HK1215263B - Gla domains as targeting agents - Google Patents

Description

GLA domains as targeting agents

background

This application claims the benefit of priority to U.S. Provisional Application Serial No. 61/787,753, filed March 15, 2013, and U.S. Provisional Application Serial No. 61/791,537, filed March 15, 2013, the entire contents of which are hereby incorporated by reference.

1. Field

The present disclosure relates to targeting phosphatidylserine (PtdS) on cell membranes using Gla domain peptides and polypeptides. The use of these peptides and polypeptides as diagnostic and therapeutic agents is disclosed.

2. Related Technologies

Phosphatidylserine (PtdS) is a negatively charged phospholipid component that is often located in the inner leaflet (cytoplasmic side) of the cell membrane. However, PtdS can be transported from the inner leaflet to the outer leaflet by scramblase (a member of the flippase family) and exposed on the cell surface. With rare exceptions, this active externalization of PtdS is a response to cell damage (van den Eijnde et al. , 2001; Erwig and Henson, 2008). For example, tissue damage signals platelets, leukocytes, and endothelial cells to quickly and reversibly redistribute PtdS, which leads to the promotion of coagulation and complement activation on the cell surface. Similarly, apoptosis signals lead to the externalization of PtdS, but in a more gradual and continuous manner. This external PtdS provides a key recognition marker that enables macrophages to ingest dying cells from surrounding tissues (Erwig and Henson, 2008). This removal process is essential for tissue homeostasis and, in a "healthy" environment, it is very effective. In fact, despite the loss of >10 ⁹ cells per day, histological detection of apoptotic cells is a rare event in normal tissues (Elltiot and Ravichandran, 2010; Elltiot et al. , 2009). However, there is evidence that in many pathological conditions, the removal process of apoptotic cells is overwhelmed, delayed, or absent (Elltiot and Ravichandran, 2010; Lahorte et al. , 2004). For example, several oncology studies suggest that a high apoptotic index is associated with higher grade tumors, increased metastasis rates, and poor patient prognosis (Naresh et al. , 2001; Loose et al. , 2007; Kurihara et al. , 2008; Kietselaer et al. , 2002). These studies and others similar to them suggest that apoptosis and external PtdS expression can be powerful markers of disease (Elltiot and Ravichandran, 2010).

There are several proteins with high affinity for anionic phospholipid surfaces, of which annexin-V is the most widely used as a PtdS targeting probe (Lahorte et al. , 2004). Due to its high affinity for PtdS-containing vesicles ( _Kd = 0.5-7nM) and a molecular weight (37 kDa) below the renal filtration threshold (approximately 60 kDa), annexin-V has shown promise as a cell apoptosis probe in the clinic (Lin et al. , 2010; Tait and Gibson, 1992). In addition, it has been used for a wide range of indications, including those in oncology, neurology, and cardiology (Lahorte et al. , 2004; Boersma et al. , 2005; Blankenberg, 2009; Reutelingsperger et al. , 2002). The use of biological probes targeting PtdS cell surface expression has been demonstrated in vitro and in vivo. Although their practicality in the clinic is promising, they are generally underdeveloped.

Summary of the Invention

Thus, according to the present disclosure, a method for targeting cell membrane phosphatidylserine (PtdS) is provided, comprising: (a) providing an isolated polypeptide comprising a γ-carboxyglutamate (Gla) domain and lacking a protease or hormone binding domain; and (b) contacting the peptide with a cell surface, wherein the polypeptide binds to PtdS on the cell membrane. The cell membrane can be a cardiac cell membrane, a neuronal cell membrane, an endothelial cell membrane, a virally infected cell membrane, an apoptotic cell membrane, a platelet membrane, or a cancer cell membrane. The polypeptide can further comprise an EGF binding domain, a Kringle domain, and/or an aromatic amino acid stacking domain. The Gla domain can be from factor II, factor VII, factor IX, factor X, protein S, or protein C.

The polypeptide may further comprise a detectable label, such as a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme, a dye, or a ligand. The polypeptide may further comprise a therapeutic agent, such as an anticancer agent, including a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a hormone, an antibody or antibody fragment, or a toxin or an antiviral agent. The therapeutic agent may be an enzyme, such as a prodrug-converting enzyme, a cytokine, a growth factor, a coagulation factor, or an anticoagulant. The polypeptide may be 300 residues or less, 200 residues or less, or 100 residues or less, including ranges of 100-200 and 100-300 residues.

The polypeptide may comprise 5-15 Gla residues, 9-13 Gla residues, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gla residues. The polypeptide may comprise more than 13 Gla residues, but less than 30% of the total Gla residues. The size of the polypeptide may be between about 4.5 and 30 kD. The polypeptide may comprise at least one disulfide bond or 2-5 disulfide bonds. The polypeptide may comprise a Protein S Gla domain. The polypeptide may comprise a Protein S Gla domain + a Protein S EGF domain, a prothrombin Gla domain, a prothrombin Gla domain + a prothrombin Kringle domain, a Protein Z Gla domain, a Protein Z Gla domain + a prothrombin Kringle domain, a Factor VII Gla domain, or a Factor VII Gla domain + a prothrombin Kringle domain. The polypeptide may further comprise an antibody Fc region. Any of the aforementioned polypeptides may contain conservative substitutions of the native sequences of the aforementioned proteins, and/or exhibit a percent homology to the described native domains.

In another embodiment, a method of treating a cancer in a subject is provided, comprising administering to the subject an isolated polypeptide comprising a gamma-carboxyglutamate (Gla) domain and lacking a protease or hormone binding domain, wherein the polypeptide is linked to a therapeutic payload. The therapeutic payload can be a chemotherapeutic agent, a radiotherapeutic agent, or a toxin. The cancer can be breast cancer, brain cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, skin cancer, rectal cancer, cervical cancer, uterine cancer, liver cancer, pancreatic cancer, head and neck cancer, or esophageal cancer.

In another embodiment, a method of treating a viral disease in a subject is provided, the method comprising administering to the subject an isolated polypeptide comprising a gamma-carboxyglutamate (Gla) domain and lacking a protease or hormone binding domain, wherein the polypeptide is linked to an antiviral agent. The viral disease can be influenza, human immunodeficiency virus, dengue virus, West Nile virus, smallpox virus, respiratory syncytial virus, Korean hemorrhagic fever virus, chickenpox, varicella-zoster virus, herpes simplex virus 1 or 2, Epstein-Barr virus, Marburg virus, hantavirus, yellow fever virus, hepatitis A, B, C or E, Ebola virus, human papillomavirus, rhinovirus, coxsackievirus, poliovirus, measles virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, HTLV-1 and -2.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

Other objects, features and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, as those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present disclosure from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by referring to one or more of these drawings in combination with the detailed description.

Figure 1 – Construction of a panel of Gla and Gla-EGF/Kringle domain proteins.

Figure 2 - Assay for the expression of Gla-domain protein constructs. Transient transfection into 293 cells using 293cellFectin. 10% gel containing reduced samples, loaded with 23.3 µl of culture medium.

Figure 3 - Assays for the expression of Gla-domain protein constructs. Transient transfection in BHK21 cells. 10% gel containing reduced samples, 20 µl (1/100 total cell pellet) loaded.

Figure 4 - Changing the signal sequence alters secretion . Transient transfection in BHK21 cells. 10% gel containing reduced sample, 13.3 µl loaded.

Figure 5 - Protein S Gla + EGF sequence .

Figure 6 - Purification of Protein S Gla + EGF . F1-F4 are column chromatography fractions. 10% gel, non-reducing conditions.

Figure 7 - Apoptosis assay on Protein S Gla + EGF. The upper and lower panels represent identical replicates, but with reduced amounts of Protein S Gla + EGF and reduced amounts of anti-His domain antibody.

Figure 8 - Apoptosis assay for Protein S Gla + EGF. The upper and lower panels represent identical replicates except for the amount of Annexin V used (which was doubled in the lower panel).

Description of Exemplary Embodiments

Like annexins, γ-carboxyglutamate (Gla)-domain proteins, such as Factors II, VII, IX, X, Protein C, and Protein S, bind to anionic membranes. Indeed, the Gla domain has been used as a model for the rational design of small molecules specific for apoptosis probes (Cohen et al. , 2009). Here, the inventors propose the use of membrane-targeting portions of these Gla domain proteins as a new class of bioprobes specific for apoptosis and disease. The use of these naturally occurring and targeted proteins could result in enhanced specificity relative to current probes, with the added advantage of a smaller size (<30 kDa). Even in larger embodiments (including EGF and/or Kringle domains), these proteins can still be smaller than Annexin V (37 kDa) and potentially as small as <5 kDa. These bioprobes can target PtdS cell surface expression in vitro and in vivo. Thus, it may be possible to develop apoptosis/disease-targeting probes that surpass Annexin V in affinity, specificity, and size, with the added potential for use as therapeutic agents. These and other aspects of the present disclosure are described in more detail below.

Where appropriate, terms used in the singular also include the plural form, and vice versa. For the purpose of interpreting this specification and its related claims, in the event that any definition set forth below conflicts with the usage of the term in any other document (including any document incorporated herein by reference), the definition set forth below will always prevail unless the contrary meaning is clearly indicated (for example, in the document in which the term is originally used). Unless otherwise stated, the use of "or" means "and/or". The application of "one" in this article means "one or more", unless otherwise stated or when the use of "one or more" is clearly inappropriate. The use of "including" and "comprising" is interchangeable and not restrictive. For example, the term "including" should mean "including, but not limited to". The word "about" means the number stated plus or minus 5%.

As used herein, "isolated peptide or polypeptide" is intended to mean a peptide or polypeptide that is substantially free of other biomolecules, including peptides or polypeptides having different sequences. In certain embodiments, the isolated peptide or polypeptide has a purity of at least about 75%, about 80%, about 90%, about 95%, about 97%, about 99%, about 99.9%, or about 100% by dry weight. In certain embodiments, purity can be measured by methods such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

" conservative substitution " used herein represents the modification of polypeptide, and it relates to the amino acid with similar biochemical properties with one or more amino acid replacements, and does not cause the loss of the biological or biochemical function of the polypeptide." conservative amino acid replacement " is the replacement of amino acid residues with amino acid residues having similar side chains. The family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with following side chains: basic side chains (for example, lysine, arginine, histidine), acidic side chains ( for example , aspartic acid, glutamic acid), uncharged polar side chains ( for example , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains ( for example , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains ( for example , threonine, valine, isoleucine) and aromatic side chains ( for example , tyrosine, phenylalanine, tryptophan, histidine). The antibody of the present disclosure can have one or more conservative amino acid replacements and still retain antigen-binding activity.

For nucleic acids and polypeptides, the term "substantial homology" indicates that two nucleic acids or two polypeptides, or designated sequences thereof, are identical in at least about 80% of the nucleotides or amino acids (typically at least about 85%, in certain embodiments, about 90%, 91%, 92%, 93%, 94%, or 95%, and in at least one embodiment, at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the nucleotides or amino acids), when optimally aligned and compared, with appropriate nucleotide or amino acid insertions or deletions. Alternatively, substantial homology exists for nucleic acids when the segments hybridize under selective hybridization conditions to the complement of the strand. Also included are nucleic acid sequences and polypeptide sequences having substantial homology to the specific nucleic acid sequences and amino acid sequences recited herein.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and percent identity determination between two sequences can be achieved using a mathematical algorithm, such as, but not limited to, the AlignX™ module of VectorNTI™ (Invitrogen Corp., Carlsbad, CA). For AlignX™, the default parameters for multiple alignment are: Gap Open Penalty: 10; Gap Extension Penalty: 0.05; Gap Separation Penalty Range: 8; % Identity of Alignment Delay: 40. (Additional details are available on the World Wide Web at invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Communities/Vector-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).

Another method for determining the best overall match between a query sequence (a sequence of the present disclosure) and a subject sequence (also known as a global sequence alignment) can be determined using the CLUSTALW computer program (Thompson et al. , Nucleic Acids Res , 1994, 2(22): 4673-4680), which is based on the algorithm of Higgins et al., Computer Applications in the Biosciences (CABIOS), 1992, 8(2): 189-191). In a sequence alignment, both the query and subject sequences are DNA sequences. The results of the global sequence alignment are calculated as percent identity. The parameters that can be used to calculate percent identity via pairwise alignment in the CLUSTALW alignment of DNA sequences are: matrix = IUB, k-tuple = 1, number of top diagonals = 5, gap penalty = 3, gap open penalty = 10, and gap extension penalty = 0.1. For multiple alignments, the following CLUSTALW parameters can be used: gap opening penalty = 10, gap extension parameter = 0.05; gap separation penalty range = 8; % identity of alignment delay = 40.

Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it is purified from other cellular components that normally accompany the nucleic acid in its natural environment. To isolate nucleic acids, standard techniques such as alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art can be used.

I. Phosphatidylserine (PtdS)

A. Structure and Synthesis

Phosphatidylserine (abbreviated PtdS, Ptd-L-Ser, or PS) is a phospholipid component that is often retained on the inner leaflet (cytosolic side) of the cell membrane by enzymes called flippases. When a cell undergoes apoptosis, phosphatidylserine is no longer confined to the cytosolic portion of the membrane but becomes exposed on the surface of the cell. The chemical formula of PtdS is C ₁₃ H ₂₄ NO ₁₀ P and has a molecular weight of 385.304. The structure is shown below:

Phosphatidylserine is biosynthesized in bacteria by the phosphatidic acid condensation of amino acid serine and CDP (cytidine diphosphate)-activation. In mammals, phosphatidylserine is produced by base exchange reaction with phosphatidylcholine and phosphatidylethanolamine. On the contrary, phosphatidylserine can also produce phosphatidylethanolamine and phosphatidylcholine, although in animals, the approach of producing phosphatidylcholine from phosphatidylserine only works in the liver.

B. Function

Early studies of phosphatidylserine distilled the chemical from cow brains. Because of concerns about mad cow disease, modern research and commercially available products are made from soy. The fatty acids attached to serine in soy products differ from those in bovine products and are also less pure. Preliminary studies in rats indicate that soy products are at least as effective as those derived from cattle.

The US FDA has granted "qualified health claim" status to phosphatidylserine, with narratives stating, "Consumption of phosphatidylserine may reduce the risk of dementia in older adults" and "Consumption of phosphatidylserine may reduce the risk of cognitive impairment in older adults."

Phosphatidylserine has been shown to accelerate recovery, prevent muscle soreness, improve well-being, and may have synergistic properties in athletes involved in period work, weight training, and endurance running. Soy-PtdS has been reported to be an effective supplement for combating exercise-induced stress (by blunting the increase in cortisol levels induced by exercise) in a dose-dependent manner (400 mg). PtdS supplementation promotes desirable hormone balance in athletes and may mitigate the physiological decline associated with training and/or overexertion. In recent studies, PtdS has been shown to enhance the mood of young people under mental stress and improve accuracy during tee shots by increasing stress resistance in golfers. The first preliminary study indicates that PtdS supplementation may be beneficial for children with attention deficit hyperactivity disorder.

Traditionally, phosphatidylserine (PtdS) supplements have been derived from bovine cortex (BC-PS); however, due to the potential for infectious disease transfer, soy-derived PS (S-PS) has been established as a potentially safe alternative. Soy-derived PS is generally recognized as safe (GRAS) and is a safe nutritional supplement for the elderly (if taken at a dose of 200 mg three times daily). Phosphatidylserine has been shown to reduce specific immune responses in mice.

PtdS can be found in meat, but is most abundant in the brain and in organ meats such as liver and kidney. Only small amounts of PS can be found in dairy products or in vegetables (except white beans).

Annexin-A5 is a naturally occurring protein with a strong binding affinity for PtdS. Labeled Annexin-A5 can visualize cells in early to mid-apoptotic states in vitro or in vivo. Another PtdS-binding protein is Mfge8. Technetium-labeled Annexin-A5 can differentiate between malignant and benign tumors, whose pathology includes high cell division and apoptosis rates in malignant tumors compared to low apoptosis rates in benign tumors.

II. Gla domain proteins

A. Gla domain

The general structure of Gla-domain proteins is as follows: a Gla domain followed by an EGF domain, then a C-terminal serine protease domain. Exceptions are prothrombin, which contains a Kringle domain in place of the EGF domain, and protein S, which lacks a serine protease domain and instead possesses a sex hormone-binding globin-like (SHBG) domain (Hansson and Stenflo, 2005). Gla-domain proteins vary in their affinity for anionic membranes. Generally, they fall into three categories: 1) high-affinity binders with a Kd of 30-50 nM, 2) intermediate-affinity binders with a _Kd of 100-200 nM, and 3) low-affinity binders with a Kd of 1000-2000 nM. High-affinity Gla-domain proteins have been shown to bind to anionic membranes with protein S, specifically binding to apoptotic cells via its interaction with PtdS (Webb et al. , 2002). Low-affinity Gla domain proteins use a second receptor to bind to the cell membrane. For example, FVII utilizes tissue factor (TF). It is believed that the Gla domain/the first EGF domain constitutes the high-affinity TF binding domain of FVII. Importantly for this approach, many studies have confirmed that the TF on the surface of cancer cells (including colorectal cancer, NSCL cancer and breast cancer) is raised, and these high TF levels have been associated with poor prognosis (Yu et al ., 2004). Although the affinity of anionic membranes to FVII is relatively low, the interaction of high-affinity TF in cancer and the raising of the TF recorded make it a potentially interesting cancer-specific probe.

B. Gla domain-containing proteins

1. Factor II

Prothrombin (also known as coagulation factor II) is proteolytically cleaved to form thrombin in the coagulation cascade, which ultimately leads to the stemming of blood loss. Thrombin, in turn, acts as a serine protease, converting soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions. It is primarily expressed in the liver.

The gene encoding prothrombin is located on chromosome 11 in the centromeric region. It consists of 14 exons and contains 24 kilobases of DNA. This gene encodes a signal region, a propeptide region, a glutamate domain, two kringle regions, and a catalytic domain. In the presence of vitamin K, the enzyme γ-glutamyl carboxylase converts the N-terminal glutamate residue to a γ-carboxyglutamate residue. This γ-carboxyglutamate residue is required for prothrombin to bind to phospholipids on the platelet membrane.

Hereditary factor II deficiency is an autosomal recessive disorder that can manifest as hypoprothrombinemia (a decrease in the total synthesis of prothrombin) or dysprothrombinemia (synthesis of dysfunctional prothrombin). Homozygous individuals are usually asymptomatic and have functional prothrombin levels of 2-25%. However, symptomatic individuals may experience easy bruising, epistaxis, soft tissue bleeding, excessive postoperative bleeding, and/or menorrhagia.

Prothrombin plays a role in chronic urticaria, autoimmune diseases, and various vascular disorders. Livedoid vasculopathy is associated with immunoglobulin (Ig) M anti-phosphatidylserine-prothrombin complex antibodies. The presence of anti-phosphatidylserine-prothrombin complex antibodies and histopathological necrotizing vasculitis in the upper to mid-dermis indicates cutaneous leukocytic vasculitis rather than cutaneous polyarteritis nodosa.

In addition to prothrombin deficiency, another prothrombin disorder is the prothrombin 20210a mutation. Familial causes of venous thromboembolism (i.e., the prothrombin 20210a mutation) result in increased plasma prothrombin levels and a concomitant increased risk of developing thrombotic disorders. Although the exact mechanism of this disorder has not been elucidated, the prothrombin 20210a mutation involves the replacement of guanine with adenine at position 20210 within the 3' untranslated region of the prothrombin gene. This mutation alters the polyadenylation site of the gene and leads to increased mRNA synthesis and subsequent protein expression.

2. Factor VII

Factor VII (formerly known as proconvertin) is one of the proteins that causes blood coagulation in the coagulation cascade. The gene for Factor VII is located on chromosome 13 (13q34). It is an enzyme of the serine protease class, and the U.S. Food and Drug Administration has approved the use of recombinant human Factor VIIa (NovoSeven) for uncontrolled bleeding in hemophiliacs. It is sometimes used for serious uncontrolled bleeding without permission, although there has been safety concerns. The biosimilar form (AryoSeven) of the recombinant activated Factor VII is manufactured by AryoGen Biopharma.

The main function of factor VII (FVII) is to initiate the coagulation process together with tissue factor (TF/factor III). Tissue factor is present on the outside of blood vessels--usually not exposed to blood flow. After vascular injury, tissue factor is exposed to blood and circulating factor VII. After being bound to TF, FVII is activated into FVIIa by different proteases, including thrombin (factor IIa), factor Xa, IXa, XIIa and the FVIIa-TF complex itself. The most important substrates of FVIIa-TF are factor X and factor IX. It has been confirmed that factor VII interacts with tissue factor (TF).

The action of this factor is blocked by tissue factor pathway inhibitor (TFPI), which is released almost immediately after coagulation begins. Factor VII is vitamin K-dependent; it is produced in the liver. Use of warfarin or similar anticoagulants reduces hepatic synthesis of FVII.

Deficiency is rare (congenital proconvertin deficiency) and is inherited recessively. Factor VII deficiency presents as a hemophilia-like bleeding disorder. It is treated with recombinant factor VIIa (NovoSeven or AryoSeven). Recombinant factor VIIa is also used for people with hemophilia (having factor VIII or IX deficiency), which has formed inhibitors for replacing coagulation factors. It has also been used in uncontrollable bleeding situations, but its effect in this situation is controversial, and there is not enough evidence to support its use outside of clinical trials. The first report of its use in bleeding was in 1999 in Israeli soldiers with uncontrollable bleeding. The risks of its use include an increase in arterial thrombosis.

3. Factor IX

Factor IX (or Christmas factor) is a serine protease of the coagulation system; it belongs to the peptidase family S1. The gene for Factor IX is located on the X chromosome (Xq27.1-q27.2) and is therefore X-linked recessive: mutations in this gene affect males much more frequently than females. Deficiency of this protein causes hemophilia B. Factor IX is produced as a zymogen (an inactive precursor). It undergoes processing to remove the signal peptide, glycosylation, and then cleavage by Factor XIa (in the contact pathway) or Factor VIIa (in the tissue factor pathway) to produce a two-chain form, in which the chains are linked by disulfide bonds. When activated to Factor IXa in the presence of Ca2 ⁺ , membrane phospholipids, and the Factor VIII cofactor, it hydrolyzes an arginine-isoleucine bond in Factor X to form Factor Xa. Factor IX is inhibited by antithrombin.

Factors VII, IX, and X all play a key role in blood coagulation and share a common domain architecture. The Factor IX protein consists of four protein domains. These are a Gla domain, two tandem copies of the EGF domain, and a C-terminal trypsin-like peptidase domain that performs catalytic cleavage. It has been shown that the N-terminal EGF domain is at least partially responsible for binding to tissue factor. Wilkinson et al. concluded that residues 88-109 of the second EGF domain mediate binding to platelets and assembly of the Factor X activation complex. The structures of all four domains have been revealed. The structures of the two EGF domains and the trypsin-like domain of the porcine protein have been determined. The structure of the Gla domain responsible for Ca(II)-dependent phospholipid binding has also been determined by NMR. Several structures of "hyperactive" mutants have been resolved, revealing the nature of Factor IX activation for other proteins in the coagulation cascade.

A deficiency of factor IX causes Christmas disease (hemophilia B). More than 100 mutations in factor IX have been described; some do not cause symptoms, but many can lead to significant bleeding disorders. Recombinant factor IX is used to treat Christmas disease and is commercially available as BeneFIX. Some rare mutations in factor IX result in elevated clotting activity and can lead to coagulation disorders such as deep vein thrombosis.

4. Factor X

Factor X (Stuart-Prower factor; prothrombinase) is an enzyme in the coagulation cascade. The human factor X gene is located on chromosome 13 (13q34). It is a serine endopeptidase (protease group S1). Factor X is synthesized in the liver, and its synthesis requires vitamin K. Factor X is activated to factor Xa by factor IX (together with its cofactor factor VIII, in a complex known as intrinsic Xase ) and factor VII (together with its cofactor tissue factor, in a complex known as extrinsic Xase ). Factor X has a half-life of 40-45 hours. It is therefore the first member of the final common pathway, or thrombin pathway. It works by cleaving prothrombin at two locations (the Arg-Thr bond, followed by the Arg-Ile bond), which produces active thrombin. This process is optimized when factor Xa forms a complex with activated cofactor V in the prothrombinase complex. Factor X is a component of fresh frozen plasma and the prothrombinase complex. The only commercially available concentrate is "Factor XP Behring" manufactured by CSL Behring.

Factor Xa is inactivated by the protein Z-dependent protease inhibitor (ZPI) (serine protease inhibitor, serpin). The presence of protein Z increases the protein's affinity for factor Xa by 1000-fold, whereas protein Z is not required for factor XI inactivation. Defects in protein Z lead to increased factor Xa activity and thrombotic tendency.

Congenital deficiency of factor X is very rare (1:500,000) and may be associated with nosebleeds (epistaxis), hemarthrosis (bleeding into joints), and gastrointestinal blood loss. In addition to congenital deficiency, low factor X levels may occasionally occur in a number of disease states. For example, factor X deficiency may be seen in amyloidosis, in which factor X is adsorbed to amyloid fibrils in the vasculature. In addition, vitamin K deficiency or antagonism by warfarin (or similar drugs) can lead to the production of inactive factor X. This is required to prevent thrombosis in warfarin therapy. As of late 2007, four of the five emerging anti-coagulant therapeutics target this enzyme. Direct Factor Xa inhibitors are popular anticoagulants.

The traditional coagulation model developed in the 1960s foresees two separate cascades: the extrinsic (tissue factor (TF)) pathway and the intrinsic pathway. These pathways converge to a common point: the formation of the Factor Xa/Va complex, which, together with calcium and bound to a phospholipid surface, generates thrombin (Factor IIa) from prothrombin (Factor II). A new model of anticoagulation (a cell-based model) appears to more fully explain the steps in coagulation. This model has three phases: 1) initiation of coagulation on cells bearing TF, 2) amplification of the procoagulant signal by thrombin generated on cells bearing TF, and 3) propagation of thrombin generation on the platelet surface. Factor Xa plays a key role in all three phases.

In stage 1, factor VII binds to the transmembrane protein TF on the cell surface and is converted into factor VIIa. The result is the factor VIIa/TF complex, which catalyzes the activation of factor X and factor IX. Factor Xa formed on the surface of cells bearing TF interacts with factor Va to form the prothrombinase complex, which produces small amounts of thrombin on the surface of cells bearing TF. In stage 2 (amplification phase), if sufficient thrombin has been produced, platelets and platelet-associated cofactors are activated. In stage 3 (thrombin generation), factor XIa activates free factor IX on the surface of activated platelets. Activated factor IXa forms a "tenase" complex with factor VIIIa. This complex activates more factor X, which in turn forms a new prothrombinase complex with factor Va. Factor Xa is the primary component of the prothrombinase complex, which converts large amounts of prothrombin (the "thrombin burst"). Each factor Xa molecule can generate 1,000 thrombin molecules. This large thrombin burst is responsible for the polymerization of fibrin to form a thrombus.

Inhibition of factor X synthesis or activity is the mechanism of action of many anticoagulants currently in use. Warfarin (a synthetic derivative of coumarin) is the most widely used oral anticoagulant in the United States, with other coumarin derivatives (phenprocoumon and acenocoumarol) used in some European countries. These agents are vitamin K antagonists (VKAs). Vitamin K is essential for the hepatic synthesis of factors II (prothrombin), VII, IX, and X. Heparin (unfractionated heparin) and its derivatives, low molecular weight heparin (LMWH), bind to the plasma cofactor antithrombin (AT) to inactivate several coagulation factors, IIa, Xa, XIa, and XIIa.

Recently, a new series of specific, directly acting inhibitors of Factor Xa have been developed. These include the drugs rivaroxaban, apixaban, betrixaban, LY517717, darexaban (YM150), edoxaban, and 813893. These agents have several theoretical advantages over current therapies. They can be administered orally. They have a rapid onset of action, and they may be more effective for Factor Xa because they inhibit free Factor Xa and Factor Xa in the prothrombinase complex.

5. Protein S

Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the endothelium. In the circulation, protein S exists in two forms: a free form and a complexed form bound to the complement protein C4b-binding protein (C4BP). In humans, protein S is encoded by the PROS1 gene. The best characterized function of protein S is its role in the anticoagulant pathway, where it plays a role as a cofactor of protein C in the inactivation of factors Va and VIIIa. Only the free form has cofactor activity.

Protein S can be bound to negatively charged phospholipids via the GLA domain of carboxylate. This property allows Protein S to work in the removal of cells undergoing apoptosis. Apoptosis is a form of cell death in which the body removes unwanted or damaged cells from tissues. Cells in apoptosis ( i.e. , during apoptosis) no longer actively process the distribution of phospholipids in their outer membranes, and therefore begin to display negatively charged phospholipids, such as phosphatidylserine, on the cell surface. In healthy cells, ATP (adenosine triphosphate)-dependent enzymes remove these from the outer leaflet of the cell membrane. These negatively charged phospholipids are recognized by phagocytes such as macrophages. Protein S can be bound to negatively charged phospholipids and work as a bridging molecule between apoptotic cells and phagocytes. The bridging properties of Protein S can enhance the phagocytosis of apoptotic cells, thereby allowing it to be "cleanly" removed without the symptoms of any tissue damage, such as inflammation.

Mutations in the PROS1 gene can lead to protein S deficiency, a rare blood disorder that can lead to an increased risk of blood clots. Protein S has been shown to interact with factor V.

6. Protein C

Protein C (also known as autoprothrombin IIA and blood coagulation factor XIV) is a zymogen (inactive) protein whose activated form plays an important role in regulating blood coagulation, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals. Activated protein C (APC) performs these actions primarily by proteolytically inactivating the proteins Factor V _a and Factor VIII _a . APC is classified as a serine protease because it contains a serine residue in its active site. In humans, protein C is encoded by the PROC gene present in chromosome 2.

The zymogen form of protein C is a vitamin K-dependent glycoprotein that circulates in plasma. Its structure is a two-chain polypeptide consisting of a light chain and a heavy chain linked by a disulfide bond. Protein C zymogen becomes activated when bound to thrombin (another protein deeply involved in coagulation), and the presence of thrombomodulin and the endothelial protein C receptor (EPCR) greatly promotes protein C activation. Due to the action of EPCR, activated protein C is primarily present near endothelial cells (i.e., those that make up the blood vessel walls), and APCs affect these cells and leukocytes (white blood cells). Because of the important role that protein C plays as an anticoagulant, those with protein C deficiency or certain types of APC resistance suffer a significantly increased risk of forming dangerous blood clots (thrombosis).

Research on the clinical use of activated protein C, also known as alpha-activated tegaserod (trademarked as Xigris), has been surrounded by controversy. The manufacturer, Eli Lilly and Company, launched an aggressive marketing campaign to promote its use in people with severe sepsis and septic shock, including funding from the 2004 Surviving Sepsis Campaign Guidelines. However, a 2011 Cochrane review found that its use cannot be recommended because it does not improve survival (and increases the risk of bleeding).

Human protein C is a vitamin K-dependent glycoprotein that is structurally similar to other vitamin K-dependent proteins that affect blood coagulation, such as prothrombin, factor VII, factor IX, and factor X. Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32-amino acid N-terminal signal peptide preceding the propeptide. Protein C is formed when the dipeptide of Lys ¹⁹⁸ and Arg ¹⁹⁹ is removed; this results in conversion to a heterodimer with N -linked carbohydrates on each chain. The protein has one light chain (21 kDa) and one heavy chain (41 kDa) linked by a disulfide bond between Cys ¹⁸³ and Cys ³¹⁹ .

Inactive protein C consists of 419 amino acids arranged in multiple domains: a Gla domain (residues 43-88); a helical aromatic segment (89-96); two epidermal growth factor (EGF)-like domains (97-132 and 136-176); an activation peptide (200-211); and a trypsin-like serine protease domain (212-450). The light chain contains the Gla and EGF-like domains and the aromatic segment. The heavy chain contains the protease domain and activation peptide. In this form, 85-90% of protein C circulates in plasma as a zymogen, awaiting activation. The remaining protein C zymogen contains a slightly modified form of the protein. Enzyme activation occurs when the thrombin molecule cleaves the activation peptide from the N-terminus of the heavy chain. The active site contains the typical catalytic triad of serine proteases (His ²⁵³ , Asp ²⁹⁹ , and Ser ⁴⁰² ).

The activation of protein C is strongly promoted by thrombomodulin and endothelial protein C receptor (EPCR), the latter of which is mainly present on endothelial cells (cells inside blood vessels). The presence of thrombomodulin can accelerate activation by several orders of magnitude, and EPCR accelerates activation by 20 times. If any of these two proteins is not present in mouse samples, then mice die from excessive blood coagulation when they are still in the embryonic state. On the endothelium, APC plays an important role in regulating blood coagulation, inflammation and cell death (apoptosis). Because thrombomodulin accelerates the activation of protein C, it can be said that the protein is not activated by thrombin, but by thrombin-thrombomodulin (or even thrombin-thrombomodulin-EPCR) complex activation. Once in active form, APC can or cannot remain bound to EPCR, and the affinity of the APC to EPCR is about the same as that of the protease.

The Gla domain is particularly useful for binding negatively charged phospholipids (for anticoagulation) and EPCR (for cytoprotection). One specific domain effectively enhances protein C's ability to inactivate factor _Va . Another is required for interaction with thrombomodulin.

The zymogen form of protein C is present in normal adult plasma at concentrations between 65-135 IU/dL. Activated protein C is present at levels approximately 2000 times lower than this. Mild protein C deficiency corresponds to plasma levels above 20 IU/dL, but below the normal range. Moderately severe deficiency describes blood concentrations between 1-20 IU/dL; severe deficiency produces protein C levels below 1 IU/dL or undetectable. Protein C levels in healthy, full-term infants average 40 IU/dL. Protein C concentrations increase until 6 months of age, when the average level is 60 IU/dL; levels remain low during childhood until they reach adult levels after puberty. The half-life of activated protein C is about 15 minutes.

The protein C pathway is a specific chemical reaction that controls the expression level of APC and its activity in vivo. Protein C is pleiotropic, with two major functions: anticoagulation and cytoprotection (its direct effect on cells). Which function protein C performs depends on whether APC remains bound to EPCR after activation; when it is not bound, APC's anticoagulation effect occurs. In this case, protein C acts as an anticoagulant as follows: irreversibly proteolytically inactivates factor V _a and factor VIII _a , converting them into factor V _i and factor VIII _i , respectively. When still bound to EPCR, activated protein C performs its cytoprotective effect, acting on the effector substrate PAR-1, i.e., protease-activated receptor-1. To a certain extent, the anticoagulant properties of APC are independent of its cytoprotective properties, because the expression of one pathway is not affected by the presence of another pathway.

By reducing the amount of either available thrombomodulin or EPCR, the activity of protein C can be downregulated. This can be achieved by inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Activated leukocytes release these inflammatory mediators during inflammation, thereby inhibiting the production of thrombomodulin and EPCR and inducing their shedding from the endothelial surface. These two effects downregulate protein C activation. Thrombin itself may also have an impact on EPCR levels. In addition, proteins released from cells can hinder protein C activation, such as eosinophils, which can explain thrombosis in high eosinophilic heart disease. Platelet factor 4 can upregulate protein C. It is speculated that this cytokine improves the activation of protein C as follows: an electrostatic bridge is formed from the Gla domain of protein C to the glycosaminoglycan (GAG) domain of thrombomodulin, thereby reducing the Michaelis constant (K _M ) of their reaction. In addition, protein C inhibitors inhibit protein C.

Hereditary protein C deficiency (in its mild form associated with simple heterozygosity) causes a significantly increased risk of venous thrombosis in adults. If the fetus is homozygous or mixed heterozygous for the deficiency, there may be presentation of purpura fulminans, severe disseminated intravascular coagulation, and simultaneous venous thromboembolism in utero; this is very serious and often fatal. Deletion of the protein C gene in mice causes fetal death around the time of birth. Fetal mice without protein C initially develop normally but experience severe bleeding, coagulopathy, fibrin deposition, and liver necrosis. The frequency of protein C deficiency in asymptomatic individuals is 1 in 200 and 1 in 500. In contrast, significant symptoms of deficiency are detectable in 1 in 20,000 individuals. No racial or ethnic bias has been detected.

When APC cannot perform its function, activated protein C resistance occurs. The disease has symptoms similar to those of protein C deficiency. The most common mutation causing activated protein C resistance in Caucasians is at the APC cleavage site for factor V. There, Arg ⁵⁰⁶ is replaced by Gln, resulting in factor V Leiden. This mutation is also known as R506Q. Mutations that result in the loss of this cleavage site actually stop APC from effectively inactivating factor V _a and factor VIII _a . As a result, a person's blood clots very easily, and they are permanently at increased risk of thrombosis. Individuals heterozygous for the factor V _Leiden mutation have a risk of venous thrombosis 5-7 times higher than in the general population. Homozygous subjects have an 80-fold higher risk. This mutation is also the most common genetic risk for venous thrombosis in Caucasians.

About 5% of APC resistance is unrelated to the above mutations and Factor V _Leiden . Other gene mutations can cause APC resistance, but none to the extent achieved by Factor V _Leiden . These mutations include various other forms of Factor V, the spontaneous production of autoantibodies targeting Factor V, and dysfunction of any cofactor of APC. In addition, some acquired disorders may reduce the effectiveness of APC in performing its function of preventing coagulation. Studies suggest that 20% to 60% of patients with thrombotic tendencies suffer from some form of APC resistance.

C. Gla domain peptides and polypeptides

The present disclosure contemplates the design, production, and use of a variety of peptides and polypeptides containing Gla domains. The structural characteristics of these molecules are as follows. First, the peptides or polypeptides possess a Gla domain comprising approximately 30-45 contiguous residues that constitute the Gla domain. Thus, even when the term "comprising" is included, the term "peptide having no more than "X" contiguous residues" should not be construed as encompassing a greater number of contiguous residues. Second, the peptides and polypeptides may contain additional non-Gla domain residues, such as an EGF domain, a Kringle domain, an Fc domain, and the like.

In general, the peptides and polypeptides will be 300 residues or less, again including 30-45 consecutive residues of the Gla domain. The total length can be 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, and up to 300 residues. Peptide lengths ranging from 50-300 residues, 100-300 residues, 150-300 residues, 200-300 residues, 50-200 residues, 100-200 residues, 150-300 residues, and 150-200 residues are contemplated. The number of consecutive Gla residues can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

The present disclosure can utilize L-configuration amino acids, D-configuration amino acids, or mixtures thereof. Although L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in certain proteins produced by exotic marine organisms (such as cone snails). They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine can act as a neurotransmitter in the brain. The L and D conventions for amino acid configurations do not refer to the optical activity of the amino acid itself, but rather to the optical activity of the isomers of glyceraldehyde that can theoretically synthesize the amino acid (D-glyceraldehyde is dextrorotatory; L-glyceraldehyde is levorotatory).

One form of an "all-D" peptide is a trans-inverted peptide. The trans-inverted modification of a naturally occurring polypeptide involves the synthetic assembly of amino acids with an α-carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids that are in reverse order with respect to the native peptide sequence. Trans-inverted analogs thus have inverted ends and a reversed orientation of the peptide bonds (NH-CO, rather than CO-NH), while maintaining approximately the same topology of the side chains as in the native peptide sequence. See U.S. Patent No. 6,261,569, incorporated herein by reference.

D. Synthesis

Advantageously, solid phase synthesis techniques (Merrifield, 1963) are used to produce peptides and polypeptides. Other peptide synthesis techniques are well known to those skilled in the art (Bodanszky et al. , 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Suitable protecting groups for use in such synthesis are found in the above text, as well as in Protective Groups in Organic Chemistry (1973). These synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to the extended peptide chain. Typically, any amino or carboxyl group of the first amino acid residue is protected with a suitable, optionally removable protecting group. A different, optionally removable protecting group is used for amino acids containing reactive side groups (such as lysine).

Using solid phase synthesis as an example, protected or derivatized amino acids are connected to an inert solid support via their unprotected carboxyl or amino groups. The protecting groups of the amino or carboxyl groups are then selectively removed, and the next amino acid in the sequence with the complementary (amino or carboxyl) group suitably protected is mixed and reacted with the residue that has been connected to the solid support. The protecting groups of the amino or carboxyl groups are then removed from the newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so on. After all desired amino acids have been connected in the appropriate order, any remaining end and side group protecting groups (and solid support) are removed sequentially or in parallel to provide final peptides. The peptides and polypeptides of the present disclosure preferably do not have benzylated or methylbenzylated amino acids. Such protecting group moieties can be used in the synthesis process, but they are removed before using the peptides and polypeptides. Additional reactions may be necessary, as described elsewhere, to restrict conformation to form intramolecular bonds.

In addition to the 20 standard amino acids that can be used, there are a vast number of "non-standard" amino acids. Two of these can be specified by the genetic code, but are quite rare in proteins. Selenocysteine is incorporated into some proteins at the UGA codon (which is usually a stop codon). Pyrrolysine is used by some methanogenic archaea in their methane-producing enzymes. It is encoded by the codon UAG. Examples of non-standard amino acids not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter γ-aminobutyric acid. Non-standard amino acids often exist as intermediates in the metabolic pathways of standard amino acids; for example, ornithine and citrulline are present in the urea cycle (part of amino acid catabolism). Non-standard amino acids are often formed by modifications of standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or through demethylation of methionine via the intermediate metabolite S-adenosylmethionine, while hydroxyproline is produced through post-translational modification of proline.

E. Connector

Linkers or cross-linking agents can be used to fuse Gla domain peptides or polypeptides to other protein sequences ( e.g. , antibody Fc domains). Bifunctional cross-linking agents have been widely used for a variety of purposes, including the preparation of affinity matrices, modification and stabilization of different structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents carrying two identical functional groups have been shown to be very effective in inducing cross-links between identical and different macromolecules or macromolecular subunits and in linking polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By exploiting the differential reactivity of the two different functional groups, cross-linking can be selectively and sequentially controlled. Bifunctional cross-linking agents can be separated according to the specificity of their functional groups (e.g., amino-, sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups). Among these, reagents targeting free amino groups have become particularly popular due to their commercial availability, ease of synthesis, and the mild reaction conditions they can be used in. Most heterobifunctional cross-linking agents contain primary amine-reactive groups and sulfhydryl-reactive groups.

In another embodiment, heterobifunctional cross-linking agents and methods of using the same are described in U.S. Patent No. 5,889,155, which is expressly incorporated herein by reference in its entirety. The cross-linking agents combine nucleophilic hydrazide residues with electrophilic maleimide residues, thereby allowing, in one embodiment, the coupling of aldehydes to free sulfhydryl groups. The cross-linking agents can be modified to cross-link different functional groups and thus can be used to cross-link polypeptides. In the case where a particular peptide does not contain residues in its native sequence that are suitable for a given cross-linking agent, conservative genetic or synthetic amino acid changes in the basic sequence can be utilized.

F. Additional peptide/polypeptide sequences

A factor drug development is to achieve an appropriate circulation half-life, which affects dosage, drug administration and efficacy, and this is particularly important for biotherapeutics. Small proteins below 60 kD are rapidly cleared by the kidneys and therefore do not reach their targets. This means that high doses are required to achieve efficacy. Modifications currently used to increase the half-life of proteins in circulation include: PEGylation; conjugation or gene fusion with proteins (e.g., transferrin (WO06096515A2), albumin, growth hormone (U.S. Patent Publication 2003104578AA)); conjugation with cellulose (Levy and Shoseyov, 2002); conjugation or fusion with Fc fragments; glycosylation and mutagenesis protocols (Carter, 2006).

In the case of PEGylation, polyethylene glycol (PEG) is conjugated to a protein, which may be, for example, a plasma protein, an antibody, or an antibody fragment. The first study on the effect of PEGylation of antibodies was conducted in the 1980s. Conjugation can be performed enzymatically or chemically, and is well established in the art (Chapman, 2002; Veronese and Pasut, 2005). Due to PEGylation, the total size can be increased, which reduces the chance of renal filtration. PEGylation further protects against proteolytic degradation and slows down clearance from the blood. In addition, it has been reported that PEGylation can reduce immunogenicity and increase solubility. The pharmacokinetics improved by the addition of PEG are due to several different mechanisms: an increase in molecular size, protection from proteolysis, reduced antigenicity, and masking of specific sequences from cell receptors. In the case of antibody fragments (Fab), a 20-fold increase in plasma half-life has been achieved by PEGylation (Chapman, 2002).

So far, there are several approved PEGylated drugs, for example, the PEG-interferon α2b (PegIntron) sold in 2000 and the α2a (Pegasys) sold in 2002. The PEGylated antibody fragment for TNFα (called Cimzia or Cerulezumab) was submitted to the FDA for approval in 2007 for the treatment of Crohn's disease and was approved on April 22, 2008. A limitation of PEGylation is the difficulty of synthesizing long monodisperse substances, particularly when it is necessary to exceed 1000 kD PEG chains. For many applications, polydisperse PEG with a chain length exceeding 10000 kD is used to produce conjugate populations with different length PEG chains, which require extensive analysis to ensure equivalent batches between production. The different lengths of PEG chains may result in different biological activities and therefore different pharmacokinetics. Another limitation of PEGylation is a decrease in affinity or activity, as has been observed with the alpha-interferon Pegasys, which has only 7% of the antiviral activity of the native protein but has improved pharmacokinetics due to an enhanced plasma half-life.

Another approach is to conjugate the drug to a long-lived protein (e.g., albumin, which is 67 kD and has a plasma half-life of 19 days in humans). Albumin is the most abundant protein in plasma and is involved in plasma pH regulation, but also acts as a carrier of substances in plasma. In the case of CD4, increased plasma half-life has been achieved after fusing it to human serum albumin (Yeh et al. , 1992). Other examples of fusion proteins are insulin, human growth hormone, transferrin, and cytokines (Duttaroy et al. , 2005; Melder et al. , 2005; Osborn et al. , 2002a; Osborn et al. , 2002b; Sung et al. , 2003), and see (U.S. Patent Publication Nos. 2003104578A1, WO06096515A2, and WO07047504A2, incorporated herein by reference in their entirety).

The effects of glycosylation on plasma half-life and protein activity have also been extensively studied. In the case of tissue plasminogen activator (tPA), the addition of new glycosylation sites reduced plasma clearance and increased potency (Keyt et al. , 1994). Glycoengineering has been successfully applied to many recombinant proteins and immunoglobulins (Elliott et al. , 2003; Raju and Scallon, 2007; Sinclair and Elliott, 2005; Umana et al. , 1999). In addition, glycosylation can affect the stability of immunoglobulins (Mimura et al. , 2000; Raju and Scallon, 2006).

Another molecule for fusion proteins is the Fc fragment of IgG (Ashkenazi and Chamow, 1997). Fc fusion protocols have been used, for example, in Trap Technology developed by Regeneron ( e.g. , IL1 trap and VEGF trap). The use of albumin to extend the half-life of peptides has been described in U.S. Patent Publication 2004001827A1, as have Fab fragments and scFv-HSA fusion proteins. The extended serum half-life of albumin has been demonstrated to be due to a recycling process mediated by FcRn (Anderson et al. , 2006; Chaudhury et al. , 2003).

Another strategy is to use directed mutagenesis techniques that target the interaction of immunoglobulins with their receptors to improve binding properties, i.e., affinity maturation in the Fc region. Due to increased affinity with FcRn, extended half-life can be achieved in vivo (Ghetie et al. , 1997; Hinton et al. , 2006; Jain et al. , 2007; Petkova et al. , 2006a; Vaccaro et al. , 2005). However, affinity maturation strategies require several rounds of mutagenesis and testing. This takes time, is expensive, and is limited by the number of amino acids that produce extended half-life when mutated. Therefore, simple alternatives are needed to improve the in vivo half-life of biotherapeutics. Therapeutic agents with extended in vivo half-life are particularly important for the treatment of chronic diseases, autoimmune disorders, inflammatory diseases, metabolic diseases, infectious diseases, eye diseases, and cancer, particularly when treatment requires long periods of time. Therefore, there remains a need to develop therapeutic agents ( eg , antibodies and Fc fusion proteins) with enhanced persistence and half-life in the circulation in order to reduce the dose and/or injection frequency of various therapeutic agents.

G. Marking

The peptides and polypeptides of the present disclosure can be conjugated to labels for diagnostic purposes, such as to identify cancer cells or virus-infected cells, including their use in histochemistry. A label according to the present disclosure is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

Labeled conjugates are generally preferred for use as diagnostic agents. Diagnostic agents are generally divided into two categories: those used for in vitro diagnosis, and those used for in vivo diagnostic protocols (commonly referred to as "directed imaging"). Many suitable imaging agents are known in the art, as are methods for linking them to peptides and polypeptides (see, e.g. , U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moiety used can be a paramagnetic ion, a radioisotope, a fluorescent dye, an NMR-detectable substance, and an X-ray imaging agent.

In the case of paramagnetic ions, mention may be made, by way of example, of ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts (such as X-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II) and, in particular, bismuth (III).

In the case of radioisotopes for therapeutic and/or diagnostic applications, mention may be made of astatine ²¹¹ , carbon ¹⁴ , chromium ⁵¹ , chlorine ³⁶ , cobalt ⁵⁷ , cobalt ⁵⁸ , copper ⁶⁷ , Eu ¹⁵² , gallium ⁶⁷ , hydrogen ³ , iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , iron ⁵⁹ , phosphorus ³² , rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , selenium ⁷⁵ , sulfur ³⁵ , technetium ^99m and/or yttrium ^{90. 125} I is often preferred for use in certain embodiments, and technetium ^99m and/or indium ¹¹¹ are also often preferred due to their low energy and suitability for long-range detection. Radiolabeled peptides and polypeptides can be produced according to methods well known in the art. For example, peptides and polypeptides can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidant (such as sodium hypochlorite) or an enzymatic oxidant (such as lactoperoxidase). Peptides can be labeled with technetium ^-99m by a ligand exchange process, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the peptide to the column. Alternatively, direct labeling techniques can be used, for example, by incubating pertechnetate, a reducing agent such as _SNCl2 , a buffer solution such as sodium-potassium phthalate solution, and the peptide. Intermediate functional groups frequently used to bind radioisotopes (which exist as metal ions) to peptides are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and/or Texas Red.

Another type of conjugate foreseen is a conjugate primarily intended for in vitro applications in which the peptide is attached to a second binding partner and/or an enzyme (enzyme tag) that produces a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. Preferred second binding partners are biotin and avidin and streptavidin compounds. The application of such labels is well known to those skilled in the art and is described in, for example, U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

Other methods for connecting or conjugating a peptide to its conjugate portion are known in the art. Some methods of connection include the use of metal chelate complexes using, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycoluril-3 (U.S. Patents 4,472,509 and 4,938,948) linked to an antibody. Peptides or polypeptides can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

IV. Diagnosis and Therapy

A. Drug Formulation and Route of Administration

In the case of foreseeing clinical use, the pharmaceutical composition must be prepared in a form suitable for the intended use. Generally, this requires preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

It is generally desirable to employ appropriate salts and buffers to stabilize the delivery vector and permit uptake by the target cells. Buffers are also employed when recombinant cells are introduced into a patient. The aqueous compositions of the present disclosure comprise an effective amount of a vector or cells dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agents are incompatible with the vectors or cells of the present disclosure, its use in therapeutic compositions is foreseen. Supplementary active ingredients may also be incorporated into the compositions.

The active compositions of the present disclosure may include typical pharmaceutical formulations. Administration of the compositions according to the present disclosure may be via any common route, provided that the target tissue can be reached via that route. Such routes include oral, nasal, buccal, rectal, vaginal, or topical routes. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intratumoral, intraperitoneal, or intravenous injection. Such compositions are typically administered as the pharmaceutically acceptable compositions described above.

The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or a pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow easy injection. It must be stable under the conditions of preparation and storage and must be protected from the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size (in the case of dispersions), and by the use of a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferred to include an isotonic agent, such as sugar or sodium chloride. The absorption of injectable compositions can be prolonged by using agents that prolong absorption (e.g., aluminum monostearate and gelatin) in the composition.

Sterile injectable solutions are prepared by incorporating the required amount of the active compound into an appropriate solvent containing various other ingredients, as required, as listed above, followed by filtered sterilization. Dispersions are generally prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

For oral administration, the peptides and polypeptides of the present disclosure can be blended with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash can be prepared containing the desired amount of the active ingredient in a suitable solvent such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient can be incorporated into an antiseptic lotion containing sodium borate, glycerol, and potassium bicarbonate. The active ingredient can also be dispersed in dentifrices, including gels, pastes, powders, and slurries. The active ingredient can be added to a paste dentifrices in a therapeutically effective amount, which can include water, a binder, an abrasive, a flavoring agent, a foaming agent, and a humectant.

The compositions of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

After preparation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms (such as injectable solutions, drug release capsules, etc.). For parenteral administration in an aqueous solution, for example, if necessary, the solution should be appropriately buffered and the liquid diluent should first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, those skilled in the art will know the sterile aqueous media that can be used, taking into account the present disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and 1000 ml of subcutaneous infusion fluid can be added or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences", 15th edition, pages 1035-1038 and 1570-1580). Some variations in dosage will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for each subject in each case. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards set by the Office of Food and Drug Administration (FDA) for biological products.

B. Disease States and Conditions

1. Cancer

Cancer is caused by the growth of clonal colonies of cells from tissues. The occurrence of cancer (called carcinogenesis) can be modeled and characterized in many ways. The relationship between the development of cancer and inflammation has long been understood. The inflammatory response is involved in the host's defense against microbial infection and also drives tissue repair and regeneration. Considerable evidence points to a link between inflammation and the risk of developing cancer, that is, chronic inflammation can lead to dysplasia. There are hundreds of different forms of human cancer, and as the understanding of the underlying genetics and biology of cancer increases, these forms are further subdivided and reclassified.

Determining the causes of cancer is complex. Many factors are known to increase cancer risk, including tobacco use, certain infections, radiation, physical inactivity, obesity, and environmental pollutants. These can directly damage genes or compound existing genetic defects within cells to cause disease. Approximately 5-10% of cancers are completely hereditary.

Cancer can be detected in many ways, including the presence of certain signs and symptoms, screening tests or medical imaging. Once possible cancer is detected, it is diagnosed by microscopic examination of tissue samples. Cancer is often treated with chemotherapy, radiation therapy and surgery. The chance of survival from the disease varies greatly with the type and position of the cancer and the degree of the disease when treatment begins. Although cancer can affect people of all ages and several types of cancer are more common in children, the risk of cancer occurring increases with age usually. In 2007, cancer caused approximately 13% (7.9 million) of all deaths worldwide. Along with more people surviving to old age, and along with a large amount of lifestyle changes occurring in developing countries, ratio is increasing.

Treatments fall into five main categories: surgery, chemotherapy, radiation, alternative medicine, and palliative care. Surgery is the mainstay of treatment for most isolated solid cancers and may play a role in palliation and prolonged survival. It is often an important part of making a definitive diagnosis and staging the tumor, as a biopsy is often required. In localized cancers, surgery usually attempts to remove the entire mass, and in some cases, along with lymph nodes in the area. For some types of cancer, this is all that is needed to eliminate the cancer.

Chemotherapy combined with surgery has been shown to be effective in many different cancer types, including breast cancer, colorectal cancer, pancreatic cancer, osteogenic sarcoma, testicular cancer, ovarian cancer, and some lung cancers. The effectiveness of chemotherapy is often limited by toxicity to other tissues in the body.

Radiation therapy refers to the use of ionizing radiation in an attempt to cure or improve cancer symptoms. It is used in about half of all cases, and the radiation can come from an internal source (in the form of brachytherapy) or an external source. Radiation is usually used in conjunction with surgery and/or chemotherapy, but can be used alone for certain types of cancer, such as early-stage head and neck cancer. For painful bone metastases, it has been found to be effective in about 70% of people.

Alternative and complementary medicine encompasses a diverse collection of health care systems, practices, and products that are not part of conventional medicine. "Complementary medicine" refers to methods and substances used alongside conventional medicine, while "alternative medicine" refers to compounds used instead of conventional medicine. Most complementary and alternative medicine for cancer has not been rigorously studied or tested. Some alternative treatments have been studied and shown to be ineffective, yet continue to be marketed and promoted.

Finally, palliative care refers to treatment that attempts to make the patient feel better, which may or may not be combined with attempts to attack the cancer. Palliative care includes actions that alleviate the physical, emotional, mental, and psychosocial suffering experienced by cancer patients. Unlike treatments that aim to directly kill cancer cells, the primary goal of palliative care is to improve the patient's quality of life.

2. Viral infection

Viruses are small infectious pathogens that can replicate only within the living cells of an organism. Viruses can infect all types of organisms, from animals and plants to bacteria and archaea. About 5,000 species of viruses have been described in detail, although there are millions of different types. Viruses are found in almost every ecosystem on Earth and are the most abundant type of biological entity.

Virus particles (called virions) consist of two or three parts: i) genetic material made from DNA or RNA (long molecules that carry genetic information); ii) a protein coat that protects these genes; and in some cases, iii) a lipid coat surrounding the protein coat (when outside the cell). Viral shapes range from simple helical and icosahedral forms to more complex structures. A typical virus is about 1/100 the size of a typical bacterium. Most viruses are too small to be seen directly with a light microscope.

Viruses are spread in many ways. Viruses in plants are often spread from plant to plant by insects (such as aphids) that feed on plant fluids; viruses in animals can be carried by blood-sucking insects. These disease-carrying organisms are called vectors. Influenza viruses are spread through coughs and sneezes. Norovirus and rotavirus (a common cause of viral gastroenteritis) are transmitted through the fecal-oral route and are passed from person to person through contact, entering the body in food or water. HIV is one of several viruses that is transmitted through sexual contact and through exposure to infected blood. The range of host cells a virus can infect is called its "host range." This can be narrow, or, in the case of a virus that can infect many species, broad.

Viral infections in animals trigger immune responses that often eliminate the infectious virus. Immune responses can also be generated by vaccines, which confer artificially acquired immunity against specific viral infections. However, some viruses, including those that cause AIDS and viral hepatitis, can evade these immune responses and lead to chronic infections. Antibiotics have no effect on viruses, but several antiviral drugs have been developed.

A variety of diseases can be promoted by viral infection, including influenza, human immunodeficiency virus, dengue virus, West Nile virus, smallpox virus, respiratory syncytial virus, Korean hemorrhagic fever virus, chickenpox, varicella-zoster virus, herpes simplex virus 1 or 2, Epstein-Barr virus, Marburg virus, hantavirus, yellow fever virus, hepatitis A, B, C, or E, Ebola virus, human papillomavirus, rhinovirus, coxsackievirus, poliovirus, measles virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, and HTLV-1 and -2.

C. Treatment

Peptides and polypeptides can be administered to mammalian subjects ( e.g. , human patients) alone or in combination with other drugs for treating the above-mentioned diseases. The required dosage depends on: the choice of administration route; the nature of the formulation, including additional agents linked to the polypeptide; the nature of the patient's disease; the size, weight, surface area, age, and sex of the subject; future combination therapy; and the judgment of the attending physician. Suitable dosages are in the range of 0.0001-100 mg/kg. Considering the variety of available compounds and the varying efficiencies of various administration routes, a wide range of required dosages is foreseen. For example, oral administration is expected to require higher doses than intravenous administration. Variations in these dosage levels can be adjusted using standard empirical practices for optimization that are well understood in the art. Administration can be single or multiple (e.g., 2, 3, 4, 6, 8, 10, 20, 50, 100, 150, or more). Encapsulating the polypeptide in a suitable delivery vehicle ( e.g. , polymeric microparticles or implantable devices) can increase the efficiency of delivery, particularly for oral delivery.

Engineered Gla domain proteins can be used as targeting agents to deliver therapeutic payloads to cancer cells, such as radionuclides, chemotherapeutic agents, or toxins. Specific chemotherapeutic agents include temozolomide, epothilone, melphalan, carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polyphenprosan, ifosfamide, chlorambucil, mechlorethamine, busulfan, cyclophosphamide, carboplatin, cisplatin, thiotepa, capecitabine, streptozocin, bicalutamide, flutamide, nilutamide, leuprolide acetate, doxorubicin hydrochloride, bleomycin sulfate, daunorubicin hydrochloride, dactinomycin, daunorubicin citrate liposomal, doxorubicin hydrochloride liposomal, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, doxorubicin, valrubicin, anastrozole, toremifene citrate, cytarabine, fluorouracil, fludarabine, floxuridine, interferon alpha-2b, plicamycin, mercaptopurine, methotrexate, interferon alpha-2a, Medroxyprogesterone acetate, estramustine sodium phosphate, estradiol, leuprolide acetate, megestrol acetate, octreotide acetate, diethylstilbestrol diphosphate, testosterone, goserelin acetate, etoposide phosphate, vincristine sulfate, etoposide, vinblastine, etoposide, vincristine sulfate, teniposide, trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane, irinotecan hydrochloride, asparaginase, gemcitabine hydrochloride, altretinoin, topotecan hydrochloride, hydroxyurea, cladribine, mitotane, procarbazine hydrochloride, vinorelbine tartrate, pentostatin sodium, mitoxantrone, pegaspargase, denileukin diftitix, altretinoin, porfibril sodium, bexarotene, paclitaxel, docetaxel, arsenic trioxide, or tretinoin. Toxins include Pseudomonas exotoxin (PE38), ricin A chain, diphtheria toxin, besides PE and RT, pokeweed antiviral protein (PAP), saporin, and gelonin. Radionuclides used in cancer therapy include Y-90, P-32, I-131, In-111, Sr-89, Re-186, Sm-153, and Sn-117m.

Agents or factors suitable for use in antiviral infection therapy include abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, Ampligen, arbidol, atazanavir, Atripla, Boceprevirertet, cidofovir, succinate, darunavir, delavirdine, didanosine, docosin, edoxuridine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfoacetic acid, ganciclovir, ibacitabine, Imunovir, iodine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, dapoxetine, lopinavir, loviride, maraviroc, morpholinoguanidine, methisazone, nelfinavir, nevirapine, nexavir, nucleoside analogs, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, raltegravir, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine, synergist (antiretroviral), tea tree oil, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, triamterene, tromantine, Truvada, valacyclovir, valacyclovir, viraviroc, vidarabine, tarivirine hydrochloride, zalcitabine, zanamivir, and zidovudine.

The skilled artisan is referred to "Remington's Pharmaceutical Sciences", 15th edition, Chapter 33, especially pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for each subject in each case. In addition, for human administration, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards of the Office of the Food and Drug Administration (FDA) for biological products.

V. Examples

The following examples are included to illustrate specific embodiments of the present disclosure. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present disclosure and, therefore, can be considered to constitute preferred modes for its practice. However, it will be understood by those skilled in the art, in light of the present disclosure, that many changes can be made to the disclosed specific embodiments and still achieve the same or similar results without departing from the spirit and scope of the present disclosure.

Example 1

By using prepared phospholipid vesicles (Shah et al. , 1998; Nelsestuen, 1999), the affinity of Gla-domain proteins for cell membranes has been determined in vitro. However, it has not yet been fully elucidated how these in vitro values can be transferred to an in vivo context. For example, the interaction between FVII and TF demonstrates that although the Gla domains of these proteins are very homologous, additional differences in their cell membrane binding specificity and affinity may be mediated by their EGF and/or Kringle domains. Unfortunately, these interactions cannot be replicated using studies based on phospholipid vesicles alone and may remain unrecognized.

Therefore, the inventors propose to prepare and test the Gla+EGF/Kringle domain and the Gla domain independently from the following protein sets: hS (high affinity binder), hZ (medium affinity binder), hPT (medium affinity - contains a kringle), hFVII (low affinity - utilizes a second "receptor" that is also upregulated in cancer), and B0178 (hFVII with increased phospholipid affinity). These proteins may have different in vivo binding characteristics that may be beneficial for their use as probes (and, if validated and selective, as therapeutic agents) and have so far been confirmed to have gone unrecognized.

The general approach is to construct recombinant proteins and test their expression. An assay is then developed to assess binding. Expression and purification methods are then optimized, followed by quality control of γ-carboxylation.

Figure 1 shows sequences from various Gla domain proteins that include carboxylation sites. Figure 2 shows the expression of several different Gla domain proteins that were engineered and transiently expressed in 293 cells. Figure 3 shows a similar study in BHK21 cells. Given that one of the best expression constructs was the Protein S + EGF construct, a signal sequence from Protein S was used with prothrombin Gla + Kringle and Protein Z + EGF. However, expression was observed only intracellularly (Figure 4).

Protein S Gla + EGF was selected for further study. The sequence is shown in Figure 5. The protein was produced in BHK21 cells using RF286 medium. 600 ml was harvested and concentrated 4-fold. Purification was performed in three steps:

1. Ni-NTA column, 10 ml, freshly packed. Load the culture medium onto the column and elute with an imidazole gradient. Perform Gla western blotting on all fractions to identify His-tagged Gla proteins S G+E.

2. Hitrap Q with CaCl ₂ step elution. Gla-positive fractions were pooled and loaded into 1 ml HitrapQ and eluted with 10 mM CaCl ₂ .

3. Hitrap Q using a _CaCl₂ gradient (0-10 mM shadow gradient). Step-purified Gla protein was applied to Q and eluted with a _CaCl₂ gradient (up to 10 mM). A total of 0.9 mg of protein was produced at a purity level of 95%. Figure 6 shows the purified fractions under reducing and non-reducing conditions. Figures 7 and 8 show different FAC-based apoptosis assays. Both demonstrate that the Protein S Gla + EGF construct is specific for cells undergoing apoptosis, just like Annexin V (Figure 7), and that Annexin V can compete with Protein S Gla + EGF binding.

In summary, protein S Gla+EGF was expressed and purified. Analysis of the purified material suggested that it was highly gamma-formylated. A FAC-based apoptosis assay demonstrated that protein S G+E (11 Gla) can bind to "apoptotic" cells, and that this binding is specific to the cells via phosphatidylserine targeting, as demonstrated by an Annexin V competition assay.

In view of the present disclosure, all compositions and/or methods disclosed and claimed herein can be prepared and performed without excessive experimentation. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the compositions and/or methods described herein and the steps or the order of the steps of the methods without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents that are chemically and physiologically related can replace the agents described herein while achieving the same or similar results. All such similar substitutions and changes apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

VI. References

To the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, the following references are specifically incorporated herein by reference:

U.S. Patent 5,440,013

U.S. Patent 5,446,128

U.S. Patent 5,475,085

U.S. Patent 5,597,457

U.S. Patent 5,618,914

U.S. Patent 5,670,155

U.S. Patent 5,672,681

U.S. Patent 5,674,976

U.S. Patent 5,710,245

U.S. Patent 5,790,421

U.S. Patent 5,840,833

U.S. Patent 5,859,184

U.S. Patent 5,889,155

U.S. Patent 5,929,237

U.S. Patent 6,093,573

U.S. Patent 6,261,569

U.S. Patent Application 2005/0015232

U.S. Patent Application 2004/001827A1

U.S. Patent Application 2003/104578A2

U.S. Patent Application 2003/104578A1

WO06096515A2

WO07047504A2

WO06096515A2

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Sequence Listing

<110> Bayer Healthcare, LLC

BAUZON, Maxine

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Ala Asn Ser Leu Leu Xaa Xaa Thr Lys Gln Gly Asn Leu Xaa Arg Xaa

1 5 10 15

Cys Ile Xaa Xaa Leu Cys Asn Lys Xaa Xaa Ala Arg Xaa Val Phe Xaa

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35 40 45

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<221> Features not yet classified

<222> (7)..(8)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (11)..(11)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (15)..(15)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (17)..(17)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (20)..(21)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (26)..(27)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (30)..(30)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (33)..(33)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (35)..(35)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (40)..(40)

<223> X is a γ-carboxyglutamic acid residue

<400> 2

Ala Gly Ser Tyr Leu Leu Xaa Xaa Leu Phe Xaa Gly Asn Leu Xaa Lys

1 5 10 15

Xaa Cys Tyr Xaa Xaa Ile Cys Val Tyr Xaa Xaa Ala Arg Xaa Val Phe

20 25 30

Xaa Asn Xaa Val Val Thr Asp Xaa Phe Trp Arg Arg Tyr Lys

35 40 45

<210> 3

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> Features not yet classified

<222> (6)..(7)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (14)..(14)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (16)..(16)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (19)..(20)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (25)..(26)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (29)..(29)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (32)..(32)

<223> X is a γ-carboxyglutamic acid residue

<400> 3

Ala Asn Thr Phe Leu Xaa Xaa Val Arg Lys Gly Asn Leu Xaa Arg Xaa

1 5 10 15

Cys Val Xaa Xaa Thr Cys Ser Tyr Xaa Xaa Ala Phe Xaa Ala Leu Xaa

20 25 30

Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr

35 40 45

<210> 4

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> Features not yet classified

<222> (6)..(7)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (14)..(14)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (16)..(16)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (19)..(20)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (25)..(26)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (29)..(29)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (35)..(35)

<223> X is a γ-carboxyglutamic acid residue

<400> 4

Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa

1 5 10 15

Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Lys

20 25 30

Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser

35 40 45

<210> 5

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> Features not yet classified

<222> (6)..(7)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (14)..(14)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (16)..(16)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (19)..(20)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (25)..(26)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (29)..(29)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (32)..(32)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (35)..(35)

<223> X is a γ-carboxyglutamic acid residue

<400> 5

Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Gln Gly Ser Leu Xaa Arg Xaa

1 5 10 15

Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Xaa

20 25 30

Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser

35 40 45

<210> 6

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> Features not yet classified

<222> (6)..(7)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (14)..(14)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (16)..(16)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (19)..(20)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (25)..(26)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (29)..(29)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (32)..(32)

<223> X is a γ-carboxyglutamic acid residue

<220>

<221> Features not yet classified

<222> (36)..(36)

<223> X is a γ-carboxyglutamic acid residue

<400> 6

Ala Asn Ser Leu Leu Xaa Xaa Thr Lys Gln Gly Asn Leu Xaa Arg Xaa

1 5 10 15

Cys Ile Xaa Xaa Leu Cys Asn Lys Xaa Xaa Ala Arg Xaa Val Phe Xaa

20 25 30

Asn Asp Pro Xaa Thr Asp Tyr Phe Tyr Pro Lys Tyr Leu Val Cys Leu

35 40 45

Arg Ser Phe Gln Thr Gly Leu Phe Thr Ala Ala Arg Gln Ser Thr Asn

50 55 60

Ala Tyr Pro Asp Leu Arg Ser Cys Val Asn Ala Ile Pro Asp Gln Cys

65 70 75 80

Ser Pro Leu Pro Cys Asn Glu Asp Gly Tyr Met Ser Cys Lys Asp Gly

85 90 95

Lys Ala Ser Phe Thr Cys Thr Cys Lys Pro Gly Trp Gln Gly Glu Lys

100 105 110

Cys Glu Phe Asp Ile Asn Glu Cys Lys Asp Pro Ser Asn Ile Asn Gly

115 120 125

Gly Cys Ser Gln Ile Cys Asp Asn Thr Pro Gly Ser Tyr His Cys Ser

130 135 140

Cys Lys Asn Gly Phe Val Met Leu Ser Asn Lys Lys Asp Cys Lys Asp

145 150 155 160

Val Asp Glu Cys Ser Leu Lys Pro Ser Ile Cys Gly Thr Ala Val Cys

165 170 175

Lys Asn Ile Pro Gly Asp Phe Glu Cys Glu Cys Pro Glu Gly Tyr Arg

180 185 190

Tyr Asn Leu Lys Ser Lys Ser Cys Glu Asp Ile Asp Glu Cys Ser Glu

195 200 205

Asn Met Cys Ala Gln Leu Cys Val Asn Tyr Pro Gly Gly Tyr Thr Cys

210 215 220

Tyr Cys Asp Gly Lys Lys Gly Phe Lys Leu Ala Gln Asp Gln Lys Ser

225 230 235 240

Cys Glu Ser Arg His His His His His

245 250

Claims (28)

1. A polypeptide for the isolation of phosphatidylserine bound to cell membranes, comprising: (a) Protein Sγ-carboxyglutamate (Gla) domain, (b) The protein's S EGF domain, and (c) Lack of protease or hormone-binding domains; The polypeptide is linked to a payload selected from markers, proteins, and therapeutic agents. 2. The polypeptide of claim 1, wherein the polypeptide is conjugated to the payload. 3. The polypeptide according to claim 1, wherein the Gla domain comprises 5-15 Gla residues. 4. The polypeptide of claim 1, wherein the size of the polypeptide is in the range of 4.5 to 30 kD. 5. The polypeptide according to claim 1, wherein the polypeptide comprises at least one disulfide bond. 6. The polypeptide according to claim 5, wherein the polypeptide comprises 2-5 disulfide bonds. 7. The polypeptide according to claim 1, wherein the polypeptide has 300 amino acids or less. 8. The polypeptide according to claim 1, wherein the polypeptide is in the range of 100-300 amino acids. 9. The polypeptide of claim 1, wherein the payload is a detectable marker. 10. The polypeptide of claim 9, wherein the detectable label is a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme, a dye, or a ligand. 11. The polypeptide of claim 1, wherein the payload is a therapeutic agent. 12. The polypeptide of claim 11, wherein the therapeutic agent is an anticancer agent. 13. The polypeptide of claim 12, wherein the anticancer agent is a chemotherapeutic agent, a radiotherapy agent, a cytokine, a hormone, an antibody or antibody fragment or a toxin. 14. The polypeptide of claim 12, wherein the anticancer agent is a chemotherapeutic agent, a radiotherapy agent, or a toxin. 15. The polypeptide according to claim 12, wherein the anticancer agent is selected from temozolomide, epothilone, melphalan, carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polyphenylpropionate, ifosfamide, chlorambucil, nitrogen mustard, carboplatin, cisplatin, thiotepa, capecitabine, streptozoline, bicalutamide, flutamide, nilumethicone, leuprolide acetate, doxorubicin hydrochloride, bleomycin sulfate, daunorubicin hydrochloride, daunorubicin, daunorubicin citrate liposomes, doxorubicin hydrochloride liposomes, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, doxorubicin, penoxorubicin, anastrozole, toremifene citrate, cytarabine, fluorouracil, fludarabine, fluorouracil, interferon α-2b, procainamide, mercaptopurine, Methotrexate, interferon alpha-2a, medroxyprogesterone acetate, estradiol sodium phosphate, estradiol, megestrol acetate, octreotide acetate, diethylstilbestrol diphosphate, testosterone, goserelin acetate, etoposide phosphate, vincristine sulfate, etoposide, vinblastine, teniposide, trastuzumab, gemtuzumab oxamicin, rituximab, exemestane, irinotecan hydrochloride, asparaginase, gemcitabine hydrochloride, hexamethylmelamine, topotecan hydrochloride, hydroxyurea, cladribine, mitotane, procarbazine hydrochloride, vinorelbine tartrate, pentostatin sodium, mitoxantrone, pegaspargase, diazepam, retinoic acid, porphyrin, bexarotine, paclitaxel, docetaxel, arsenic trioxide or retinoic acid; toxins, and radionuclides used in cancer treatment. 16. The polypeptide of claim 11, wherein the therapeutic agent is an antiviral agent. 17. The polypeptide of claim 16, wherein the antiviral agent is selected from: abacavir, acyclovir, adefovir, amantadine, ampradil, arbidol, atazanavir, acetaminophen, pravelin, cidofovir, dalunavir, duravir, doxorubicin, inosine, doxorubicin, edaurenidine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitor, famciclovir, fomivir, fosanavir, phosphonoformic acid, phosphonoacetic acid, ganciclovir, ibatabin, isoprosine, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, interferon, lamivudine, lopinavir Viagra, cyclovir, maraviro, morpholine guanidine, meticazone, nelfinavir, nevirapine, rasavir, nucleoside analogs, oseltamivir, pegylated interferon alpha-2a, penciclovir, pelamyrvir, proconazole, podophyllotoxin, protease inhibitors, raltegravir, reverse transcriptase inhibitors, ribavirin, amantadine, ritonavir, prazolamydine, saquinavir, stavudine, telaprevir, tenofovir, tenofovir disoproxil fumarate, telanavir, trifluralin, trifluralin, triamcinolone, trevada, valacyclovir, valganciclovir, velivirol, vidarabine, tarivirin, zanamivir, or zidovudine. 18. The polypeptide of claim 17, wherein the interferon is interferon type III, interferon type II or interferon type I. 19. The polypeptide of claim 11, wherein the therapeutic agent is an enzyme. 20. The polypeptide of claim 11, wherein the therapeutic agent is a cytokine, growth factor, coagulation factor, or anticoagulant. 21. The polypeptide of claim 1, wherein the polypeptide further comprises an antibody Fc region. 22. A pharmaceutical preparation comprising the polypeptide according to claim 1. 23. Use of the polypeptide of claim 1 or the pharmaceutical preparation of claim 22 in the manufacture of a medicament for treating cancer. 24. The polypeptide or pharmaceutical preparation according to claim 23, wherein the cancer is breast cancer, brain cancer, gastric cancer, lung cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, skin cancer, rectal cancer, cervical cancer, uterine cancer, liver cancer, pancreatic cancer, head and neck cancer, or esophageal cancer. 25. Use of the polypeptide of claim 1 or the pharmaceutical preparation of claim 22 in the manufacture of a medicament for treating viral diseases. 26. The polypeptide or pharmaceutical preparation of claim 25, wherein the viral disease is influenza, human immunodeficiency virus, dengue virus, West Nile virus, smallpox virus, respiratory syncytial virus, Korean hemorrhagic fever virus, varicella, varicella-zoster virus, herpes simplex virus 1 or 2, Epstein-Barr virus, Marburg virus, Hantavirus, yellow fever virus, hepatitis A, B, C or E, Ebola virus, human papillomavirus, rhinovirus, Coxsackie virus, poliovirus, measles virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, HTLV-1 or -2. 27. The polypeptide of claim 15, wherein the toxin is a Pseudomonas exotoxin (PE38), ricin A chain, diphtheria toxin, pokeweed antiviral protein (PAP), saponin, or white tree toxin. 28. The polypeptide of claim 15, wherein the radionuclide for cancer treatment is Y-90, P-32, I-131, In-111, Sr-89, Re-186, Sm-153 or Sn-117m.