the CDRs may be derived from one source or more than one source.

Antibodies containing engineered Fc regions are selected based on their ability to bind to cell surface molecules or soluble molecules that bind to cell surface molecules. Thus, for example, antibodies can be selected based on their ability to bind to such cell surface molecules, such as cytokine receptors (e.g., IL-2R, TNF-aR, IL-15R, etc.), adhesion molecules (e.g., E-selectin, P-selectin, L-selectin, VCAM, ICAM, etc.), cell differentiation or activation antigens (e.g., CD3, CD4, CD8, CD20, CD25, CD40, etc.), and others. Alternatively, the antibody may be selected based on its ability to bind to soluble molecules that bind to cell surface molecules. These soluble molecules include, but are not limited to: cytokines and chemokines (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-5, IL-6, etc.), growth factors (e.g., EGF, PGDF, GM-CSF, HGF, IGF, BMP-1, etc.), molecules that induce cell differentiation (e.g., EPO, TPO, SCF, PTN, etc.), and others.

In general, the construction of the antibodies disclosed herein is accomplished by using accepted procedures employed in genetic engineering techniques. For example, such techniques are well known in the art: isolating DNA, preparing and selecting vectors for expressing DNA, purifying and analyzing nucleic acids, specific methods for making recombinant vector DNA, cleaving DNA with restriction enzymes, ligating DNA, introducing DNA comprising vector DNA into host cells by stable or transient methods, culturing host cells in selective or non-selective media to select and maintain cells expressing DNA.

The monoclonal antibodies disclosed herein can be derived using hybridoma methods known in the art or other recombinant DNA methods well known in the art. In the hybridoma method, a mouse or other suitable host animal is immunized with DNA, peptide, or protein that causes lymphocytes to produce antibodies.

Alternatively, lymphocytes may be immunized in vitro. The lymphocytes produced in response to the antigen are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. The hybridoma cells are then seeded and grown in a suitable culture medium that preferably contains one or more agents that inhibit the growth or survival of the unfused parent myeloma cells. Preferred myeloma cells are those which: they fuse efficiently, support stable antibody production by selected antibody-producing cells, and are insensitive to media such as HAT media (sigma chemical company, st. louis, mo., catalogno. h-0262).

Antibodies containing engineered Fc regions may also be used as separately administered compositions given in conjunction with a therapeutic agent. For diagnostic purposes, the antibody may be labeled or unlabeled.

Unlabeled antibodies can be used in conjunction with other labeled antibodies (secondary antibodies) that react with engineered antibodies, such as antibodies specific for human immunoglobulin constant regions. Alternatively, the antibody may be directly labeled. A wide variety of labels may be used, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), and the like. A variety of immunoassays are available and are well known to those skilled in the art.

According to one embodiment, the present invention provides a method of producing a fusion protein, the method comprising: (i) introducing a DNA molecule encoding the fusion protein into a mammalian host cell, (ii) growing the cell in its growth medium under conditions in which the fusion protein is expressed; and (iii) harvesting the resulting fusion protein.

In other exemplary embodiments, pharmaceutical compositions comprising the above-described fusion proteins or antibody molecules or antibody fragments are provided. Also provided are methods of treating or preventing certain conditions by administering the pharmaceutical compositions. For example, the following methods are provided: (i) reducing the symptoms of an autoimmune disease, preventing/treating an autoimmune disease, (ii) inhibiting transplant rejection, (iii) treating/preventing endotoxin induced shock comprising administering a therapeutically effective amount of a fusion protein that hybridizes Fc and p40 protein or a variant/fragment thereof.

The composition may include a pharmaceutical carrier. The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the antibody to a patient. Sterile water, ethanol, fats, waxes, and inert solids may be included in the carrier. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be incorporated into the pharmaceutical composition.

The antibody composition can be administered to a subject in a variety of ways. For example, the pharmaceutical composition may be administered parenterally, e.g., subcutaneously, intramuscularly or intravenously. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to achieve approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the fusion protein, antibody or antibody fragment in these formulations can vary widely, for example, from less than about 0.5%, typically at or at least about 1% up to 15 or 20% by weight, and is selected based on fluid volume, viscosity, etc., primarily according to the particular mode of administration selected.

The invention also provides isolated nucleic acid molecules encoding the fusion proteins, and expression vectors carrying the nucleic acid molecules. The nucleic acid can be delivered directly to a subject in need of a polypeptide encoded by the nucleic acid. Alternatively, the polynucleotide is produced by expressing the nucleic acid in a culture medium and then administered to the subject.

The term "peptide", "polypeptide" or "protein" refers to a molecule of 2 to 40 amino acids, and preferably a molecule of 3 to 20 amino acids and most preferably a molecule of 6 to 15 amino acids. Exemplary peptides can be randomly generated by any of the methods cited above, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins.

As used herein, the term "drug" refers to a substance that exhibits therapeutic activity when administered to a human or animal, examples of which include, but are not limited to: polypeptides, compounds, extracts and nucleic acids. Preferred are polypeptide drugs.

As used herein, the terms "physiologically active polypeptide", "biologically active molecule", "physiologically active protein", "active polypeptide", "polypeptide drug" and "protein drug" are interchangeable in their meaning and are characterized in that they exhibit various physiological functions in vivo in the form of physiological activity.

Polypeptide drugs have the disadvantage of not maintaining physiological effects for a long period of time due to their property of being easily denatured or degraded by proteolytic enzymes present in the body. However, when a polypeptide drug is linked (or conjugated) to an immunoglobulin Fc fragment according to an embodiment of the present invention to form a fusion protein, the drug has increased structural stability and serum half-life. Also, the polypeptides linked to the Fc fragment have much less reduction in physiological activity than other known polypeptide pharmaceutical formulations. Accordingly, a polypeptide comprising a fusion of a polypeptide drug and an Fc fragment, or a conjugate of a polypeptide drug and an Fc fragment of the present invention is characterized by significantly improved in vivo bioavailability compared to the in vivo bioavailability of conventional polypeptide drugs. This is also clearly described by the embodiments of the present invention. That is, IFN- α, G-CSF, EPO, p40, TNF receptors, and other protein drugs, when linked to the Fc fragment of the present invention, exhibit increased bioavailability in vivo as compared to their native or other traditional fusion forms.

It will be appreciated that the present invention develops conventional recombinant DNA methodologies to produce Fc fusion proteins, antibodies comprising an engineered Fc region according to the present invention, and antibody fragments for use in the practice of the present invention. The Fc fusion construct is preferably produced at the DNA level, and the produced DNA is integrated into an expression vector and expressed to produce the fusion protein, antibody or antibody fragment of the invention.

As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence capable of being incorporated into a host and recombined with and integrated into the host cell genome, or capable of autonomous replication as an episome. Such vectors include linear nucleic acids, plasmids, antibiotics, cosmids, RNA vectors, viral vectors, and the like. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses. As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an Fc fusion protein product or an antibody or antibody fragment.

Useful expression vectors are RcCMV (Invitrogen, Carlsbad) or variants thereof. Useful expression vectors should carry the human Cytomegalovirus (CMV) promoter to facilitate constitutive transcription of the gene of interest in mammalian cells, and the bovine growth hormone polyadenylation signal sequence to increase the steady state level of RNA following transcription. In an embodiment of the invention, the expression vector is pAD11, which is a modified vector for RcCMV. Examples of expression vectors carrying nucleotide sequences encoding biologically active molecular drugs may include, without limitation: pAD11EPO-hFc-1, pAD11G-CSF-hFc-1, pAD11p40N303Q-hFc-1, pAD11EPO-hFc-6, pAD11G-CSF-hFc-6, pAD11p40N303Q-hFc-6, pAD11EPO-hFc-5, pAD11G-CSF-hFc-5, pAD11p40N303Q-hFc-5 or pAD11TNFR-hFc-5, as described in more detail in the examples.

Suitable host cells may be transformed or transfected with the DNA sequences of the present invention, and used for expression and/or secretion of the protein of interest. Preferred host cells currently used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells, HeLa cells, and COS cells.

One expression system that has been used to produce high levels of expressed fusion proteins or antibodies or antibody fragments in mammalian cells is a DNA construct encoding a secretory expression cassette in the 5 'to 3' direction, which includes a signal sequence and an immunoglobulin Fc region, as well as proteins of interest, such as p40, EPO, G-CSF, TNF receptors. Several target proteins have been successfully expressed in this system and include, for example: IL2, CD26, Tat, Rev, OSF-2, ss; IG-H3, IgE receptor, PSMA, and gp 120. These expression constructs are disclosed in U.S. Pat. Nos. 5,541,087 and 5,726,044 to Lo et al, the contents of which are incorporated herein by reference.

The fusion protein or antibody molecule or antibody fragment of the invention may or may not include a signal sequence when expressed. As used herein, the term "signal sequence" is understood to mean a signal that directs a biologically active molecular drug; a fragment of the fusion protein that is secreted and subsequently post-translationally cleaved in a host cell. The signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates transport of a protein across the endoplasmic reticulum membrane. Signal sequences for use in the present invention include antibody light chain signal sequences, such as antibody 14.18(Gillies et al, J.Immunol.Meth.1989.125: 191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al, Nature1980.286: 676-683), and any other signal sequence known in the art (see, e.g., Watson et al, nucleic acids research 1984.12: 5145-5164).

Signal sequences are well characterized in the art and are known to contain 16 to 30 amino acid residues, and may contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region comprises 4 to 12 hydrophobic residues that anchor the signal peptide across the membrane lipid bilayer during transport of the nascent polypeptide. Upon initiation, the signal peptide is normally cleaved within the endoplasmic reticulum by a cellular enzyme known as a signal peptidase. The potential cleavage sites for signal peptides generally follow the "(-3, -1) rule". Thus, typical signal peptides have small, neutral amino acid residues in positions-1 and-3 and lack proline residues in this region.

Signal peptidases cleave this signal peptide between-1 and +1 amino acids. Thus, the signal sequence may be cleaved from the amino terminus of the fusion protein during secretion. This results in the secretion of an Fc fusion protein consisting of an immunoglobulin Fc region and a protein of interest. Detailed discussion of the signal peptide sequence is described by von Heijne (1986) nucleic acids Res.14: 4683A.

As will be apparent to those skilled in the art, the suitability of a particular signal sequence for a secretion expression cassette may require some routine experimentation.

Such experiments include determining the ability of the signal sequence to direct the secretion of the Fc fusion protein, as well as determining the optimal configuration (conformation), genome or cDNA of the sequence to be used to achieve efficient secretion of the Fc fusion protein. In addition, one skilled in the art can prepare synthetic signal peptides according to the rules provided by von Heijne (1986) and test the efficacy of such synthetic signal sequences by routine experimentation. The signal sequence may also be referred to as a "signal peptide", "leader sequence" or "leader peptide".

The fusion of a signal sequence and an immunoglobulin Fc region is sometimes referred to as a secretory expression cassette. An exemplary secretory expression cassette for use in the practice of the present invention is a polynucleotide encoding, in the 5 'to 3' direction, the signal sequence of an immunoglobulin light chain gene and the Fcy1 region of a human immunoglobulin y1 gene. The Fcy1 region of the immunoglobulin Fcy1 gene preferably includes at least a portion of an immunoglobulin hinge domain and at least a CH3 domain, or more preferably at least a portion of the hinge domain, CH2 domain, and CH3 domain. As used herein, a "portion" of an immunoglobulin hinge region is understood to mean a portion of an immunoglobulin hinge that comprises at least one, preferably two cysteine residues that are capable of forming interchain disulfide bonds. The DNA encoding the secretory expression cassette may be in its genomic configuration or its cDNA configuration. In certain instances, it is advantageous to generate the Fc region from a human immunoglobulin Fcy2 heavy chain sequence. Although Fc fusion based on human immunoglobulin y1 and y2 sequences behaves similarly to mice, Fc fusion based on the y2 sequence may exhibit superior pharmacokinetics in humans.

In another embodiment, the DNA sequence encodes a proteolytic cleavage site inserted between the secretory expression cassette and the protein of interest. The cleavage site provides for proteolytic cleavage of the encoded fusion protein, which thereby separates the Fc domain from the protein of interest. As used herein, "proteolytic cleavage site" is understood to mean an amino acid sequence that is preferentially cleaved by a proteolytic enzyme or other proteolytic cleavage agent. Useful proteolytic cleavage sites include amino acid sequences recognized by proteolytic enzymes such as trypsin, plasmin, or enterokinase K. Many cleavage site/cleavage agent pairs are known (see, e.g., U.S. Pat. No. 5,726,044).

Further, substitutions or deletions of constructs of these constant regions, in which one or more amino acid residues of the constant region domain are substituted or deleted, are also useful. One example is the introduction of amino acid substitutions into the above CH2 region to produce Fc variants with reduced affinity for Fc receptors (Cole et al (1997) j.immunol.159: 3613). Those skilled in the art can prepare these constructs using well known molecular biology techniques.

Non-limiting examples of protein drugs that can be conjugated to the immunoglobulin Fc fragment of the present invention include human growth hormone, bone morphogenic protein-1 (BMP-1), growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptors (e.g., interferon- α, - β and- γ, water soluble type I interferon receptor, etc.), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), glucagon-like peptides (e.g., GLP-1, etc.), G protein coupled receptors, interleukins (e.g., interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, etc.), -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, etc.) and interleukin receptors (e.g., IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase, α -galactosidase-A, acarbose (agalsidase) α and β, α -L-iduronate, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase (imiglucerase), lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc.), interleukins and cytokine binding proteins (e.g., IL-18bp, TNF-binding proteins, etc.), macrophage activating factor, macrophage peptides, B-cytokines, T-cytokines, protein A, allergy inhibitors, necrosis glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor inhibitors (tumor inhibitors), cancer metastasis growth factor (metastasis growth factor), alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietin; hemoglobin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor XIII, plasminogen activating factor, fibrin-binding peptide (fibrin-bindingpeptide), urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiotensin, bone growth factor, bone stimulating protein (bonestingprotein), calcitonin, insulin, cardionin, cartilage-inducing factor, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone releasing hormone, nerve growth factor (e.g., nerve growth factor, ciliary neurotrophic factor, axon regenerating factor-1 (axogenisosfactor-1) Brain natriuretic peptide, glial cell line-derived neurotrophic factor, netrin, neutrophil inhibitory factor, neurotrophic factor, neutrophilin, etc.), parathyroid hormone, relaxin, secretin, growth regulator, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid stimulating hormone, autotoxin (autoxin), lactoferrin, myostatin, receptors (e.g., TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activator receptor, etc.), receptor antagonists (e.g., IL1-Ra, etc.), cell surface antigens (e.g., CD2, 3, 4, 5,7, 11a, 11B, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), viral vaccine antigens, and the like, Monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., scFv, Fab ', F (ab') 2, and Fd), and viral-derived immune antigens. The antibody fragment may be a Fab, Fab ', F (ab ') 2, Fd or scFv capable of linking to a specific antigen, and preferably a Fab '. The Fab fragment comprises the Variable (VL) and Constant (CL) domains of the light chain and the Variable (VH) and first constant (CH1) domain of the heavy chain. Fab' fragments differ from Fab fragments in that several amino acid residues from the hinge region, including one or more cysteine residues, are added to the carboxy terminus of the CH1 domain. The Fd fragment contains only the VH and CH1 domains, and the F (ab ') 2 fragment is produced as a pair of Fab' fragments by disulfide linkage or chemical reaction. scFv (single chain Fv) fragments comprise VL and VH domains which are connected to each other by a peptide linker and thus exist as a single polypeptide chain.

In particular, preferred as the bioactive molecule are those requiring frequent administration after administration to a human body for the treatment or prevention of diseases, including human growth hormone, interferons (interferon- α, - β, - γ, etc.), granulocyte colony-stimulating factor (G-CSF), Erythropoietin (EPO), TFN receptor, p40 and antibody fragments. In addition, certain derivatives are included within the scope of the biologically active molecules of the invention as long as they have substantially the same or improved function, structure, activity or stability as compared to the native form of the biologically active molecule. In the present invention, the most preferred polypeptide drug is interferon-a.

In another aspect of the invention, IgG-Fc and IgG-CH fusion proteins are, for example, synthesized as monomers that can assemble to form dimers. Typically, the dimers are often linked by disulfide bonds at the IgG hinge region. The conditioned medium of cells secreting IgG fusion proteins may comprise a mixture of IgG fusion protein monomers and dimers. For use as a human therapy, it may be desirable to use a homogeneous population of IgG fusion protein monomers or dimers, rather than a mixture of the two forms.

Also provided are methods of obtaining substantially pure preparations of dimeric active polypeptide-IgG fusion proteins. The method is generally implemented as follows: obtaining host cells capable of expressing IgG fusion proteins, collecting the conditioned medium, and purifying dimeric fusion proteins from monomeric fusion proteins, aggregates (aggregates) and contaminating proteins by column chromatography. Suitable host cells for expression of the IgG fusion protein include yeast, insect, mammalian or other eukaryotic cells. In one embodiment, the host cell may be a mammalian cell, in particular a COS, CHO or BHK cell.

Novel fusion proteins of a polypeptide drug and an Fc fragment are also provided. In one embodiment, a polypeptide drug such as EPO, p40, G-CSF, or TNF receptor is directly linked to the hybrid Fc fragment without the insertion of a peptide linker. In another embodiment, the polypeptide drugs are linked to each other by a peptide linker of 1 to 50 amino acids, and more preferably by a peptide linker of 1 to 7 amino acids. Particularly useful linkers for this purpose include immunologically inactive peptides consisting of Gly and Ser residues (e.g., glyglyserrglyglgyglgyglgyglgyglgyglgyglgyglgyglgyglgygorserryglgygorssesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesesese: 25 at position 282-314.

In the case when a linker is used, the linker and the polypeptide drug may be in a certain orientation. That is, the linker may be attached to the N-terminus, C-terminus, or free group of the hybrid Fc fragment, and may also be attached to the N-terminus, C-terminus, or free group of the polypeptide drug. When the linker is a peptide linker, the linkage may occur at a certain linkage site. When the polypeptide drug and the hybrid Fc are expressed separately and then linked to each other, the coupling may be performed using any one of a number of coupling agents known in the art. Non-limiting examples of coupling agents include 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, imidoesters including disuccinimide esters such as 3, 3' -dithiobis (succinimide propionate), and difunctional maleimides such as bis-N-maleimido-1, 8-octane.

The present invention also provides a method for producing the polypeptide drug-hybrid Fc fragment.

The present invention also provides methods of treating conditions that are alleviated by administration of a polypeptide agent. These methods comprise administering to a mammal having a condition, which may or may not be directly related to a disease of interest, an effective amount of a polypeptide of the invention. For example, a nucleic acid, such as DNA or RNA, encoding a desired polypeptide drug-hybrid Fc fragment fusion protein can be administered to a subject, preferably a mammal, as a therapeutic agent. In addition, cells comprising a nucleic acid encoding a polypeptide drug-hybrid Fc fragment fusion protein can be administered to a subject, preferably a mammal, as a therapeutic agent. In addition, the polypeptide drug-hybrid Fc fragment fusion construct can be administered to a subject, preferably a mammal, as a therapeutic agent. These chimeric polypeptides may be administered by intravenous, subcutaneous, oral, buccal (buccally), sublingual, nasal, parenteral, rectal, vaginal or pulmonary routes.

The EPO (including variants/fragments thereof) -fc fusion proteins of the invention can be used to increase and maintain hematocrit in mammals.

p40 is a subunit of IL-12. IL-12 is a75 kDa heterodimeric cytokine that has several functions in vivo. For example, IL-12 stimulates activated T and NK cell proliferation and promotes Th 1-type helper cell responses. IL-12 through binding to activated T and NK cells on the plasma membrane of IL-12 receptor play its biological effect, and IL-12 binding to IL-12 receptor ability has been attributed to IL-12 p40 subunit. Thus, the p40 (including variants/fragments thereof) -fc fusion proteins of the invention may be used (i) to reduce symptoms of, prevent/treat autoimmune disease, (ii) to inhibit transplant rejection, or (iii) to treat/prevent endotoxin-induced shock. Furthermore, the p40 (including variants/fragments thereof) -fc fusion protein of the invention may be used for the treatment/prevention/amelioration of the symptoms of rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, multiple sclerosis or psoriasis. Variants and fragments are known in the art, including but not limited to WO97/20062, the contents of which are incorporated herein by reference. One embodiment of a p40 variant includes, but is not limited to, p40 comprising an Asn303Gln substitution.

Granulocyte colony stimulating factor (G-CSF) is a protein essential for proliferation and differentiation of granulocytes, particularly neutrophils. Granulocytes engulf and phagocytose microbial invasion and cell debris, and are therefore crucial for the infection response. Chemotherapy destroys granulocytes and/or reduces the production of granulocytes. Thus, the G-CSF (including variants/fragments thereof) -fc fusion proteins of the present invention may be used to treat/prevent/ameliorate the symptoms of chemotherapy-induced neutropenia myelosuppression following bone marrow transplantation, acute leukemia, aplastic anemia, myelodysplastic syndrome, severe chronic neutropenia, or transplanted peripheral blood stem cell activation.

The fusion proteins of the present invention are not only useful as therapeutic agents, but those skilled in the art will recognize that the fusion proteins can be used to produce antibodies for diagnostic use. Likewise, appropriate administration of DNA or RNA, e.g., in vectors or other delivery systems for these uses, is also included in the methods of the invention.

The compositions of the present invention may be administered by any route that is compatible with the particular molecule. It will be appreciated that the compositions of the invention are provided to the animal by suitable means, either directly (e.g., topically, such as by injection, implantation, or topical application to a tissue site) or systemically (e.g., parenterally or orally). Where the composition is provided parenterally, such as by intravenous, subcutaneous, ocular, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital (intraorbital), intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intravesicular, intranasal, or by aerosol administration, the composition preferably comprises a partially aqueous or physiologically compatible liquid suspension or solution. Thus, the carrier or vehicle is physiologically acceptable so that it does not otherwise adversely affect the electrolyte and/or volume balance of the patient, other than to deliver the desired composition to the patient. Thus, the liquid medium of the medicament may comprise physiological saline.

The DNA constructs (or gene constructs) of the invention may also be used as part of a gene therapy approach to deliver nucleic acids encoding polypeptide drugs or fusion protein constructs thereof.

The invention features expression vectors for transfecting and expressing a polypeptide drug of interest or a fusion protein construct thereof in a particular cell type in vivo to recombine or supplement the function of the desired polypeptide drug. The expression construct, or fusion protein construct thereof, of the desired polypeptide drug can be administered in any biologically effective carrier, such as any formulation or composition capable of effectively delivering the gene encoding the desired polypeptide drug, or fusion protein construct thereof, to the cell in vivo.

The method comprises inserting a target gene into a viral vector, which includes recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-1, or recombinant bacteria or eukaryotic plasmids. For humans, the preferred dosage of nucleic acid encoding the fusion protein of the invention per administration is in the range of 0.1mg to 100mg, more preferably 1mg to 10mg, and most preferably 2mg to 10 mg. It will be appreciated that the optimum dose and manner of administration may be determined by routine experimentation well known to those skilled in the art.

For humans, the preferred dose of fusion protein per administration is in the range of 0.1mg to 1,000mg, more preferably 1mg to 100mg, and most preferably 5mg to 20 mg. However, it will be appreciated that the optimum dosage will also depend on the condition being treated and the presence of side effects. However, the optimum dosage can be determined using routine experimentation. Administration of the fusion protein may be by periodic bolus injection (periodicbolus injections), or by continuous intravenous, subcutaneous, or intraperitoneal administration from an external reservoir (e.g., from an intravenous infusion bag) or internally (e.g., from a bioerodible implant).

Furthermore, it is to be understood that the fusion proteins of the present invention may also be administered to a recipient in need thereof, together with a variety of different biologically active molecules. However, it will be appreciated that the optimal combination, mode of administration, dosage of fusion protein and other molecules may be determined by routine experimentation well known in the art.

MODE OF THE INVENTION

The invention is further illustrated by the following non-limiting examples.

< example 1> preparation of expression vectors for hFc-1, hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 fusion proteins.

The hFc-1 comprises 9 amino acids (90-98) at the C-terminal of the IgG1CH1 region, the hinge region (99-113) of IgG1, 6 amino acids (111-116) at the N-terminal of the IgG2CH2 region, 103 amino acids (118-220) of the IgG4CH2 region, and 107 amino acids (221-327) of the IgG4CH3 region (FIGS. 1 and 2). The amino acid sequence of hFc-1 is shown in seq id no: 18, respectively. In order to obtain codon-optimized nucleotides each encoding respectively hFc-1(SEQ ID NO: 1), human EPO (SEQ ID NO: 7), human G-CSF (SEQ ID NO: 8) and human p40N303Q (derived from a mutant in which Asn is substituted for amino acid Gln at position 303 of the human p40 subunit) (the nucleotide sequence of p40N303Q is shown as SEQ ID NO: 9 and the amino acid sequence of human p40 is shown as SEQ ID NO: 17), these nucleotide molecules were synthesized by the custom service (cumStoervice) of TOPGene technologies (Quebec, Canada) (www.topgenetech.com). In order to increase the protein expression level, it is very helpful to optimize the codon usage of the gene. The manner in which codons are used varies between organisms. Some codons are used more frequently in one organism and rarely in another. This bias in codon usage has been attributed to the translational efficiency, the ability of the organism to synthesize the encoded protein. To insert each fusion gene into the expression vector pAD11(SEQ ID NO: 10), an EcoRI site was generated 5 'to the ATG sequence of EPO, G-CSF and p40N303Q and an XbaI site was generated 3' to the stop codon of hFc-1. The expression vector pAD11 was obtained from the RcCMV backbone (available from Invitrogen, Carlsbad). pAD11 includes a promoter derived from Cytomegalovirus (CMV), a poly (A) sequence derived from bovine growth hormone, a globin intervening sequence (gIVS) derived from rabbit beta globin (MolCellBiol, 19888: 4395), and the like. To prepare the pAD11 vector, there were several modifications of the RcCMV vector (Invitrogen). The neomycin resistance region was removed by treatment with XhoI enzyme and gIVS was added 3' of the CMV promoter region. In addition, a mouse dihydrofolate reductase (DHFR) gene (Pubmed, NM010049) was added 5' to the CMV promoter. In combination with several elements including these, the pAD11 vector was formed after a number of expression experiments. In our unpublished results, the pAD11 vector showed an approximately 12-fold increase in expression levels compared to the RcCMV vector (Invitrogen). To generate a ligation site within the reading frame (frame) between the 3 'end of EPO, G-CSF and p40N303Q and the 5' end of hFc-1, NheI sites were generated at the 3 'end of the coding sequence of EPO, G-CSF and p40N303Q and at the 5' end of the coding sequence of hFc-1. After subcloning using each restriction enzyme site, the final expression vector of hFc-1 fused to EPO, G-CSF or p40N303Q was generated and then named pAD11EPO-hFc-1, pAD11G-CSF-hFc-1 and pAD11p40N303Q-hFc-1, respectively.

The amino acid sequences of hFc-2, hFc-3, hFc-4, hFc-5, and hFc-6 are shown in SEQ ID NOs: 19-23. The hFc-6 comprises 9 amino acids (90-98) of the IgDCH1 domain at the C-terminal, 64 amino acids (99-162) of the IgD hinge region, 8 amino acids (shtqplgv163-170) of the IgDCH2 domain at the N-terminal, 100 amino acids (121-220) of the IgG4CH2 domain and 107 amino acids (221-327) of the IgG4CH3 domain (FIGS. 1 and 2). To obtain codon-optimized nucleotide molecules encoding hFc-6(SEQ ID NO: 6), genes were synthesized by a custom service of TOPGene technologies (www.topgenetech.com). To prepare the fusion in frame between the 3 'end of EPO, G-CSF or p40N303Q and the 5' end of hFc-6, the NheI site (gctagc: Ala-Ser) included in the N-terminal coding region (90 and 91 amino acids) of hFc-6 was used. Also, to insert each hFc-6 fusion gene into the pAD11 vector, an XbaI site was created at the 3' end of the hFc-6 gene. After subcloning using each restriction enzyme site, the final expression vector of hFc-6 fused with EPO, G-CSF and p40N303Q was produced and then named pAD11EPO-hFc-6, pAD11G-CSF-hFc-6 and pAD11p40N303Q-hFc-6, respectively. hFc-2, hFc-3, hFc-4 and hFc-5 have identical CH2 and CH3 regions, but they have differently sized IgD hinges (FIGS. 1 and 2). The hFc-2(SEQ ID NO: 19), hFc-3(SEQ ID NO: 20), hFc-4(SEQ ID NO: 21) and hFc-5(SEQ ID NO: 22) respectively comprise 5 amino acids (158-162), 10 amino acids (153-162), 20 amino acids (143-162) and 30 amino acids (133-162) of the C-terminal IgD hinge (FIGS. 1 and 2). To prepare fusion genes between EPO, G-CSF, p40N303Q or TNFR (tumor necrosis factor receptor II) (SEQ ID NO: 30) and the nucleic acid molecules encoding these hFcs (SEQ ID NOS: 2-5), the gene fragment that was the smallest in the total size of the fusion gene was synthesized by the customization service of TOPGene technologies (www.topgenetech.com). Synthetic fragments of each EPO, G-CSF, p40N303Q or TNFR fused to a nucleotide molecule encoding the hinge and N-terminal CH2 region of each hFc-2, hFc-3, hFc-4 or hFc-5 include sequences ranging from the entire EPO, G-CSF, p40N303Q or TNFR sequence to the same enzyme site, the BstEII site (GGTGACC) located at amino acid residue 138-140 of the CH2 region in IgG4(SEQ ID NO: 13). The subcloned vector containing several gene fragments was cleaved with EcoRI and BstEII at the 5 'and 3' ends, respectively, and then ligated to the CH2-CH3 regions of hFc-6. Finally, each of the fusion genes was subcloned into pAD11 using EcoRI and XbaI sites, and then named pAD11EPO-hFc-2, pAD11EPO-hFc-3, pAD11EPO-hFc-4, pAD11EPO-hFc-5, pAD11G-CSF-hFc-2, pAD11G-CSF-hFc-3, pAD11G-CSF-hFc-4, pAD11G-CSF-hFc-5, pAD11p40N303Q-hFc-2, pAD11p40N303Q-hFc-3, pAD11p40N303Q-hFc-4, pAD11p40N303Q-hFc-5, and pAD11TNFR-hFc-5, respectively.

< example 2> preparation of expression vectors for thFc-1 and thFc-2 coupled to IFN-b

the thFc-1 comprises 23 amino acids (MDAMLRGLCCVLLLCGAVFVSPS) of the signal sequence of human tissue plasminogen activator (tPA), 15 amino acids (99-113) of the hinge region of IgG1, 6 amino acids (111-116) of the N-terminal IgG2CH2 region, 103 amino acids (118-220) of the IgG4CH2 region, and 107 amino acids (221-327) of the IgG4CH3 region (fig. 3). the amino acid sequence of thFc-1 is shown in SEQ ID NO: 28 (c). the thFc-2 comprises 23 amino acids (MDAMLRGLCCVLLLCGAVFVSPS) of the tPA signal sequence, 15 amino acids (148-162) of the IgD hinge region, 8 amino acids (163-170) of the N-terminal IgDCH2 region, 100 amino acids (121-220) of the IgG4CH2 region and 107 amino acids (221-327) of the IgG4CH3 region (FIG. 3). the amino acid sequence of thFc-2 is shown in SEQ ID NO: 29 (b). These nucleotide molecules were synthesized by the custom service of TOPGene technologies (Quebec, Canada) (www.topgenetech.com) in order to obtain codon-optimized nucleotides encoding thFc-1(SEQ ID NO: 26) or thFc-2(SEQ ID NO: 27) coupled to the N-terminus of human IFN- β from which its signal sequence was deleted. To insert each fusion gene into the expression vector pAD11(SEQ ID NO: 10), an EcoRI site was created at the 5 'end of either thFc-1 or thFc-2 and a NotI site was created at the 3' end of the IFN- β stop codon. After subcloning using each restriction enzyme site, the final expression vectors were named pAD11thFc-1-AL (0) -IFN- β and pAD11thFc-2-AL (0) -IFN- β, respectively.

To prepare a thFc conjugated to IFN- β via albumin linkers of different sizes or a Gly-Ser linker, a gene fragment ranging from the PstI site of the CH3 region of thFc-1, which is conjugated to IFN- β from which its signal sequence was deleted via different albumin linkers (3aa, 8aa, 13aa, 18aa, 23aa, and 33aa) or a Gly-Ser linker (15aa), was synthesized by the custom service of TOPGene technologies (www.topgenetech.com) (FIG. 4). To insert 7 different sized gene fragments into the expression vectors pAD11thFc-1-AL (0) -IFN- β and pAD11thFc-2-AL (0) -IFN- β, PstI sites were generated at their 5 'ends and NotI sites were generated at the 3' end of the IFN- β stop codon. After subcloning using each restriction enzyme site, the final expression vectors were designated pAD11thFc-1-AL (1) -IFN- β, pAD11thFc-1-AL (2) -IFN- β, pAD11thFc-1-AL (3) -IFN- β, pAD11thFc-1-AL (4) -IFN- β, pAD11thFc-1-AL (5) -IFN- β, pAD11thFc-1-AL (6) -IFN- β, pAD11thFc-1-GS-IFN- β, and pAD11thFc-2-AL (1) -IFN- β, pAD11thFc-2-AL (2) -IFN- β, pAD11thFc-2-AL (3) -IFN- β, pAD11thFc-2-AL (4) -IFN- β, pAD11thFc-1-AL (4) -IFN- β, pAD11thFc-2-AL (4) -IFN- β, and pAD11thFc-1-AL (4) -IFN- β, respectively, pAD11thFc-2-AL (5) -IFN- β, pAD11thFc-2-AL (6) -IFN- β pAD11, and thFc-2-GS-IFN- β.

< example 3> expression of human EPO-hFcs, human G-CSF-hFcs, human p40N303Q-hFcs, human TNFR-hFc-5 and thFcs-IFN-beta proteins

COS-7 cells were used for the expression assay and cultured with DMEM medium (Invitrogen, Carlsbad) supplemented with 10% fetal bovine serum (Hyclone, SouthLogan) and antibiotics (Invitrogen, Carlsbad). Vectors encoding EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5, thFcs-IFN-beta were transfected into 5X 10 cells using conventional electroporation⁶COS-7 cells. At 48h post-transfection, supernatants and cells were harvested. To detect the expression of the fusion protein per vector, all samples were passed through several kits (R)&System D, Minneapolis, # DEP00, for EPO; biosource, Camarillo, # KHC2032 for G-CSF; r&System D, Minneapolis, # DY1240 for p40N 303Q; r&System D, Minneapolis, # DRT200 for TNFR, pblbiomedia laboratories, #41410-1A for IFN- β) for ELISA assays and western blot analysis by anti-human IgG antibodies (santa cruz biotechnology, santa cruz). As a result, all vectors showed the correct expression pattern in both supernatant and cell lysates (data not shown).

< example 4> purification of hFc-fusion protein

CHO/DHFR cultured with a-MEM (Invitrogen, Carlsbad), 10% dialyzed fetal bovine serum (JRHBiosciences, Kansas), HT supplements (HTsupplements) (Invitrogen, Carlsbad) and antibiotics (Invitrogen, Carlsbad)^-/-Cells (chinese hamster ovary cells, DG44, ATCC). According to the conventional CaPO₄Co-precipitation method, expression vector was transfected into CHO cells. At 48h post transfection, CHO cells were isolated from the plates and diluted in multiples (1/2, 1/5, 1/20, 1/50, 1/100, 1/200, 1/500). The diluted cells were plated onto 100mm petri dishes and cultured with medium without HT supplement. During the screening process, fresh medium without HT supplement was provided to the cells without passage. Colonies were generated 2-3 weeks after plating and single colonies were transferred to 48-well plates. Positive colonies were screened after ELISA assays for EPO, G-CSF, p40N303Q and TNFR detection. Each colony showing the highest expression was cultured on a large scale (5L) using serum-free medium (JRHBiosciences, Kansas). The harvested serum-free supernatant was used for purification of each fusion protein. For purification, the HiTrap recombinant protein AFF (Amershambiosciences, Piscataway) column was equilibrated with 20mM sodium phosphate (pH 7.0). The filtered supernatant was added to the column and eluted with 0.1M sodium citrate (ph 3.0). After dialysis three more times with a membrane (MWCO1214K, Spectrapor, rancho dominguez), the eluted protein was finally obtained. The concentration of all protein samples was determined by BCA kit (Pierce Biotechnology, Rockford) for measuring total protein and ELISA kit for measuring EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5 and thFcs-IFN- β.

< example 5> FcgRI and C1q binding assay

To investigate whether hFc-5 fusion proteins bound to FcgRI and C1q, MabThera (Rituximab, Roche), hIgG1(Calbiochem, Cat #, 400120),(etanercept, Amgen), EPO-hFc-5, G-CSF-hFc-5 and p40N303Q-hFc-5 were serially diluted (from 2-fold from 2ug/ml to 16ng/ml) and coated on 8-well strips (COSTAR, new york)4 degrees overnight (overnight 4). To make a standard curve, FcgRI (R)&D, Cat # BAF1257) or C1q (AbDserotech, Cat #.2221-5504) were also serially diluted (2-fold from 2ug/ml to 32ng/ml) and coated on 8-well strips (COSTAR, NewYork) overnight at4 degrees. After washing each band of the sample with washing solution (PBS containing 0.05% tween) at room temperature and blocking with 10% FBS in PBS for 1 hour, FcgRI or C1q was added to each well at 2ug/ml, followed by incubation at room temperature (roomthermature, RT) for 2 hours. All belts were washed with washing solution. For the C1q binding assay, HRP-conjugated anti-C1 q (AbDserotech, cat # 2221-5004P) was added to each well at 2.5ug/ml and then incubated for 30min at room temperature in the dark. For FcgRI binding assays, biotinylated anti-FcgRI (R) was used&D, cat # 1257-FC) was added to each well at 2ug/ml and then incubated for 1 hour at room temperature. After washing them with the washing solution, 3,000-fold dilution of Streptavidine-HRP (BD, cat # 554066) was added to each band, followed by incubation for 30 minutes at room temperature under dark conditions. After washing the strip, TMB solution (1: 1 mixture of TMB peroxide substrate and peroxide substrate solution B, KPL, cat # 50-76-01, cat # 50-65-00) was added for color development and 2NH was added₂SO₄For stopping the color development. As shown in figures 6(a) and 6(b),and hIgG1 showed good binding to FcgRI and C1q, but not EPO-hFc-5, G-CSF-hFc-5 and p40N 303Q-hFc-5.

< example 6> in vitro biological Activity of purified hFc fusion protein

To investigate the in vitro biological activity of EPO-hFc proteins, the human F35E cell line was cultured in RPMI1640 medium (Cambrex, charles city) supplemented with 10% FBS, antibiotics and 5IU/ml recombinant human EPO (DongA, republic of korea). By mixing 2X 10⁴The individual cells were seeded into assay wells of a 96-well cell culture plate (Corning, Netherlands) to perform bioassay. Serially diluted samples (from 5-fold from 0, 0.064mIU/ml to 25IU/ml) of EPO, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6, EPO-IgG1Fc or Aranesp (darbepoetinalfa, Amgen) were added to these wells and the plates were incubated at 5% CO in the wet state₂Incubate at 37 ℃ for 72 hours in an incubator. MTT assay was performed by using a cell growth colorimetric assay kit (Sigma-aldrich. korea) according to the manufacturer's protocol. The human F35E cell line showed a strong proliferative response to rhepo as demonstrated by a dose-dependent manner in cell number and uptake values. Coupled to IgG1Fc or hFcs as shown in FIG. 7(a)And the EPO protein showed loss of biological activity compared to the EPO protein. However, EPO-hFc-1, EPO-hFc-5 and EPO-hFc-6 showed significantly higher biological activities than EPO-IgG1 Fc. Furthermore, EPO-hFc-5 and EPO-hFc-6 showed slightly higher levels thanThis indicates that these hFc fusion proteins appear to be better than EPO proteins in maintaining biological activity

To investigate the in vitro bioactivity of the G-CSF-hFc protein, recombinant mouse IL-3 (R) was added with 10% FBS, antibiotics and 100units/ml&Dsystem, Minneapolis) in RPMI1640 medium (Cambrex, Charles City). By mixing 2X 10⁴Cells were seeded into wells of 96-well cell culture plates (Corning, Netherlands) to establish bioassays. Serial dilutions (3-fold) of G-CSF-hFc-5 and Neula (Amgen)From 0 to 10,000pg/ml) was added to the wells and the plates were incubated in a humidified 5% CO₂Incubate at 37 ℃ for 72 hours in an incubator. Protein samples were assayed in triplicate wells and the experiment was repeated 5 times. At 72 hours after incubation, MTT assay was performed by using a cell growth colorimetric assay kit (Sigma-aldrich. korea) according to the manufacturer's method. As illustrated in FIG. 7(b), G-CSF-hFc-5 showed slightly higher thanThe in vitro biological activity of (a).

To study the in vitro bioactivity of the p40N303Q-hFc protein, 2ug/ml of anti-human CD3 antibody (R) was used in RPMI1640 medium (Cambrex, Charles City) supplemented with 10% FBS and antibiotics&Dsystem, # MAB100) with or without 10ng/ml human p40 (R)&Dsystem) or p40N303Q-hFc-5 Peripheral Blood Mononuclear Cells (PBMCs) from rheumatoid arthritis patients were incubated. After 6 days, cells positive for CD4 and IL-17 were determined by FACS analysis. As shown in fig. 7(c), for CD4⁺/IL-17⁺The generation of cells, p40N303Q-hFc-5, showed stronger inhibitory effect than p40 protein, indicating the inhibitory function of p40N303Q-hFc-5 on Th17 polarization.

To study the in vitro biological activity of the TNFR-hFc protein, murine L929 cells were cultured in RPMI1640 medium (Cambrex, Charles City) supplemented with 10% FBS and antibiotics. By mixing 3X 10⁴Individual cells were seeded into wells of 96-well cell culture plates (Corning, Netherlands) for cytopathic inhibition assays, and then treated with 1ng/ml TNF-a. TNFR-hFc-5 andsamples of serial dilutions (from 15.6 to 1,000ng/ml 2-fold) of (etanercept, Amgen) were added to the wells and the plates were incubated in wet 5% CO₂The culture was carried out in an incubator at 37 ℃ for 48 hours. After incubation, MTT assay was performed by using a cell growth colorimetric assay kit (Sigma-aldrich, Korea) according to the manufacturer's method. As illustrated in fig. 7(d), TNFR-hFc-5 showed slightly higher thanThe in vitro biological activity of (a).

To investigate the in vitro biological activity of the thFc-1-AL (0) -IFN- β and of the thFc-1-AL (3) -IFN- β proteins, WISH cells (ATCC, CCL-25) were cultured in DMEM/F12(Cambrex, Charles City) supplemented with 10% FBS and antibiotics. By mixing 3X 10⁴Individual cells were seeded into wells of 96-well cell culture plates (Corning, Netherlands) for cytopathic inhibition assays and then treated with 1,500 PFU/well of VSV (ATCC, VR-158). Samples of serial dilutions (2-fold from 40IU/ml) of recombinant IFN- β (WHO standard, NIBSC00/572), thFc-1-AL (0) -IFN- β, and thFc-1-AL (3) -IFN- β proteins were added to these wells and the plates were incubated in humidified 5% CO₂Incubate at 37 ℃ for 48 hours in an incubator. After incubation, MTT assay was performed by using a cell growth colorimetric assay kit (Sigma-aldrich. korea) according to the manufacturer's method. As illustrated in FIG. 7(e), thFc-1-AL (3) -IFN- β showed about 20-fold higher in vitro biological activity than thFc-1-AL (0) -IFN- β, indicating the important role of the albumin analog (albominliker) in maintaining the biological activity of IFN- β fused to Fc.

< example 7> in vivo half-life of purified hFc fusion protein

To compare the half-lives of EPO-hFc-1, EPO-hFc-5 and Aranesp, 15 macaques were treated with these proteins at a dose of 2,400IU/kg via a single Subcutaneous (SC) injection or a single Intravenous (IV) injection. Blood samples were obtained from each monkey before injection and at 1, 3, 6, 12, 24, 30, 48, 54, 72, 78, 96, 120, 168, 336, 504 and 672 hours after injection. The blood samples were incubated at room temperature for 30min to allow coagulation. After centrifugation at 3000rpm for 10min, serum was obtained for each sample and stored in a deep freezer. All samples obtained at each point were passed through an EPOELISA kit (R)&D, cat #. DEP00) was subjected to quantitative determination of EPO. As shown in FIG. 8(a), all monkey individuals injected with EPO-hFc-1 or EPO-hFc-5 via SC or IV route showed a higher indication than those injected via SC or IV routeShooting deviceThe monkey individual has a longer half-life.

To study the pharmacokinetics of G-CSF-hFc-1, 100ug/kg of LEUCOSTIM (filgrastim, DongA, Reublica Korea) was used as a control and 100ug/kg of G-CSF-hFc-1 was administered via SC or IV route to 2 male Sprague Dawley rats (Charles river laboratories, Wilmington) per group. Blood was obtained before injection and 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120 and 192 hours after injection. After incubation at room temperature for 30min, serum was obtained by centrifugation at 3,000rpm for 10min and stored in a deep freezer. Samples were quantified using a G-CSF kit (Biosource, Camarillo, # KHC2032) with several dilution factors such as 1/2, 1/5, 1/50, 1/250, 1/500. As shown in FIG. 8(b), G-CSF-hFc-1 injected via SC or IV route showed a ratioLonger half-life, G-CSF-hFc-1 and G-CSF have in vivo t-after SC administration of 8.76h and 2.36h, respectively_1/2And in vivo t of 10.42h and 1.78h, respectively, after IV administration_1/2. Thus, is obtained byIn contrast, G-CSF-hFc-1 showed a 3.7-fold increase following SC injection and a 5.9-fold increase following IV injection.

To study p40N303Q-hFc-5 andpharmacokinetics of (3) macaques per group were treated with a single SC injection at a dose of 100 ug/kg. Blood samples were obtained from each monkey at pre-injection and 8, 24, 48, 72, 96, 120, 168, 336, 504 and 672 hours post-injection. The blood was incubated at room temperature for 30min to allow it to clot. After centrifugation at 3000rpm for 10min, serum was obtained for each sample and stored in a deep freezer. By means of ELISA kits (R in each case)&Dsystem，Minneapolis, # DY1240 and # DRT200), quantitative assays of human p40 and human TNFRII were performed on all samples obtained at each point. As shown in FIG. 8(c), p40N303Q-hFc-5 shows a ratioLong half-life (average 199h to 127h) -although p40N303Q-hFc-5 shows a ratioLow Cmax values (mean 3ng/ml to 7 ng/ml).

To study TNFR-hFc-5 and3 male sprague dawley rats (charles river laboratories, Wilmington) per group were treated with a single SC injection at a dose of 500 ug/kg. Blood samples were obtained from each rat before and 2,4, 8, 12, 24, 30, 48, 72 and 120 hours after injection. The blood samples were incubated at room temperature for 30min to allow coagulation. After centrifugation at 3,000rpm for 10min, serum was obtained for each sample and stored in a deep freezer. By ELISA kit (R)&Dsystem, Minneapolis, # DRT200) performed a quantitative assay for human TNFRII on all samples obtained at each spot. As shown in FIG. 8(d), TNFR-hFc-5 showed slightly higher levels thanAUC levels of (average 198.1 to 172.9ug h/ml) -although TNFR-hFc-5 was shown to be comparable toSimilar half-life (average 28.6h to 29.4 h).

< example 8> in vivo biological Activity of purified hFc fusion protein

To compare EPO-hFc-5 withIn vivo growth ofPhysical Activity 3 macaques per group were treated with a single IV injection at a dose of 2,400 IU/kg. Blood samples were obtained from each monkey before injection and at 1, 3, 6, 12, 24, 30, 48, 54, 72, 78, 96, 120, 168, 336, 504 and 672 hours after injection. Measurement of the number of various blood cells including reticulocytes to evaluate EPO-hFc-5 andthe in vivo biological activity of (a). As shown in FIG. 9(a), EPO-hFc-5 showed slightly higher levels in both SC and IV pathways in increasing monkey reticulocytes than did EPO-hFc-5In vitro potency of (a).

To investigate the in vivo biological activity of G-CSF-hFc-1, one will(filgrastim, DongA, republic korea) as a control and G-CSF-hFc-1 was administered via SC or IV route to two male sprague dawley rats (charles river laboratories, Wilmington) per group at a dose of 100 ug/kg. Blood was obtained using EDTA tubes before and 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120, and 192 hours after injection. Each blood sample was treated with RBC lysate (BDBioscience, Korea) for 4 minutes and total WBCs (white blood cells) diluted in FACS buffer were counted three times using a hemocytometer. The granulocyte count was measured using a FACS machine by measuring cell size using FSC (forward scatter) and particles using SSC (side scatter). Treated via SC and IV routes as shown in FIG. 9(b)Peak numbers of WBCs and granulocytes were elicited 24 hours post-injection, whereas G-CFS-hFc-1 elicited peak numbers of WBCs and granulocytes 72 hours post SC injection and 48 hours post IV injection. From 24h to 120h after injection, withCompared with the G-CSF-hFc-1, the biological activity in vivo is more durable.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Industrial applicability

The present invention discloses fusion proteins comprising a biologically active molecule and an immunoglobulin (Ig) Fc domain linked to the biologically active molecule. The Fc domain is a hybrid human Fc domain of (i) IgG1, IgG2, or IgG4, or (ii) IgG4 and IgD. Hybrid Fc is used as a carrier for biologically active molecules.

Claims (11)

1. A polypeptide represented by the formula:

N'-(Z1)_p-Y-Z2-Z3-Z4-C'

wherein N 'is the N-terminus and C' is the C-terminus of the polypeptide;

z1 is an amino acid sequence consisting of amino acid residues at positions 90 to 98 of (i) SEQ ID NO:11 or (ii) SEQ ID NO: 14;

y is an amino acid sequence consisting of amino acid residues in positions 133 to 162 of SEQ ID NO. 14;

z2 is an amino acid sequence consisting of the amino acid residues in positions 163 to 170 of SEQ ID NO: 14;

Z3-Z4 is an amino acid sequence consisting of a contiguous amino acid sequence of amino acid residues at positions 121 to 220 of SEQ ID NO. 13 and amino acid residues 221 to 327 of SEQ ID NO. 13; and

p is an integer of 0 or 1.

2. The polypeptide of claim 1, wherein p is 0.

3. A chimeric polypeptide consisting of the polypeptide of any one of claims 1 and 2 and a biologically active polypeptide, wherein said biologically active polypeptide is fused to the N-terminus or C-terminus of said polypeptide, and wherein said biologically active polypeptide exhibits an increased circulatory half-life compared to the circulatory half-life of the native form of said biologically active polypeptide, and said biologically active polypeptide is EPO, G-CSF, TNF receptor or p 40.

4. The chimeric polypeptide of claim 3, wherein the biologically active polypeptide is a p40 variant, said p40 variant comprising an Asn303Gln substitution.

5. The chimeric polypeptide of claim 3, wherein the polypeptide and the biologically active polypeptide are coupled to each other via an albumin linker or a synthetic linker.

6. The chimeric polypeptide of claim 5, wherein:

(a) the albumin linker consists of the amino acid sequence 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333, or 306 to 338 of seq id No. 25; or

(b) The synthetic linker is a peptide represented by SEQ ID NO: 32.

7. A nucleic acid molecule encoding a polypeptide according to any one of the preceding claims 1-6.

8. An expression vector comprising the nucleic acid molecule according to claim 7.

9. A method of producing a polypeptide according to any one of claims 1-6, wherein said method comprises the steps of: (i) introducing the nucleic acid molecule of claim 8 into a mammalian host cell, (ii) growing said cell in its growth medium under conditions in which said polypeptide can be expressed; and (iii) harvesting the expressed polypeptide.

10. Use of the chimeric polypeptide of claim 3 in the manufacture of a medicament for increasing the circulating half-life of the biologically active polypeptide.

11. The use of claim 10, wherein the chimeric polypeptide is administered intravenously, subcutaneously, orally, buccally, sublingually, nasally, parenterally, rectally, vaginally, or via a pulmonary route.