Anti-psgl-1 antibodies and uses thereof   

Abstract: Provided herein, in one aspect, are antibodies that immunospecifically bind to PSGL-1, polynucleotides comprising nucleotide sequences encoding such antibodies, and expression vectors and host cells for producing such antibodies. Also provided herein are kits and pharmaceutical compositions comprising antibodies that specifically bind to PSGL-1, as well as methods of treating a disorder or disease caused by or associated with increased proliferation and/or numbers of activated T cells using the antibodies described herein.

This application claims the benefit of the filing date of U.S. provisional patent application 61/496,249, filed Jun. 13, 2011 entitled “ANTI-PSGL-1 ANTIBODIES AND USES THEREOF”. The entire teachings and contents of the referenced provisional application are incorporated herein by reference.

Provided herein are antibodies that specifically bind to P-selectin glycoprotein ligand-1 (PSGL-1), polynucleotides encoding such antibodies, expression vectors comprising such polynucleotides, host cells comprising such expression vectors, and related compositions. Also provided herein are methods for treating a disorder or a disorder caused by or associated with increased proliferation and/or numbers of activated T cells, such as psoriasis, using anti-PSGL-1 antibodies.

Inflammatory responses to infection or injury are initiated by the adherence of leukocytes to the vascular wall (McEver et al., 1997, J. Clin. Invest., 100 (3): 485-492). Selectins represents a family of glycoproteins which mediate the first leukocyte-endothelial cell and leukocyte-platelet interactions during inflammation. The selectin family, which consists of L-selectin, E-selectin, and P-selectin, comprise an NH2-terminal lectin domain, followed by an EGF-like domain, a series of consensus repeats, a transmembrane domain, and a short cytoplasmic tail. The lectin domains of selectins interact with specific glycoconjugate ligands in order to facilitate cell adhesion. L-selectin, expressed on most leukocytes, binds to ligands on some endothelial cells and other leukocytes. E-selectin, expressed on cytokine activated endothelial cells, binds to ligands on most leukocytes. P-selectin, expressed on activated platelets and endothelial cells, also binds to ligands on most leukocytes.

P-selectin glycoprotein ligand-1 (“PSGL-1”), also known as SELPLG or CD162 (cluster of differentiation 162) is a human mucin-type glycoprotein ligand for all three selectins (Constantin, Gabriela, 2004, Drugs News Perspect, 17(9): 579-585; McEver et al., 1997, J. Clin. Invest., 100 (3): 485-492). PSGL-1 is a disulfide-bonded homodimer with two 120-kD subunits and is expressed on the surface of monocytes, lymphocytes, granulocytes, and in some CD34+ stem cells. PSGL-1 is likely to contribute to pathological leukocyte recruitment in many inflammatory disorders since it facilitates the adhesive interactions of selectins, suggesting that inhibitors of PSGL-1, such as antibodies to PSGL-1, are potentially useful anti-inflammatory drugs.

Several anti-PSGL-1 antibodies have been developed (see, e.g., International Application Pub. No. WO 2005/110475, published Nov. 24, 2005; International Application Pub. No. WO 2003/013603, published Feb. 20, 2003; Constantin, Gabriela, 2004, Drugs News Perspect, 17(9): 579-585).

SUMMARY

In one aspect, provided herein are antibodies and antibody derived antigen-binding fragments that specifically bind to PSGL-1. In one embodiment, provided herein is a monoclonal antibody which immunospecifically binds to human PSGL-1 comprising: (i) a variable light (“VL”) chain region comprising the amino acid sequence of SEQ ID NO: 3; (ii) a heavy chain comprising a variable heavy (“VH”) chain region comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a human IgG4 constant region which contains a Serine to Proline substitution at amino acid 228 of the heavy chain numbered according to the EU (gamma-G1 immunoglobulin) index (See, Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85). In a specific embodiment, provided herein is a monoclonal antibody which immunospecifically binds to human PSGL-1 comprising: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. In another specific embodiment, provided herein is a monoclonal antibody which immunospecifically binds to human PSGL-1 comprising: (i) a heavy chain consisting of SEQ ID NO: 2; and (ii) a light chain consisting of SEQ ID NO: 1. In another specific embodiment, provided herein is a monoclonal antibody which immunospecifically binds to human PSGL-1 comprising a heavy chain that comprises the amino acid sequence of SEQ ID NO: 2, and a complementary light chain. In a specific embodiment, any one of the foregoing monoclonal antibodies is purified.

In another aspect, provided herein is a pharmaceutical composition comprising any one of the foregoing monoclonal antibodies and a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody that is purified.

In one embodiment, provided herein is a pharmaceutical preparation comprising any one of the foregoing monoclonal antibodies in an aqueous solution comprising sodium citrate, sodium chloride, and citric acid monohydrate. In a specific embodiment, the pharmaceutical preparation is an aqueous solution comprising 9.1 mM sodium citrate dihydrate, 150 mM sodium chloride, and 0.9 mM citric acid. In another specific embodiment, the monoclonal antibody is present at a concentration of 0.267 mM in the pharmaceutical preparation. In a specific embodiment, the pharmaceutical preparation is an aqueous solution comprising 2.676 g/L sodium, 8.766 g/L sodium chloride, 0.2 g/L polysorbate 80, and 0.189 g/L citric acid. In another specific embodiment, the monoclonal antibody is present at a concentration of 40 g/L in the pharmaceutical preparation. In a specific embodiment, all the foregoing aqueous solutions are at pH 6.0.

In another aspect, provided herein are polynucleotides encoding an antibody described herein or an antigen-binding fragment thereof. In a specific embodiment, provided herein is an isolated polynucleotide encoding an antibody heavy chain comprising SEQ ID NO: 2. In an embodiment, an expression vector comprises the foregoing isolated polynucleotide. In certain embodiments, the expression vector further comprises a polynucleotide encoding an antibody light chain comprising SEQ ID NO:1. In a specific embodiment, the expression vector is a mammalian expression vector. In another aspect, provided herein is a host cell comprising a foregoing expression vector. In a specific embodiment, the host cell comprises (a) a first nucleic acid encoding SEQ ID NO: 2, operably linked to a promoter functional in said host cell; and (b) a second nucleic acid encoding SEQ ID NO: 1 operably linked to a promoter functional in said host cell. In another specific embodiment, the first and second nucleic acids of the host cell are in the same expression vector or in different expression vectors.

In another aspect, provided herein is a method of producing any of the foregoing monoclonal antibodies comprising culturing any of the foregoing host cells such that said first and second nucleic acids are expressed by said cell, and said heavy and light chains assemble together to form said antibody.

In another aspect, provided herein is an antibody heavy chain comprising SEQ ID NO: 2 or an antibody heavy chain fragment comprising amino acids 1 to 228 of SEQ ID NO:2. In a specific embodiment, provided herein is an antibody heavy chain comprising SEQ ID NO: 2. In another specific embodiment, provided herein is an antibody heavy chain fragment comprising amino acids 1 to 228 of SEQ ID NO:2. Also provided herein is a method of producing the foregoing heavy chain comprising culturing any of the foregoing host cells such that said heavy chain is expressed by the cell, and isolating said heavy chain. In a specific embodiment, a method of producing any of the foregoing antibodies comprises producing the heavy chain according to the forgoing method for producing the heavy chain, isolating said heavy chain, and complexing said heavy chain to an antibody light chain comprising SEQ ID NO:1.

In another aspect, provided herein is a kit comprising a first container containing any of the foregoing monoclonal antibodies. In a specific embodiment, the first container is a vial containing said monoclonal antibody as a lyophilized sterile powder under vacuum, and the kit further comprises a second container comprising a pharmaceutically acceptable fluid. Also provided herein is an injection device containing any of the foregoing monoclonal antibodies. In a specific embodiment, the injection device is a syringe.

In another aspect, provided herein are methods for preventing and/or treating a disease or disorder associated with or caused (in whole or part) by increased and/or numbers of activated T cells relative to healthy individuals or individuals not having the particular disease or disorder. In a specific embodiment, provided herein is a method for treating an inflammatory disorder, comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing monoclonal antibodies. In another specific embodiment, a method for treating an inflammatory disorder, comprises administering to a subject in need thereof a therapeutically effective amount of any of the foregoing pharmaceutical compositions. In a specific embodiment, the inflammatory disorder is an autoimmune disease. In another specific embodiment, the inflammatory disorder is psoriasis. In another specific embodiment, the inflammatory disorder is plaque psoriasis. In another specific embodiment, the plaque psoriasis is moderate to severe. In another specific embodiment, the inflammatory disorder is erythrodermic psoriasis. In another specific embodiment, the inflammatory disorder is psoriatic arthritis. In another specific embodiment, the inflammatory disorder is rheumatoid arthritis. In another specific embodiment, the inflammatory disorder is Crohn\'s disease. In another specific embodiment, the inflammatory disorder is ankylosing spondylitis. In another specific embodiment, the inflammatory disorder is diabetes.

FIG. 1 depicts non-reducing capillary gel electrophoresis (CGE) for h15A7 and 15A7H. The arrow indicates the peak for the half antibody molecule.

FIG. 2 depicts binding of 15A7H and control antibody to activated human CD4+ T-cells. EC50 values (nM) were derived using a one site 4-parameter fit model by plotting the mean fluorescence intensity (MFI) versus antibody concentration.

FIG. 3 depicts pooled dose response of 15A7H antibody in trans-vivo DTH in 4 donor PBMCs. The pooled mean±SEM of 4 donors after treatment with 0.03, 0.1, 0.3, 1 and 10 mg/kg antibody, or vehicle. pbmc: PBMC only, V: vehicle, 0.03, 0.1, 0.3, 1 and 10 mg/kg of 15A7H antibody. Percent inhibitions of foot pad thickness in response to 0.03, 0.1, 0.3, 1, and 10 mg/kg of 15A7H antibody treatment.

FIG. 4 depicts twenty four hour plasma concentrations of 15A7H antibody in C57BL/6 mice following a single intraperitoneal injection. Pooled plasma levels of the 2 antibodies (mean±SEM, n=4) in the experimental animals are plotted against their % inhibition of footpad thickness in the trans-vivo DTH assay.

FIG. 5 depicts 15A7H plasma concentrations versus time in C57BL/6 mice. Four satellite mice were dosed i.p. with 0.03, 0.1, 0.3, 1, and 10 mg/kg of 15A7H antibody. Blood samples were collected at 1, 3, 5 and 24 hrs.

FIG. 6 depicts CDC activity of 15A7H and control antibodies, which were tested at different concentrations (5, 0.5, 0.05, 0.005 and 0.0005 μg/ml).

FIG. 7A depicts the heavy chain amino acid sequence of antibody 15A7H (SEQ ID NO:2). The variable heavy chain region (SEQ ID NO:4) is in bold. The CDRs (CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO:9), and CDR3 (SEQ ID NO:10)) are boxed. The framework regions (FR1 (SEQ ID NO:17), FR2 (SEQ ID NO:18), FR3 (SEQ ID NO:19), and FR4 (SEQ ID NO:20)) are also indicated. The constant region is indicated. The hinge region amino acid sequence (SEQ ID NO:12) is boxed. The substituted proline at amino acid position 228 in the hinge region is italicized.

FIG. 7B depicts the light chain amino acid sequence of antibody 15A7H (SEQ ID NO:1). The variable light chain region (SEQ ID NO:3) is in bold. The CDRs (CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7)) are boxed. The framework regions (FR1 (SEQ ID NO:13), FR2 (SEQ ID NO:14), FR3 (SEQ ID NO:15), and FR4 (SEQ ID NO:16)) are also indicated. The constant region is indicated.

TABLE 1 List of SEQ ID NOs and their corresponding sequences SEQ ID NO. Corresponding Sequence SEQ ID NO: 1 15A7H Light Chain amino acid sequence SEQ ID NO: 2 15A7H Heavy Chain amino acid sequence SEQ ID NO: 3 15A7H Variable Light Chain Region (VL) amino acid sequence SEQ ID NO: 4 15A7H Variable Heavy Chain Region (VH) amino acid sequence SEQ ID NO: 5 15A7H VL CDR1 amino acid sequence SEQ ID NO: 6 15A7H VL CDR2 amino acid sequence SEQ ID NO: 7 15A7H VL CDR3 amino acid sequence SEQ ID NO: 8 15A7H VH CDR1 amino acid sequence SEQ ID NO: 9 15A7H VH CDR2 amino acid sequence SEQ ID NO: 10 15A7H VH CDR3 amino acid sequence SEQ ID NO: 11 Full length human PSGL-1 amino acid sequence SEQ ID NO: 12 IgG4 hinge region amino acid sequence SEQ ID NO: 13 15A7H VL FR1 SEQ ID NO: 14 15A7H VL FR2 SEQ ID NO: 15 15A7H VL FR3 SEQ ID NO: 16 15A7H VL FR4 SEQ ID NO: 17 15A7H VH FR1 SEQ ID NO: 18 15A7H VH FR2 SEQ ID NO: 19 15A7H VH FR3 SEQ ID NO: 20 15A7H VH FR4 SEQ ID NO: 21 IgG4 wild-type hinge region amino acid sequence

DETAILED DESCRIPTION

Provided herein are antibodies that specifically bind to PSGL-1. Also provided are isolated nucleic acids encoding such antibodies. Further provided are vectors and host cells comprising nucleic acids encoding such antibodies or antigen-binding fragments thereof. Also provided are methods of making such antibodies, cells, e.g. CHO cells, antibodies produced by such cells and purification of produced antibodies. Also provided herein is a method of treating and/or preventing a disorder or disease described herein (e.g., an inflammatory condition) comprising administering an antibody or an antibody derived antigen-binding fragment described herein that immunospecifically binds to PSGL-1. In a specific embodiment, the antibody is the IgG4 monoclonal antibody 15A7H described in Examples 1-4, infra.

6.1 Antibodies

Provided herein are monoclonal antibodies that immunospecifically bind to human P-selectin glycoprotein ligand-1 (“PSGL-1”). In a specific embodiment, provided herein is a monoclonal antibody which immunospecifically binds to human PSGL-1 comprising: (i) a variable light (“VL”) chain region comprising the amino acid sequence of SEQ ID NO: 3; (ii) a heavy chain comprising a variable heavy (“VH”) chain region comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a human IgG4 constant region which contains a Serine to Proline amino acid substitution at amino acid 228 of the heavy chain numbered according to the EU index. Non-limiting examples of human constant regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Preferably, the antibody binds to PSGL-1 and selectively induces apoptosis of activated T cells (relative to other cells that express PSGL-1). In a specific embodiment, binding of the antibody to PSGL-1 does not interfere with the interaction of P-selectin with PSGL-1, a function associated with PSGL-1 and a requirement for efficient localization of activated T cells and neutrophils to target tissues.

In a specific embodiment, an antibody which immunospecifically binds to PSGL-1 is a full-length immunoglobulin G of class 4 (IgG4), and preferably comprises a heavy chain sequence of SEQ ID NO:2, and even more preferably comprises a heavy chain sequence of SEQ ID NO:2 and a light chain sequence of SEQ ID NO:1 (the latter being monoclonal antibody 15A7H).

Also provided herein are antigen-binding fragments of an antibody, comprising the variable region sequences SEQ ID NO:3 and SEQ ID NO:4 and at least a portion of a human heavy chain constant region containing the human IgG4 hinge region up through and including a Serine to Proline substitution at amino acid 228 of a human heavy chain numbered according to the EU index. In a specific embodiment, an antibody derived antigen-binding fragment herein is a F(ab′)2 fragment.

An antibody or an antibody derived antigen-binding fragment described herein is preferably isolated, most preferably purified.

As used herein and unless otherwise specified, the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies and antibody derived antigen-binding fragments and are used interchangeably herein, and refer to the binding of an antibody or antibody derived antigen-binding fragment via its antigen combining site to its epitope, as would be understood by one skilled in the art. In one specific embodiment, an antibody or an antibody derived antigen-binding fragment that specifically binds to an antigen also can bind to other peptides or polypeptides, albeit generally with lower affinity as determined by, e.g., immunoassays, Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, Id.), or other assays known in the art. In a specific embodiment, an antibody or an antibody derived antigen-binding fragment that immunospecifically binds to an antigen binds to the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the Ka when the antibody or the antibody derived antigen-binding fragment binds to another antigen. In another specific embodiment, an antibody or an antibody derived antigen-binding fragment that immunospecifically binds to an antigen do not cross react by binding with other proteins. In specific embodiments, an antibody or an antibody derived antigen-binding fragment described herein specifically binds to a native isoform or native variant of PSGL-1 (that is a naturally occurring isoform or variant of PSGL-1 in an animal that can be isolated from an animal, preferably a human). In particular embodiments, an antibody or an antibody derived antigen-binding fragment described herein immunospecifically binds to human PSGL-1 or a fragment thereof. In specific embodiments, an antibody or an antibody derived antigen-binding fragment described herein specifically binds to human PSGL-1 and/or cynomologous PSGL-1 or a fragment thereof.

The amino acid sequence of SEQ ID NO:11 depicts the full length human PSGL-1, GenBank™ accession number AAA74577.1, GI:902797. In specific embodiments, an antibody described herein immunospecifically binds to PSGL-1 as determined, e.g., by ELISA or other antigen-binding assay known in the art, or described herein.

In specific aspects, provided herein is an antibody that specifically binds human and/or cynomologous PSGL-1 and that is an immunoglobulin G (having a gamma-heavy region) of class 4 (an IgG4) that is a tetramer of two identical disulfide-bonded dimers, each comprising a heavy chain and a light chain. The antibody preferably comprises a heavy chain of SEQ ID NO:2. More preferably, the antibody comprises a heavy chain of SEQ ID NO:2 and a light chain of SEQ ID NO:1. With respect to the light chain, in a specific embodiment, the light chain of an antibody described herein is a kappa light chain.

In specific embodiments, an antibody described herein comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:2. In specific embodiments, an antibody described herein comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:1, and comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:2. In specific embodiments, an antibody described herein comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:1. In specific embodiments, an antibody described herein comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:2.

In a specific embodiment, the antibodies provided herein are IgG4 monoclonal antibodies that specifically bind to PSGL-1. IgG4 antibodies are known to undergo a process called Fab arm exchange, also known as IgG4 shuffling, in which increased susceptibility of native IgG4 hinge disulfide bonds to reduction allows the heavy chains to separate and randomly re-associate to produce a mixed population of IgG4 molecules with randomized heavy-chain and light-chain pairs (Aalberse et al., 1999. Int Arch Allergy Immunol 118:187-189; Labrijn, et al., 2009, Nat Biotechnol 27:767-771; Schuurman et al., 2001. Mol Immunol 38:1-8; van der Neut Kolfschoten et al., 2007. Science 317:1554-1557).

It has been demonstrated that a Serine to Proline mutation at position 241 using Kabat numbering (Kabat et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) or at position 228 using the EU index (Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85) in the hinge region of human IgG4 results in considerable reduction of intra-chain disulfide bond formation, resulting in the reduction of IgG4 “half-antibody” molecules and reduced heterogeneity/shuffling of IgG4 molecules (Bloom et al. 1997, Protein Sci, 6:407-415; Angal et al., 1993, Molecular Immunology, 30(1): 105-108)). There are also published reports that this hinge mutation may decrease IgG4 shuffling and increase the half-life of the IgG4 molecules in vivo (Labrijn, et al., 2009, Nat Biotechnol 27:767-771; Stubenrauch, et al., 2010, Drug Metab Dispos 38:84-91). Van der Neut Kolfschoten et al., reported that the CH3 domain of IgG4 and not the core hinge is predominantly involved in the Fab arm exchange reaction (see Van der Neut Kolfschoten et al, 2007, Science, 317:1554-1557 (“Van der Neut Kolfschoten”) at page 1555, col. 2). Van der Neut Kolfschaten reported that exchanging the CH3 domain of IgG1 for the CH3 domain of IgG4 activated Fab arm exchange for the IgG1, while exchanging the CH3 domain of IgG4 abrogated Fab arm exchange for the IgG4 (see, p. 1555 and FIG. 2D).

In a specific embodiment, provided herein are IgG4 antibodies or antigen-binding fragments thereof, that specifically bind to PSGL-1, and that contain one or more amino acid substitutions in the IgG4 hinge region, wherein said antibody or antigen-binding fragment thereof retains specific binding to said PSGL-1 and wherein IgG4 shuffling is reduced relative to an antibody comprising an IgG4 hinge region not comprising said one or more amino acid substitutions. In a specific embodiment, the IgG4 hinge region only comprises a single amino acid substitution. An example of a “human IgG4 hinge region,” is the region on the heavy chain of an IgG4 antibody between the CH1 and CH2 domains consisting of the amino acid sequence of SEQ ID NO:12, as set forth in Angal et al., 1993, Molecular Immunology, 30(1): 105-108.

In a specific embodiment, a reduction in IgG4 shuffling is determined by detecting of a lower amount of half antibody molecules or of arm exchange produced from an antibody described herein which contains one or more amino acid substitutions in the hinge region, as compared to the amount of half antibody molecules or of arm exchange produced from an IgG4 molecule containing an IgG4 hinge region not comprising said one or more amino acid substitutions. Any assay well-known in the art can be used to detect half antibody production and bispecific antibody molecules. See, e.g., Van der Neut Kolfschoten et al, 2007, Science, 317:1554-1557, for examples of assays to detect production of bi-specific antibodies.

In a specific embodiment, provided herein are IgG4 monoclonal antibodies that specifically bind to PSGL-1, comprising a Serine to Proline amino acid substitution at amino acid position 228 of the heavy chain numbered according to the EU index.

In a specific embodiment, an antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising a Proline at position 228 of the heavy chain numbered according to the EU index.

In a specific embodiment, a monoclonal antibody, which immunospecifically binds to human PSGL-1 comprises: (i) a light chain having the amino acid sequence of SEQ ID NO:1; and (ii) a heavy chain comprising a human IgG4 constant region containing one or more amino acid substitutions in the IgG4 hinge region, wherein said antibody retains specific binding to said PSGL-1 and wherein IgG4 shuffling is reduced relative to an antibody comprising an IgG4 hinge region not comprising said one or more amino acid substitutions.

In a specific embodiment, a monoclonal antibody, which immunospecifically binds to human PSGL-1 comprises (i) a light chain having the amino acid sequence of SEQ ID NO:1; and (ii) a heavy chain comprising a human IgG4 constant region comprising a Serine to Proline amino acid substitution at amino acid position 228 of the heavy chain numbered according to the EU index or position 241 according to the Kabat numbering system (Kabat et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85).

In a specific embodiment, a monoclonal antibody, which immunospecifically binds to human PSGL-1 comprises: (i) a VL chain region comprising the amino acid sequence of SEQ ID NO:3; (ii) a VH chain region comprising the amino acid sequence of SEQ ID NO:4; and (iii) a human IgG4 constant region containing an IgG4 hinge region comprising one or more amino acid substitutions in the hinge region, wherein said antibody retains specific binding to PSGL-1 and wherein the IgG4 shuffling is reduced relative to an antibody comprising an IgG4 hinge region not comprising said one or more amino acid substitutions.

In a specific embodiment, a monoclonal antibody which immunospecifically binds to human PSGL-1 comprises: (i) a heavy chain comprising a VH chain region comprising the amino acid sequence of SEQ ID NO:4, and (ii) a human IgG4 heavy chain constant region comprising a Serine to Proline amino acid substitution at amino acid position 228 of the heavy chain numbered according to the EU index.

In a specific embodiment, a monoclonal antibody, which immunospecifically binds to human PSGL-1 comprises: (i) a VL chain region comprising the amino acid sequence of SEQ ID NO:3; (ii) a heavy chain comprising a VH chain region comprising the amino acid sequence of SEQ ID NO:4; and (iii) a human IgG4 heavy chain constant region comprising a Serine to Proline amino acid substitution at amino acid position 228 of the heavy chain numbered according to the EU index.

In certain embodiments, an IgG4 monoclonal antibody as described herein comprises the VH CDRs having the amino acid sequences described herein (e.g., see Table 3) and VL CDRs having the amino acid sequences described herein (e.g., see Table 2), wherein the antibody immunospecifically binds to PSGL-1 and has a hinge region mutation that reduces IgG4 shuffling (e.g., a Serine to Proline amino acid substitution at amino acid 228 of the heavy chain numbered according to the EU index). In a specific embodiment, the IgG4 monoclonal antibody immunospecifically binds PSGL-1 and comprises a heavy chain comprising (a) a VH chain region comprising SEQ ID NOs: 8, 9 and 10; and (b) a human IgG4 heavy chain constant region containing a Serine to Proline amino acid substitution at amino acid 228 of the heavy chain numbered according to the EU index. More preferably, the antibody further comprises a light chain comprising a VL chain region comprising SEQ ID NOs: 5, 6, and 7.

Table 2, below, presents the VL CDRs (in particular, VL CDR1, VL CDR2, and VL CDR3) of the amino acid sequence of 15A7H. Table 3, below, presents the VH CDRs (in particular, VH CDR1, VH CDR2, and VH CDR3) of the amino acid sequence of 15A7H. In specific embodiments, an antibody described herein which immunospecifically binds to human PSGL-1 (SEQ ID NO:11) comprises the VL CDR sequences in Table 2. In specific embodiments, an antibody described herein which immunospecifically binds to human PSGL-1 (SEQ ID NO:11), comprises the VH CDR sequences selected from those in Table 3.

TABLE 2 VL CDR Amino Acid Sequences

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