Methods of Treating T-Cell-Mediated Inflammatory Disease or Cancer Using Anti-PSGL-1 Antibodies in Combination with JAK Inhibitors

Abstract

Provided herein is a method of treating or preventing a T cell-mediated inflammatory disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds human PSGL-1 in combination with a Janus kinase (JAK) inhibitor. In some embodiments, the T cell-mediated inflammatory disease is GVHD (e.g., acute GVHD or chronic GVHD).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 280,463 (filed November 17, 2021), which is incorporated by reference in its entirety herein.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (606592001640seqlist.xml, size: 38,629 bytes, created on: November 14, 2022) are incorporated by reference in their entirety into this specification.

[0003] Provided herein is a method for treating or preventing a T cell-mediated inflammatory disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds to human PSGL-1 in combination with a Janus kinase (JAK) inhibitor. [Background technology]

[0004] T cell-mediated inflammatory diseases (e.g., graft-versus-host disease (GVHD), skin disorders, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, and autoimmune thyroid disorders), as well as cancers (e.g., leukemia and lymphoma) represent serious health conditions. As an example, acute GvHD (aGvHD) is a major complication of hematopoietic cell transplantation (HCT) accounting for most of the non-relapse mortality. Standard first-line treatment for aGvHD consists of 1-2 mg / kg prednisone or equivalent doses of steroids, with complete response (CR) rates ranging from 25-41%. Patients are considered steroid-resistant if (a) they progress after 3 days of treatment with the equivalent of 2 mg / kg / day of methylprednisolone (MP), (b) they fail to improve after 7 days of treatment with the equivalent of 2 mg / kg / day of MP, (c) they progress to new organs after treatment with the equivalent of 1 mg / kg / day of MP for skin and upper gastrointestinal GVHD, or (d) they relapse during or after steroid tapering (Przepiorka et al., (2019) The oncologist, 24:1-7).

[0005] The Janus kinase / signal transducer and activator of transcription (JAK / STAT) signaling pathway is known to be involved in the development and progression of many hematological and solid cancers [Waldmann and Chen (2017) Annu Rev Immunol 35:533-550; Vainchenker and Constantinescu (2013) Oncogene 32(21):2601-2613; Thomas et al., (2015) Br J Cancer, 113:365-71; O'Shea et al., (2013) N Engl J Med 368:161-70] and is also essential for the effects of inflammatory cytokines on the immune system (Villarino et al., (2017) Nat Immunol, 18:374-384). STATs and their upstream activators, JAKs, have been widely studied as targets for the treatment of cancer and inflammatory diseases (Qureshy et al., (2020) J Cancer Metastasis Treat 6: 27-44; Hosseini et al., (2020) J Cell Physiol. 235(9): 5903-5924). Many JAK inhibitors have been or are actively being tested in clinical trials as monotherapy or in combination with other drugs in patients with inflammatory diseases or cancer. At least five of these inhibitors are already Food and Drug Administration (FDA) approved for the treatment of inflammatory diseases or cancer, including rheumatoid arthritis, ulcerative colitis, GvHD (Qureshy et al., (2020) J Cancer Metastasis Treat 6: 27-44; Damsky et al., (2021) Journal of Allergy and Clinical Immunology 147(3): 814-826), and myelofibrosis.

[0006] Ruxolitinib is a JAK1 / 2 inhibitor that has recently been approved for the treatment of steroid-refractory acute GVHD (sr-aGvHD) and chronic GVHD (cGVHD). Despite the approval of ruxolitinib and several types of second-line therapy, the long-term outcome of sr-aGvHD is very poor, with mortality rates ranging from 70-80% (Levine et al., (2010) BBMT, 16(12): 1693-1699). The outcome for patients who fail second-line therapy (treatment-refractory) is even more dismal.

[0007] Acute GvHD is caused by donor-derived alloreactive T cells that recognize and destroy patient tissues, including the skin, GI track, and liver. Targeting and controlling or eliminating the highly proliferative and highly activated alloreactive T cells is the fundamental strategy for treating GvHD.

[0008] PSGL-1, long known as an adhesion molecule involved in leukocyte trafficking, has been recognized as an immune checkpoint regulator that downregulates chronically proliferating / activated T cells (Chen et al., (2004) Blood, 104(10):3233-3242; Huang et al., (2005) Eur J Immunol, 35(7):2239-2249). Neiflizumab (AbGn-168H), a humanized IgG4κ monoclonal antibody (mAb) against PSGL-1, preferentially induces apoptosis (programmed cell death) of late-activated T cells upon binding to PSGL-1. Neiflizumab has been tested in GVHD with encouraging clinical outcomes.

[0009] Several studies have revealed that PSGL-1 may be a promising target for immunotherapy treatment of some hematological malignancies. For example, gene expression profiling revealed that PSGL-1 is strongly expressed by cells of multiple myeloma and anaplastic large T-cell lymphoma, and further evaluation of the effects induced by in vitro treatment also showed that it is a promising candidate for humoral immunotherapy in these malignancies (Tripodo et al., (2009) Curr Cancer Drug Targets, 9(5): 617-625; Azab et al., (2012) Blood, 119(6): 1468-1478; Belmonte et al., (2021) Cancers, 13(12): 2958-2973).

[0010] There remains a need for more effective treatments for T cell-mediated inflammatory diseases and cancer. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Przepiorka et al.,(2019)The oncologist,24:1-7 [Non-Patent Document 2] Waldmann and Chen(2017)Annu Rev Immunol 35:533-550 [Non-Patent Document 3] Vainchenker and Constantinescu(2013)Oncogene 32(21):2601-2613 [Non-Patent Document 4] Thomas et al.,(2015)Br J Cancer,113:365-71 [Non-Patent Document 5] O’Shea et al.,(2013)N Engl J Med 368:161-70

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Summary of the Invention

Means for Solving the Problems

[0012] To meet this need, provided herein are methods of treating or preventing T cell-mediated inflammatory diseases or cancer using anti-PSGL-1 antibodies in combination with JAK inhibitors. These methods are based, at least in part, on the demonstration herein that anti-PSGL-1 antibodies have been found to act synergistically in combination with JAK inhibitors to promote induction of apoptosis in activated T cells and improve outcomes in patients with GVHD.

[0013] Thus, in one aspect, provided herein is a method for treating or preventing a T cell-mediated inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds human PSGL-1 in combination with a Janus kinase (JAK) inhibitor. In another aspect, provided herein is a method for treating or preventing cancer (e.g., a T cell-mediated cancer such as T cell leukemia or T cell lymphoma), comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds human PSGL-1 in combination with a Janus kinase (JAK) inhibitor. In some embodiments, the antibody that specifically binds human PSGL-1 comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain. In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), a CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and a CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprises a CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and a CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7). In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence AYYIH (SEQ ID NO: 24), a CDR-H2 comprising the amino acid sequence RVNPNTGGTSYNPKFKG (SEQ ID NO: 25), and a CDR-H3 comprising the amino acid sequence SGSPYYRYDD (SEQ ID NO: 26), and the VL domain comprises a CDR-L1 comprising the amino acid sequence RASSTVNSTYLH (SEQ ID NO: 28), a CDR-L2 comprising the amino acid sequence GSSNLAS (SEQ ID NO: 29), and a CDR-L3 comprising the amino acid sequence QQYSGYPLT (SEQ ID NO: 30).In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence TNAMN (SEQ ID NO: 32), a CDR-H2 comprising the amino acid sequence RIRSKSNNYATYYADSVKD (SEQ ID NO: 33), and a CDR-H3 comprising the amino acid sequence GGSYWYFDV (SEQ ID NO: 34), and the VL domain comprises a CDR-L1 comprising the amino acid sequence RSSQSIVNSNGNTYLE (SEQ ID NO: 36), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 37), and a CDR-L3 comprising the amino acid sequence FQGSHVPWT (SEQ ID NO: 38).

[0014] In some embodiments, the JAK inhibitor inhibits JAK1 and / or JAK2. In some embodiments, the JAK inhibitor inhibits JAK1 and / or JAK3. In some embodiments, the JAK inhibitor is ruxolitinib or tofacitinib. In some embodiments, the antibody is a humanized antibody. In some embodiments, the VH domain comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSS (SEQ ID NO: 4). In some embodiments, the VL domain comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIK (SEQ ID NO: 3). In some embodiments, the heavy chain further comprises an antibody constant domain. In some embodiments, the constant domain is a human IgG4 constant domain. In some embodiments, the constant domain is a human IgG4 constant domain comprising an S228P amino acid substitution at position 228, numbering according to EU numbering. In some embodiments, the light chain is a human kappa or lambda light chain. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH domain comprises the VH domain sequence of amino acid sequence SEQ ID NO:27, and / or the VL domain comprises the VL domain sequence of amino acid sequence SEQ ID NO:31. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:27, and / or the light chain comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the VH domain comprises the VH domain sequence of amino acid sequence SEQ ID NO:35, and / or the VL domain comprises the VL domain sequence of amino acid sequence SEQ ID NO:39. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:35, and / or the light chain comprises the amino acid sequence of SEQ ID NO:39.In some embodiments, an antibody that specifically binds to human PSGL-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:1.

[0015] In some embodiments, the T cell mediated inflammatory disease is graft versus host disease (GVHD). In some embodiments, the T cell mediated inflammatory disease is acute GVHD or chronic GVHD. In some embodiments, the T cell mediated inflammatory disease is steroid-resistant acute GVHD (SR-aGVHD) or treatment-resistant acute GVHD (TR-aGVHD). In some embodiments, the T cell mediated inflammatory disease is selected from the group consisting of skin disorders, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, and autoimmune thyroid disease. In some embodiments, the cancer is a T cell mediated cancer. In some embodiments, the cancer is a T cell leukemia or T cell lymphoma. In some embodiments, the human PSGL-1 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 22. In some embodiments, the subject is a human. In some embodiments, the antibody is administered by intravenous infusion. In some embodiments, the JAK inhibitor is administered orally. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the JAK inhibitor is administered to the subject before, after, or simultaneously with administration of the antibody. In some embodiments, the subject has been treated with a corticosteroid prior to administration of the antibody and the JAK inhibitor. In some embodiments, administration of the antibody and the JAK inhibitor results in a reduction in one or more symptoms of a T cell-mediated inflammatory disease in the subject.

[0016] In another aspect, provided herein is an antibody that specifically binds human PSGL-1 for use in a method for treating or preventing a T cell-mediated inflammatory disease or cancer in a subject (e.g., according to any one of the above embodiments). In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the antibody in combination with a Janus kinase (JAK) inhibitor. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, the VH domain comprising CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprising CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7). In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence AYYIH (SEQ ID NO: 24), CDR-H2 comprising the amino acid sequence RVNPNTGGTSYNPKFKG (SEQ ID NO: 25), and CDR-H3 comprising the amino acid sequence SGSPYYRYDD (SEQ ID NO: 26), and the VL domain comprises CDR-L1 comprising the amino acid sequence RASSTVNSTYLH (SEQ ID NO: 28), CDR-L2 comprising the amino acid sequence GSSNLAS (SEQ ID NO: 29), and CDR-L3 comprising the amino acid sequence QQYSGYPLT (SEQ ID NO: 30).In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence TNAMN (SEQ ID NO: 32), CDR-H2 comprising the amino acid sequence RIRSKSNNYATYYADSVKD (SEQ ID NO: 33), and CDR-H3 comprising the amino acid sequence GGSYWYFDV (SEQ ID NO: 34), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVNSNGNTYLE (SEQ ID NO: 36), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 37), and CDR-L3 comprising the amino acid sequence FQGSHVPWT (SEQ ID NO: 38).

[0017] In another aspect, provided herein is the use of an antibody that specifically binds human PSGL-1 in the manufacture of a medicament for treating or preventing a T cell-mediated inflammatory disease or cancer, wherein the antibody is administered in combination with a Janus kinase (JAK) inhibitor.In another aspect, provided herein is the use of an antibody that specifically binds human PSGL-1 and a Janus kinase (JAK) inhibitor in the manufacture of a medicament for treating or preventing a T cell-mediated inflammatory disease or cancer. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7). In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence AYYIH (SEQ ID NO: 24), CDR-H2 comprising the amino acid sequence RVNPNTGGTSYNPKFKG (SEQ ID NO: 25), and CDR-H3 comprising the amino acid sequence SGSPYYRYDD (SEQ ID NO: 26), and the VL domain comprises CDR-L1 comprising the amino acid sequence RASSTVNSTYLH (SEQ ID NO: 28), CDR-L2 comprising the amino acid sequence GSSNLAS (SEQ ID NO: 29), and CDR-L3 comprising the amino acid sequence QQYSGYPLT (SEQ ID NO: 30).In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence TNAMN (SEQ ID NO: 32), CDR-H2 comprising the amino acid sequence RIRSKSNNYATYYADSVKD (SEQ ID NO: 33), and CDR-H3 comprising the amino acid sequence GGSYWYFDV (SEQ ID NO: 34), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVNSNGNTYLE (SEQ ID NO: 36), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 37), and CDR-L3 comprising the amino acid sequence FQGSHVPWT (SEQ ID NO: 38).

[0018] In another aspect, provided herein is a kit comprising an antibody that specifically binds human PSGL-1 and a package insert containing instructions for using the antibody in combination with a Janus kinase (JAK) inhibitor to treat or prevent a T cell-mediated inflammatory disease in a subject. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, the VH domain comprising CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprising CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7). In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence AYYIH (SEQ ID NO: 24), CDR-H2 comprising the amino acid sequence RVNPNTGGTSYNPKFKG (SEQ ID NO: 25), and CDR-H3 comprising the amino acid sequence SGSPYYRYDD (SEQ ID NO: 26), and the VL domain comprises CDR-L1 comprising the amino acid sequence RASSTVNSTYLH (SEQ ID NO: 28), CDR-L2 comprising the amino acid sequence GSSNLAS (SEQ ID NO: 29), and CDR-L3 comprising the amino acid sequence QQYSGYPLT (SEQ ID NO: 30).In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence TNAMN (SEQ ID NO: 32), CDR-H2 comprising the amino acid sequence RIRSKSNNYATYYADSVKD (SEQ ID NO: 33), and CDR-H3 comprising the amino acid sequence GGSYWYFDV (SEQ ID NO: 34), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVNSNGNTYLE (SEQ ID NO: 36), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 37), and CDR-L3 comprising the amino acid sequence FQGSHVPWT (SEQ ID NO: 38). In some embodiments, the kit or article of manufacture further comprises a JAK inhibitor.

[0019] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1 shows the results of a T cell apoptosis assay testing the synergistic effect of a combination of ruxolitinib ("RUXO") and anti-PSGL-1 antibody 15A7H. Ruxolitinib and antibody 15A7H were tested alone or in combination at the indicated concentrations (RUXO: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 1A; Donor 2; FIG. 1B; Donor 3, FIG. 1C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib at a control antibody concentration of 3 μg / mL was also tested. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 1 herein. [Figure 1B]FIG. 1 shows the results of a T cell apoptosis assay testing the synergistic effect of a combination of ruxolitinib ("RUXO") and anti-PSGL-1 antibody 15A7H. Ruxolitinib and antibody 15A7H were tested alone or in combination at the indicated concentrations (RUXO: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 1A; Donor 2; FIG. 1B; Donor 3, FIG. 1C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib at a control antibody concentration of 3 μg / mL was also tested. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 1 herein. [Figure 1C] FIG. 1 shows the results of a T cell apoptosis assay testing the synergistic effect of a combination of ruxolitinib ("RUXO") and anti-PSGL-1 antibody 15A7H. Ruxolitinib and antibody 15A7H were tested alone or in combination at the indicated concentrations (RUXO: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 1A; Donor 2; FIG. 1B; Donor 3, FIG. 1C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib at a control antibody concentration of 3 μg / mL was also tested. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 1 herein.

[0021] [Diagram 2]1 shows the results of an experiment testing the combinatorial effect of the combination of ruxolitinib ("RUXO") and anti-PSGL-1 antibody 15A7H on activated T cell apoptosis, assessed using Chou-Talalay median effect analysis as described in Example 1 herein. The indicated fixed ratios (3.3:1, 1.1:1, and 0.37:1) of ruxolitinib and antibody 15A7H were tested for their effect on activated T cell apoptosis in cells derived from the indicated donors (N089, N084, N102, and N103). The x-axis shows the observed apoptotic fraction compared to untreated control cells, and the y-axis shows the corresponding combination index (CI). Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CI was <1, 1, and >1, respectively.

[0022] [Figure 3A] 3A-3C show the results of a T cell apoptosis assay testing the combination of rapamycin ("RAPA") and anti-PSGL-1 antibody 15A7H. Rapamycin and antibody 15A7H were tested alone or in combination at the indicated concentrations (RAPA: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 3A; Donor 2; FIG. 3B; Donor 3, FIG. 3C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib was also tested at a control antibody concentration of 3 μg / mL. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 2 herein. [Figure 3B]3A-3C show the results of a T cell apoptosis assay testing the combination of rapamycin ("RAPA") and anti-PSGL-1 antibody 15A7H. Rapamycin and antibody 15A7H were tested alone or in combination at the indicated concentrations (RAPA: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 3A; Donor 2; FIG. 3B; Donor 3, FIG. 3C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib was also tested at a control antibody concentration of 3 μg / mL. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 2 herein. [Figure 3C] 3A-3C show the results of a T cell apoptosis assay testing the combination of rapamycin ("RAPA") and anti-PSGL-1 antibody 15A7H. Rapamycin and antibody 15A7H were tested alone or in combination at the indicated concentrations (RAPA: 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM; antibody 15A7H: 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL) in activated T cells derived from peripheral blood mononuclear cells (PBMCs) of three donors (Donor 1, FIG. 3A; Donor 2; FIG. 3B; Donor 3, FIG. 3C). An isotype control antibody ("Isotype Control") alone or in combination with ruxolitinib was also tested at a control antibody concentration of 3 μg / mL. The level of cell apoptosis, shown on the y-axis, was assessed as described in Example 2 herein.

[0023] [Figure 4A]4A-4C show the results of experiments testing the combinatorial effect of combinations of ruxolitinib ("RUXO") with anti-PSGL-1 antibodies LH10 (FIG. 4A), c43B6 (FIG. 4B), or h9F9 (FIG. 4C) on activated T cell apoptosis, assessed using Chou-Talalay median effect analysis as described in Example 3 herein. The indicated fixed ratios of ruxolitinib and anti-PSGL-1 antibodies (3.3:1, 1.1:1, and 0.37:1) were tested for their effect on activated T cell apoptosis in cells derived from the indicated donors (N98, M1, and N136). The x-axis shows the observed apoptotic fraction compared to untreated control cells, and the y-axis shows the corresponding Log10 combination index (CI). Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CI was <1, 1, and >1, respectively. [Figure 4B] 4A-4C show the results of experiments testing the combinatorial effect of combinations of ruxolitinib ("RUXO") with anti-PSGL-1 antibodies LH10 (FIG. 4A), c43B6 (FIG. 4B), or h9F9 (FIG. 4C) on activated T cell apoptosis, assessed using Chou-Talalay median effect analysis as described in Example 3 herein. The indicated fixed ratios of ruxolitinib and anti-PSGL-1 antibodies (3.3:1, 1.1:1, and 0.37:1) were tested for their effect on activated T cell apoptosis in cells derived from the indicated donors (N98, M1, and N136). The x-axis shows the observed apoptotic fraction compared to untreated control cells, and the y-axis shows the corresponding Log10 combination index (CI). Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CI was <1, 1, and >1, respectively. [Figure 4C]4A-4C show the results of experiments testing the combinatorial effect of combinations of ruxolitinib ("RUXO") with anti-PSGL-1 antibodies LH10 (FIG. 4A), c43B6 (FIG. 4B), or h9F9 (FIG. 4C) on activated T cell apoptosis, assessed using Chou-Talalay median effect analysis as described in Example 3 herein. The indicated fixed ratios of ruxolitinib and anti-PSGL-1 antibodies (3.3:1, 1.1:1, and 0.37:1) were tested for their effect on activated T cell apoptosis in cells derived from the indicated donors (N98, M1, and N136). The x-axis shows the observed apoptotic fraction compared to untreated control cells, and the y-axis shows the corresponding Log10 combination index (CI). Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CI was <1, 1, and >1, respectively.

[0024] [Diagram 5] 1 shows the results of experiments testing the combinatorial effect of combinations of tofacitinib ("TOFA") with anti-PSGL-1 antibodies 15A7H or LH10 on activated T cell apoptosis, assessed using Chou-Talalay median effect analysis as described in Example 4 herein. The indicated fixed 1:1 ratios of tofacitinib and antibodies 15A7H or LH10 were tested for their effect on activated T cell apoptosis in cells derived from the indicated donors (M1, N93, and N95). The x-axis shows the observed apoptotic fraction compared to untreated control cells, and the y-axis shows the corresponding Log10 combination index (CI). Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CI was <1, 1, and >1, respectively.

[0025] [Figure 6]Results from experiments testing the effect of ruxolitinib, anti-PSGL-1 antibody 15A7H, or a combination of both in a xenogeneic mouse model of graft-versus-host disease (GvHD) are shown. Progressive severe immunodeficiency (ASID) mice were irradiated and transplanted with human PBMCs to induce xenogeneic GvHD. Treatment began 72 hours after human PBMC engraftment (assigned as day 1). Ruxolitinib or vehicle was administered orally at 45 mg / kg (45mpk) twice daily for 15 days, followed by anti-PSGL-1 15A7H antibody or vehicle at 10 mg / kg (10mpk) intravenously every 3-4 days for a total of 5 doses. Mice were assessed daily for survival. Survival over time after treatment in all four treatment groups is shown (n=5-7 per group). The combination treatment group showed the best survival rate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Provided herein is a method for treating or preventing a T cell-mediated inflammatory disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds to human PSGL-1 in combination with a Janus kinase (JAK) inhibitor. In some embodiments, the antibody that specifically binds to human PSGL-1 comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, the VH domain comprising CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprising CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7). Further provided herein are related uses and kits / articles of manufacture.

[0027] I. Definition An antibody is an immunoglobulin molecule capable of specifically binding to a target (e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, as well as polypeptides including fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain variable fragments (scFv), single chain diabodies (scDbs), tandem single chain variable fragment (scFv) units (tandem scFvs are referred to as taFvs), and variants or other configurations thereof, fusion proteins containing antibody moieties, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site.

[0028] Antibodies include antibodies of any class, e.g., IgG, IgA, IgM (or subclasses thereof), and do not have to be of a particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0029] The antibodies of the present disclosure are further intended to include bispecific, multispecific, chimeric, humanized, and recombinantly constructed molecules that have affinity for a polypeptide conferred by at least one CDR region of the antibody. Single domain antibodies, either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain, are known in the art. See, e.g., Holt et al., Trends Biotechnol. 21:484-490, 2003. Also known in the art are methods for making antibodies that contain either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain that contains three of the six naturally occurring complementarity determining regions from an antibody. See, e.g., Muyldermans, Rev. Mol. Biotechnol. 74:277-302, 2001.

[0030] As used herein, "monoclonal antibody" refers to a substantially homogeneous antibody, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are generally highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations (which typically contain different antibodies directed against different determinants (epitopes)), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may, for example, be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554.

[0031] As used herein, "chimeric antibody" refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. An intact chimeric antibody contains two copies of a chimeric light chain and two copies of a chimeric heavy chain. The generation of chimeric antibodies is known in the art (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory). Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one species of mammal, while the constant portions are homologous to the sequences of antibodies from the other species. One distinct advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, for example using readily available hybridomas or B cells from non-human host organisms, and combined with constant regions derived from, for example, human cell preparations. The variable regions have the advantage of being easily prepared and the specificity is not affected by their source, while the constant regions, being human, are less likely to elicit an immune response from a human subject when the antibody is injected than constant regions derived from non-human sources. However, the definition is not limited to this particular example. In some embodiments, the amino acid modifications are made in the variable and / or constant regions.

[0032] As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (e.g., mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region or domain (e.g., an Fc domain), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO99 / 58572. Other forms of humanized antibodies have one or more (1, 2, 3, 4, 5, 6) CDRs that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0033] As used herein, "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody generated by a human and / or an antibody made using any technique for making human antibodies known in the art or disclosed herein. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. Human antibodies can be generated using a variety of techniques known in the art. In one embodiment, human antibodies are selected from phage libraries that express human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes that produce antibodies against target antigens (such B lymphocytes may be harvested from an individual or immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147(1):86-95; and U.S. Patent No. 5,750,373.

[0034] The "variable region" of an antibody (the term "variable domain" may be used interchangeably herein) refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. The heavy and light chain variable regions (VH and VL domains, respectively) each consist of four framework regions (FR) connected to three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on sequence variability between species (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either approach or a combination of both approaches.

[0035] Several HVR delimitations are in use and are encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), while Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. Residues from each of these HVRs are shown below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0036] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra, or Edelman, GM et al. (1969) Proc. Natl. Acad. Sci. USA 63:78-85).

[0037] As used herein, "Fv" may refer to the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment typically consists of a dimer of one heavy and one light chain variable region domain in tight non-covalent association. Folding of these two domains results in six hypervariable loops (three loops each from the H and L chains), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0038] The "constant region" of an antibody (sometimes referred to herein as "constant domain" interchangeably) refers to the constant region of the antibody light chain (CL) or the constant region of the antibody heavy chain (CH), either alone or in combination. Generally, the constant region of an antibody provides structural stability and other biological functions (e.g., antibody chain assembly, secretion, transplacental mobility, and complement binding), but is not involved in binding to an antigen. The amino acid sequence in the gene of the constant region and the corresponding exon sequence depend on the species from which it is derived, but the variation in amino acid sequence that results in allotypes is relatively limited for a particular constant region within a species. The variable region of each chain is linked to the constant region by a linking polypeptide sequence. The linking sequence is encoded by a "J" sequence for the light chain gene and a combination of "D" and "J" sequences for the heavy chain gene. Depending on the antibody isotype, the heavy chain constant region can include a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and / or a CH4 domain. In certain embodiments, the heavy chain constant region comprises a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0039] The term "Fc region" (which may be used interchangeably herein with the term "Fc domain") is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain may vary. In some embodiments, the Fc region may include one or more amino acids of the hinge region. In some embodiments, a human IgG heavy chain Fc region is defined to extend from the amino acid residue at EU position 216 to the carboxyl-terminus thereof. Native sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0040] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is a V protein that is linked together into a single polypeptide chain. H and V LPreferably, the sFv polypeptide comprises a V domain which enables the sFv to form the desired structure for antigen binding. H and V L It further comprises a polypeptide linker between the domains. For a review of sFv, see Pluckthun (The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0041] The term "diabody" refers to H Domain and V L These refer to antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (e.g., about 5-12 residues) between the V domains to achieve interchain but not intrachain pairing of the V domains, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are fragments that combine the V domains of two antibodies. H Domain is V L Diabodies are heterodimers of two "crossover" sFv fragments whose domains are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0042] "Percentage (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software (e.g., BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0043] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including, for example, unnatural amino acids, and other modifications known in the art. Because the polypeptides of the present disclosure are based on a tetravalent antibody, it is understood that the polypeptides can occur as single chains or associated chains.

[0044] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and / or RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps" that replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any hydroxyl groups normally present in the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, activated to prepare additional linkages to additional nucleotides, or conjugated to solid supports. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties from 1 to 20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides can also include analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs (e.g., methyl riboside). One or more phosphodiester linkages may be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages within a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0045] As used herein, "vector" refers to a construct capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0046] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. An expression control sequence can be a promoter (e.g., a constitutive or inducible promoter) or an enhancer. An expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0047] As used herein, an "effective amount" or "therapeutically effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce a beneficial, desired, and / or therapeutic result. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifested during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, improving the quality of life of people suffering from the disease, reducing the dose of other medications required to treat the disease, enhancing the effect of other medications, for example via targeting, delaying disease progression, and / or extending survival. For example, when treating an individual awaiting transplant, an effective amount of the drug can reduce to some extent the levels of alloantibodies and / or PRA in the individual. When treating an individual undergoing a transplant or transfusion, an effective amount of a drug may have some effect and / or mitigating effect on one or more of the symptoms or conditions associated with the transplant or transfusion (e.g., T cell-mediated inflammatory disease). An effective amount may be administered in one or more administrations. For purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. An effective dosage may be administered in one or more administrations. For purposes of this disclosure, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., a JAK inhibitor). Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if a desired result can or is achieved in conjunction with one or more other agents.

[0048] As used herein, "with" or "in combination with" refers to the administration of one therapeutic modality in addition to another. Thus, "with" or "in combination with" refers to the administration of one therapeutic modality before, during, or after the administration of the other therapeutic modality to an individual.

[0049] As used herein, "treatment" or "treating" refers to an approach to obtain beneficial or desired results, including desirable clinical results. Beneficial, desired, and / or therapeutic clinical results include, but are not limited to, one or more of the following: reduction or suppression of one or more symptoms of inflammation or autoimmunity (e.g., due to a T cell-mediated inflammatory disease), increasing the likelihood of a successful outcome for the patient, and / or mitigating one or more contraindications or adverse outcomes associated with medical treatment (e.g., those associated with transplantation or transfusion), reducing symptoms attributable to the disease, improving the quality of life for people suffering from the disease, reducing the dosage of other medications required to treat the disease, slowing the progression of the disease, and / or extending the survival of an individual.

[0050] As used herein, "delaying the onset of disease" means to prolong, hinder, delay, stabilize, and / or postpone the onset of disease (e.g., cancer). The length of time of this delay may vary depending on the history of the disease and / or the patient being treated. As will be apparent to one of skill in the art, sufficient or significant delay may actually include prevention, in that the individual does not develop the disease. For example, symptoms of inflammatory disease (e.g., T cell-mediated inflammatory disease) may be delayed.

[0051] An "individual" or "subject" refers to a mammal, more preferably a human, and includes, but is not limited to, farm animals, sport animals, pets (e.g., cats, dogs, or horses), primates, mice, and rats.

[0052] As used herein, the term "specifically recognize" or "specifically bind" refers to a measurable and reproducible interaction, e.g., attraction or binding between a target and an antibody (e.g., a full-length antibody, an antibody fragment, or an antibody VH-VL binding unit), that determines the presence of a target in the presence of a population of heterogeneous molecules (including biological molecules). For example, an antibody, antibody fragment, or antibody VH-VL binding unit that specifically or preferentially binds to an epitope is an antibody that binds to this epitope more readily and / or with a higher affinity, avidity, and / or longer duration than it binds to other epitopes of the target or to non-target epitopes. It is also understood by reading this definition that, for example, an antibody, antibody fragment, or antibody VH-VL binding unit that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. The association constant of an antibody, antibody fragment, or antibody VH-VL binding unit that specifically binds to a target is about 10 3 M -1 Or about 10 4 M -1 or more, in some cases about 10 5 M -1 Or about 10 6 M -1 or more, in other cases about 10 6 M -1 Or about 10 7 M -1 or more, about 10 8 M -1 ~about 10 9 M -1 , or about 10 10 M -1 ~about 10 11 M -1or more. A variety of immunoassay formats can be used to select antibodies, antibody fragments, or antibody VH-VL binding units that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that can be used to quantify specific immunoreactivity, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0053] "Package insert" refers to instructions customarily included in a commercial package of a pharmaceutical product, including information about the indication customarily included in a commercial package of a pharmaceutical product, including information such as indications, usage, dosage, administration, contraindications, other medications with which the packaged product is combined, and / or warnings regarding the use of such medications.

[0054] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to an "antibody" is a reference to one to many antibodies (e.g., molar amounts) and includes equivalents thereof known to those of skill in the art, and so forth.

[0055] Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter in itself. For example, statement of "about X" includes statement of "X."

[0056] It is to be understood that the embodiments and variations of the present disclosure described herein include those that "consist of" and / or "consist essentially of."

[0057] II. Anti-PSGL-1 Antibodies and Methods of Use Certain aspects of the present disclosure relate to a method for treating or preventing a T cell-mediated disease by administering a therapeutically effective amount of an antibody that specifically binds human PSGL-1 in combination with a Janus kinase (JAK) inhibitor of the present disclosure. Any anti-PSGL-1 antibody of the present disclosure can be used in the methods disclosed herein. In some embodiments, the T cell-mediated disease is a T cell-mediated inflammatory disease. In some embodiments, the T cell-mediated disease is a T cell-mediated cancer (e.g., T cell leukemia or lymphoma).

[0058] In some embodiments, provided herein are anti-PSGL-1 antibodies comprising at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the anti-PSGL-1 antibodies comprise (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:7, e.g., as shown in Table A.

[0059] In some embodiments, provided herein are anti-PSGL-1 antibodies comprising at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-PSGL-1 antibodies comprise (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, e.g., as shown in Table A.

[0060] In some embodiments, provided herein is an anti-PSGL-1 antibody comprising: (a) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:7; and (b) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10, e.g., as shown in Table A.

[0061] In some embodiments, the anti-PSGL-1 antibody comprises at least one of (a) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-PSGL-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-PSGL-1 antibody comprises (a) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, e.g., as shown in Table A.

[0062] In some embodiments, the present specification provides an anti-PSGL-1 antibody comprising: (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a light chain variable domain (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, e.g., as shown in Table A.

[0063] In some embodiments, provided herein is an anti-PSGL-1 antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-PSGL-1 antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VL domain derived from the light chain sequence of SEQ ID NO: 31.

[0064] In some embodiments, provided herein is an anti-PSGL-1 antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-PSGL-1 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VH domain derived from the heavy chain sequence of SEQ ID NO: 27.

[0065] In some embodiments, the anti-PSGL-1 antibody comprises a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:24, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:26, and a VL domain comprising (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:30, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VH domain derived from the heavy chain sequence of SEQ ID NO:27 and / or a VL domain derived from the light chain sequence of SEQ ID NO:31. In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:27 and / or a light chain comprising the amino acid sequence of SEQ ID NO:31.

[0066] In some embodiments, provided herein is an anti-PSGL-1 antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-PSGL-1 antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VL domain derived from the light chain sequence of SEQ ID NO: 39.

[0067] In some embodiments, provided herein is an anti-PSGL-1 antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-PSGL-1 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 34, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VH domain derived from the heavy chain sequence of SEQ ID NO: 35.

[0068] In some embodiments, the anti-PSGL-1 antibody comprises a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 34, and a VL domain comprising (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises a VH domain derived from the heavy chain sequence of SEQ ID NO: 35 and / or a VL domain derived from the light chain sequence of SEQ ID NO: 39. In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0069] Exemplary anti-PSGL-1 antibody sequences and exemplary human PSGL-1 polypeptide sequences are shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises the six CDR sequences of antibody 15A7H, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises the VH and / or VL domain sequence(s) of antibody 15A7H, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody (e.g., a tetravalent anti-PSGL-1 antibody) comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-PSGL-1 antibody comprises the six CDR sequences of antibody c43B6, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises the VH and / or VL domain sequence(s) of antibody c43B6, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises the six CDR sequences of antibody h9F9, e.g., as shown in Table A. In some embodiments, the anti-PSGL-1 antibody comprises the VH and / or VL domain sequence(s) of antibody h9F9, e.g., as shown in Table A. In some embodiments, the antibody is humanized or chimeric. In some embodiments, the antibody is a tetravalent anti-PSGL-1 antibody. [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8] [Table A-9] [Table A-10]

[0070] In some embodiments, the anti-PSGL-1 antibody is a humanized anti-PSGL1 antibody. In some embodiments, the anti-PSGL-1 antibody comprises the CDR of any embodiment provided herein and further comprises a human immunoglobulin framework or a human consensus framework. In some embodiments, the humanized anti-PSGL1 antibody comprises (a) a heavy chain variable domain (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10, and (b) a light chain variable domain (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:7, for example, as shown in Table A.

[0071] In some embodiments, the anti-PSGL-1 antibody comprises at least one, two, three, four, five, six, seven, or eight framework regions (FRs) selected from: (a) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15; (d) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16; (e) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17; (f) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18; (g) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19; and (h) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20, e.g., as shown in Table A.

[0072] In some embodiments, the anti-PSGL-1 antibody comprises at least one, two, three, or all four VL FR sequences selected from (a) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, and (d) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PSGL-1 antibody comprises at least one, two, three, or all four VH FR sequences selected from (a) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18, (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19, and (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20.

[0073] In some embodiments, the anti-PSGL-1 antibody comprises (a) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, and (d) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PSGL-1 antibody comprises (a) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18, (c) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19, and (d) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20.

[0074] In some embodiments, the anti-PSGL-1 antibody comprises: (a) a VL domain comprising at least one, at least two, at least three, or all four VL FR sequences selected from (i) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14; (iii) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15; and (iv) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16; and (b) a VH domain comprising at least one, at least two, at least three, or all four VH FR sequences selected from (i) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18; (iii) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19; and (iv) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20.

[0075] In some embodiments, the anti-PSGL-1 antibody comprises (a) a VL domain comprising (i) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14, (iii) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, and (iv) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16, and (b) a VH domain comprising (i) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18, (iii) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19, and (iv) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20.

[0076] In some embodiments, the anti-PSGL-1 antibody comprises (a) a VL domain comprising: (i) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13; (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (iii) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14; (iv) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; (v) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15; (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and (vii) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PSGL-1 antibody comprises (a) a VH domain comprising: (i) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8; (iii) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18; (iv) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9; (v) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19; (vi) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10; and (vii) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20.

[0077] In some embodiments, the anti-PSGL-1 antibody comprises (a) a VL domain comprising (i) a VL FR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (iii) a VL FR2 comprising the amino acid sequence of SEQ ID NO: 14, (iv) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, (v) a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and (vii) a VL FR4 comprising the amino acid sequence of SEQ ID NO: 16; and (b) (viii) a VH FR1 comprising the amino acid sequence of SEQ ID NO: 17, (ix) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, (x) a VH FR2 comprising the amino acid sequence of SEQ ID NO: 18, (xi) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, (xii) a VH FR3 comprising the amino acid sequence of SEQ ID NO: 19, (xiii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and (xiv) a VH FR4 comprising the amino acid sequence of SEQ ID NO: 20. and a VH domain comprising FR4.

[0078] In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain variable domain (VH) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 4 contains substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-PSGL-1 antibody comprising the sequence retains the ability to bind to PSGL-1. In some embodiments, a total of 1-10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 4. In some embodiments, a total of 1-5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 4. In some embodiments, the substitutions, insertions, or deletions occur within regions outside the CDRs (i.e., within the FRs). In some embodiments, the anti-PSGL-1 antibody comprises a VH sequence of SEQ ID NO: 4, including post-translational modifications of that sequence. In some embodiments, the VH comprises one, two, or three CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10. [ka]

[0079] In some embodiments, the anti-PSGL-1 antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 3 contains substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-PSGL-1 antibody comprising the sequence retains the ability to bind to PSGL-1. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 3. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 3. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside the CDRs (i.e., within the FRs). In some embodiments, the anti-PSGL-1 antibody comprises a VL sequence of SEQ ID NO: 3, including post-translational modifications of that sequence. In some embodiments, the VL comprises one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. [ka]

[0080] In some embodiments, the anti-PSGL-1 antibody comprises a VH of any embodiment provided herein and a VL of any embodiment provided herein. In some embodiments, the anti-PSGL-1 antibody comprises the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 3, respectively, including post-translational modifications of these sequences.

[0081] In some embodiments, the anti-PSGL-1 antibody comprises an anti-PSGL-1 antibody that binds to the same epitope as the anti-PSGL-1 antibody provided herein. For example, in some embodiments, the anti-PSGL-1 antibody binds to the same epitope as an anti-PSGL-1 antibody comprising a VH comprising the sequence of SEQ ID NO:4 and a VL comprising the sequence of SEQ ID NO:3.

[0082] In some embodiments, the anti-PSGL-1 antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the anti-PSGL-1 antibody is an antigen-binding fragment (e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment) of an anti-PSGL-1 antibody described herein. In some embodiments, the anti-PSGL-1 antibody comprises a substantially full-length anti-PSGL-1 antibody (e.g., an IgG4 antibody), or other antibody classes or isotypes described herein.

[0083] In some embodiments, the anti-PSGL-1 antibody is of the IgG, IgM, or IgA class. In some embodiments, the anti-PSGL-1 antibody has a human IgG1, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-PSGL-1 antibody has a human IgG4 constant domain. In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain, the heavy chain comprising a S228P amino acid substitution at residue position 228 according to EU numbering. In some embodiments, the anti-PSGL-1 antibody comprises an IgG4 hinge domain comprising a S228P amino acid substitution at residue position 228 according to EU numbering. In some embodiments, the anti-PSGL-1 antibody comprises an IgG4 hinge domain comprising the amino acid sequence ESKYGPPCPPCPA (SEQ ID NO: 12).

[0084] In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-PSGL-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 1. [ka]

[0085] In some embodiments, the anti-PSGL-1 antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the anti-PSGL-1 antibody 15A7H according to the numbering of Kabat et al. In some embodiments, the anti-PSGL-1 antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the anti-PSGL-1 antibody 15A7H. In some embodiments, the anti-PSGL-1 antibody of the present disclosure comprises the VH domain and / or the VL domain of the anti-PSGL-1 antibody 15A7H. In some embodiments, the anti-PSGL-1 antibody of the present disclosure comprises the heavy chain and / or the light chain of the anti-PSGL-1 antibody 15A7H. In some embodiments, the anti-PSGL-1 antibodies of the present disclosure comprise the heavy and light chains of the anti-PSGL-1 antibody 15A7H. The anti-PSGL-1 antibody 15A7H is described in US20130209449 and US20130011391. Additional anti-PSGL-1 antibodies that can be used in the methods provided herein are described in U.S. Patent Nos. 7,604,800 and 8,361,472.

[0086] In some embodiments, the anti-PSGL-1 antibody of the present disclosure is a tetravalent anti-PSGL-1 antibody. Exemplary tetravalent anti-PSGL-1 antibodies are disclosed in WO2017120534.

[0087] Human PSGL-1 is also referred to as selectin P ligand, SELPG, CLA, CD162, or PSGL1. In some embodiments, the antibodies of the disclosure bind to a polypeptide encoded by the human SELPG gene, e.g., as set forth in NCBI RefSeq Gene ID No. 6404. In some embodiments, an antibody of the disclosure binds to a polypeptide comprising the amino acid sequence MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTQPVPTEAQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAMEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPTEMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP (SEQ ID NO: 11).In some embodiments, an antibody of the disclosure binds to a polypeptide comprising the amino acid sequence MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAMEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPTEMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP (SEQ ID NO: 22). The amino acid sequence of SEQ ID NO:11 represents full-length human PSGL-1 (GenBank™ Accession Nos. AAA74577.1, GL902797), and the amino acid sequence of SEQ ID NO:22 represents the shorter 402 amino acid human PSGL-1 protein (GenBank™ Accession No. XP_005269133). In certain embodiments, the antibodies described herein specifically bind to human PSGL-1, e.g., as determined by ELISA or other antigen binding assays known in the art or described herein.

[0088] The present disclosure encompasses modifications to the antibodies or polypeptides described herein, including functionally equivalent antibodies that do not significantly affect these properties, as well as variants with enhanced or decreased activity and / or affinity. Modification of polypeptides is routinely practiced in the art and need not be described in detail herein. Examples of modified polypeptides include conservative substitution of amino acid residues, deletion or addition of one or more amino acids that do not significantly and adversely change the functional activity, or the use of chemical analogs.

[0089] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0090] Substitution variants are those in which at least one amino acid residue in an antibody molecule is removed and a different residue is inserted in its place. The most interesting sites for substitution mutagenesis are hypervariable regions, but FR modifications are also contemplated. Conservative substitutions are shown in the table below under the heading of "conservative substitutions". If such substitutions result in changes in biological activity, more drastic changes can be introduced, such as those referred to as "exemplary substitutions" in the table below, or as further described below for amino acid classes, and the products screened. [Table 2]

[0091] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone (e.g., as a sheet or helix conformation) in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are grouped into groups based on common side chain properties. (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile (2) Uncharged polar: Cys, Ser, Thr, Asn, Gln (3) Acidic (negatively charged): Asp, Glu (4) Basic (positively charged): Lys, Arg (5) Residues that influence chain direction: Gly, Pro (6) Aromatic: Trp, Tyr, Phe, His

[0092] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0093] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also be substituted (typically with serine) to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment (e.g., an Fv fragment).

[0094] In some embodiments, the anti-PSGL-1 antibody of the present disclosure comprises an antibody constant domain. In some embodiments, the antibody constant domain is a human antibody constant domain. In certain embodiments, the antibody constant domain is a human IgG4 constant domain. In some embodiments, the human IgG4 constant domain comprises a serine to proline amino acid substitution at amino acid position 228 (also known as position 241 using the Kabat numbering) of the heavy chain as numbered according to the EU index.

[0095] In some embodiments, one or more amino acid residues in the heavy and / or light chain constant regions of the antibody are modified. In some embodiments, the Fc region of the antibody is modified to enhance or decrease the ADCC and / or CDC activity of the antibody. See Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Presta et al., Biochem. Soc. Trans. 30:487-490 (2002).

[0096] The present disclosure also provides polynucleotides, including polynucleotides encoding any of the tetravalent antibodies and / or polypeptides described herein. In some embodiments, the polypeptide comprises a light chain and a heavy chain variable region sequence. In some embodiments, the polynucleotide is an isolated polynucleotide (e.g., isolated from a host cell or from one or more different polynucleotides).

[0097] Those skilled in the art understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein. Some such polynucleotides have minimal homology to the nucleotide sequence of any native gene. Thus, polynucleotides that vary due to differences in codon usage are expressly contemplated by this disclosure. Additionally, alleles of genes that include the polynucleotide sequences provided herein are within the scope of this disclosure. An allele is an endogenous gene that has been altered as a result of one or more mutations (e.g., deletion, addition, and / or substitution of nucleotides). The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0098] The polynucleotides of the present disclosure can be obtained by chemical synthesis, recombinant methods, or PCR.Methods for chemically synthesizing polynucleotides are well known in the art and do not need to be described in detail herein.Those skilled in the art can use the sequences provided herein and commercially available DNA synthesizers to generate desired DNA sequences.

[0099] To prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by direct uptake of the exogenous polynucleotide, introduction by endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (e.g., a plasmid) or can be integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al. (1989).

[0100] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

[0101] The disclosure also provides vectors (e.g., cloning or expression vectors) comprising a nucleic acid sequence encoding any of the polypeptides (including antibodies) described herein. Suitable cloning vectors can be constructed according to standard techniques or can be selected from the large number of cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended for use, but useful cloning vectors generally have the ability to replicate autonomously, may have a single target for a particular restriction endonuclease, and / or may have a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (e.g., pSA3 and pAT28). These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0102] Generally, an expression vector is a replicable polynucleotide construct that contains a polynucleotide according to the present disclosure. An expression vector can be replicable in a host cell as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and the expression vector(s) disclosed in PCT Publication No. WO87 / 04462. Vector components generally can include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and suitable transcription control elements (e.g., promoters, enhancers, or terminators). For expression (i.e., translation), one or more translation control elements (e.g., ribosome binding sites, translation initiation sites, or stop codons) are also usually required.

[0103] Methods for making antibodies and antibody-derived polypeptides are known in the art and disclosed herein. Anti-PSGL antibodies (e.g., antibodies that specifically bind to human PSGL-1) can be identified using well-established methods, from which variable domains (e.g., VH and / or VL domains) can be used in the antibodies of the present disclosure. Exemplary anti-human PSGL-1 antibodies, as well as methods for screening, producing, and purifying such antibodies, are described in International Publication No. WO2012 / 174001.

[0104] A wide variety of recombinant host-vector expression systems for eukaryotic cells are known and can be used in the present disclosure. For example, Saccharomyces cerevisiae (i.e., common baker's yeast) is the most commonly used among eukaryotic microorganisms, although several other strains (e.g., Pichia pastoris) are available. Cell lines derived from multicellular organisms, such as Sp2 / 0 or Chinese hamster ovary (CHO) (available from ATCC), can also be used as hosts. Exemplary vector plasmids suitable for transformation of eukaryotic cells include, for example, pSV2neo and pSV2gpt (ATCC), pSVL and pSVK3 (Pharmacia), and pBPV-1 / pML2d (International Biotechnology, Inc.).

[0105] Eukaryotic host cells useful in the present disclosure are, for example, hybridoma, myeloma, plasmacytoma, or lymphoma cells, although other eukaryotic host cells may be suitably utilized so long as the mammalian host cell is capable of recognizing the transcribed and translated DNA sequence for protein expression, processing the leader peptide by cleavage of the leader sequence and secretion of the protein, and effecting post-translational modification of the protein (e.g., glycosylation).

[0106] Thus, the present disclosure provides host cells (e.g., eukaryotic host cells) transformed with a recombinant expression vector comprising a DNA construct disclosed herein and capable of expressing a tetravalent antibody or polypeptide of the present disclosure. In some embodiments, the transformed host cells of the present disclosure comprise at least one DNA construct comprising a polynucleotide of the present disclosure, or a polynucleotide that expresses a monomeric, dimeric, or tetravalent antibody of the present disclosure, and transcriptional and translational regulatory sequences positioned relative to the coding DNA sequence to direct expression of the antibody or polypeptide.

[0107] Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtillis) and yeast (e.g., S. cerevisae, S. pombe, or K. lactis).

[0108] The host cells used in the present disclosure can be transformed in a variety of ways using standard transfection procedures well known in the art. Standard transfection procedures that can be used include electroporation, protoplast fusion, and calcium phosphate precipitation techniques. Such techniques are generally described by F. Toneguzzo et al. (1986), Mol. Cell. Biol., 6:703-706; G. Chu et al., Nucleic Acid Res. (1987), 15:1311-1325; D. Rice et al., Proc. Natl. Acad. Sci. USA (1979), 79:7862-7865; and V. Oi et al., Proc. Natl. Acad. Sci. USA (1983), 80:825-829. A vector containing a polynucleotide of interest can be introduced into a host cell by any of several suitable means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, particle bombardment, lipofection, and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide introduction often depends on characteristics of the host cell.

[0109] Certain aspects of the present disclosure relate to antibody variable domains and / or antibody fragments (which can be used, for example, as components of the tetravalent antibodies described herein). Antibody fragments can include the active binding regions of an antibody (e.g., Fab, F(ab')2, scFv, Fv fragments, etc.). Antibody fragments can be generated and / or isolated using a variety of methods known in the art and incorporated into the tetravalent antibodies of the present disclosure, for example, by standard recombinant techniques known in the art based on the concepts described herein.

[0110] Single chain Fv fragments can be produced, for example, as described in Iliades et al., 1997, FEBS Letters, 409:437-441. The linking of such single chain fragments using various linkers is described in Kortt et al., 1997, Protein Engineering, 10:423-433. Various techniques for the recombinant production and manipulation of antibodies are well known in the art. Such fragments can be produced from the monoclonal antibodies described herein using techniques well established in the art (Rousseaux et al. (1986), in Methods Enzymol., 121:663-69 Academic Press).

[0111] Methods for preparing antibody fragments are well known in the art. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to obtain a 100 Kd fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce a 50 Kd Fab' monovalent fragment. Alternatively, enzymatic cleavage with papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, in U.S. Patent Nos. 4,036,945 and 4,331,647 and references contained therein, which are incorporated herein by reference. See also, Nisonoff et al. (1960), Arch Biochem. Biophys. 89:230; Porter (1959), Biochem. J. 73:119; Smyth (1967), Methods in Enzymology 11:421-426. Alternatively, Fab can be produced by inserting DNA encoding the Fab of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.

[0112] Modifications also include glycosylated and non-glycosylated polypeptides, as well as peptides with other post-translational modifications (e.g., glycosylation with different sugars, acetylation, and phosphorylation). Antibodies are glycosylated at conserved positions in the constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecular interactions between moieties of the glycoprotein, which can affect the conformation and presented three-dimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides may also function to target a given glycoprotein to a specific molecule based on specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells in which expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, was modulated by tetracycline have been reported to have improved ADCC activity (Umana et al., 1999, Mature Biotech. 17:176-180).

[0113] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment to the asparagine side chain of the carbohydrate moiety. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

[0114] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0115] The glycosylation pattern of an antibody can also be modified without modifying the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Since the cell types used for recombinant glycoproteins (e.g., antibody expression) as potential therapeutics are rarely native cells, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al., 1997, J. Biol. Chem. 272:9062-9070).

[0116] In addition to the choice of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes, etc. Various methods have been proposed to modify the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335, 5,510,261, and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from glycoproteins using, for example, Endo H, N-glycosidase F, Endoglycosidase F1, Endoglycosidase F2, or Endoglycosidase F3. Furthermore, recombinant host cells can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

[0117] In some embodiments, the antibodies of the present disclosure are modified using coupling techniques known in the art, including but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to attach labels for immunoassays.

[0118] In some embodiments, the antibodies of the present disclosure can be conjugated (e.g., linked) to an agent (e.g., a therapeutic agent or label). Examples of therapeutic agents include radioactive moieties, cytotoxins, and chemotherapeutic molecules.

[0119] III. JAK Inhibitors Certain aspects of the disclosure relate to JAK inhibitors. Any JAK inhibitor of the disclosure can be used in the methods disclosed herein, for example, by administration in combination with an anti-PSGL-1 antibody of the disclosure.

[0120] In some embodiments, the JAK inhibitor inhibits JAK1 and / or JAK2. In some embodiments, the JAK inhibitor inhibits JAK1 and / or JAK3. In some embodiments, the JAK inhibitor inhibits JAK1 / JAK2 inhibitors, JAK2 / FLT3 inhibitors, JAK2 V617F inhibitors, JAK2 inhibitors, JAK1 inhibitors, or JAK2 / Src inhibitors (including pharma- ceutically acceptable salts thereof). Exemplary, non-limiting descriptions of JAK inhibitors can be found in WO2007070514, WO2008157208, and WO2019171326.

[0121] In some embodiments, the JAK inhibitor is ruxolitinib or a pharma- ceutically acceptable salt thereof.As known in the art, ruxolitinib is a JAK1 / JAK2 inhibitor (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, also referred to as 3(R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, having the following formula: [ka]

[0122] In some embodiments, ruxolitinib refers to ruxolitinib phosphate. In some embodiments, ruxolitinib is in unit dosage form (e.g., tablet). In some embodiments, ruxolitinib is administered orally.

[0123] In some embodiments, the JAK inhibitor is tofacitinib or a pharma- ceutically acceptable salt thereof.

[0124] IV. METHODS AND USES Certain aspects of the disclosure relate to methods and uses of the antibodies described herein, for example, by administration to a subject in combination with a JAK inhibitor of the disclosure, to treat or prevent a T cell mediated disease (e.g., a T cell mediated inflammatory disease) or cancer (e.g., a T cell leukemia or lymphoma). In some embodiments, the subject is a human.

[0125] As described herein, PSGL-1 is known to be involved in inflammation and T cell biology. Antibodies of the present disclosure that specifically bind human PSGL-1 can be used, for example, in combination with a JAK inhibitor of the present disclosure, in the treatment of individuals having disorders associated with T cell function (e.g., T cell-mediated inflammatory disorders) or individuals requiring medical procedures that may result in an inflammatory condition (e.g., an immunological response) or individuals in whom such a condition is prior to management (e.g., transplantation or transfusion), among others.

[0126] In some embodiments, the T cell-mediated inflammatory disease is graft-versus-host disease (GVHD) (e.g., acute or chronic GVHD). In some embodiments, the T cell-mediated inflammatory disease is steroid-resistant acute GVHD (SR-aGVHD) or treatment-resistant acute GVHD (TR-aGVHD). See, e.g., Przepiorka et al., (2019) The Oncologist, 24:1-7 and Mothy et al., (2020) Blood, 136(17):1903-1906.

[0127] In some embodiments, the T cell mediated inflammatory disease is selected from the group consisting of skin disorders, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, and autoimmune thyroid disease.

[0128] Other aspects of the disclosure relate to methods and uses of the antibodies described herein, for example, by administration to a subject in combination with a JAK inhibitor of the disclosure, for treating or preventing a T-cell leukemia or T-cell lymphoma (e.g., adult T-cell leukemia / lymphoma (ATLL), anaplastic large cell lymphoma (ALCL), or cutaneous T-cell lymphoma (CTCL)). In some embodiments, the subject is a human.

[0129] In some embodiments, the JAK inhibitor is administered to the subject before, after, or simultaneously with administration of the antibody.

[0130] In some embodiments, the subject has received one or more systemic treatments for a T cell mediated inflammatory disease (e.g., GVHD) of the present disclosure prior to administration of the antibody and / or JAK inhibitor. In some embodiments, the subject has been treated with a corticosteroid prior to administration of the antibody and / or JAK inhibitor.

[0131] In some embodiments, the disorder or disease treated by the methods described herein may be a T cell mediated disease. Non-limiting examples of disorders and diseases that can be treated or one or more of the symptoms of which can be improved or prevented using the antibodies and JAK inhibitors described herein include psoriasis, Crohn's disease, ankylosing spondylitis, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), diabetes, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), Sjogren's syndrome, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug rash, leprosy reversal. Examples of conditions that may be treated include allergies such as leprosy, erythema nodosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, erythroblastic anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, graft-versus-host disease (GVHD) (e.g., acute or chronic GVHD), transplant rejection, vitiligo, alopecia areata, cytokine release syndrome, hidradenitis suppurativa, sarcoidosis, primary biliary cirrhosis, posterior uveitis, interstitial pulmonary fibrosis, atopic allergies, AIDS, and T-cell neoplasms such as leukemia or lymphoma.

[0132] In another embodiment, the disease or disorder treated according to the methods described herein is psoriasis vulgaris.Psoriasis vulgaris or plaque psoriasis is the most common form of psoriasis, characterized by raised, sharply demarcated erythematous patches of skin covered with silvery white scales.Lesions tend to affect the extensor surfaces of the limbs, the lumbosacral region, and the scalp.The corresponding histopathological findings include prominent inflammatory cell infiltration of the dermis and epidermis, increased dilated blood vessels, and extensive epidermal thickening with disorganized differentiation of keratinocytes and hyperkeratosis.Approximately one-third of patients with psoriasis vulgaris are classified as having moderate or severe disease, making them candidates for therapies beyond mere topical treatment.

[0133] In another embodiment, the disease treated according to the methods described herein is chronic plaque psoriasis. Symptoms of plaque psoriasis include, but are not limited to, single or multiple raised red patches of skin ranging in size from a coin or larger that occur anywhere on the body, including, but not limited to, the knees, elbows, lumbosacral region, scalp, and nails.

[0134] In another embodiment, the disorder treated according to the methods described herein is guttate psoriasis. Symptoms of guttate psoriasis include, but are not limited to, flares of droplet-shaped, scaly patches on the skin following infection (e.g., streptococcal throat infections).

[0135] In another embodiment, the disease or disorder treated according to the methods described herein is inverse psoriasis.Symptoms of inverse psoriasis include, but are not limited to, smooth, usually moist (unlike the scales associated with plaque psoriasis), red, inflamed skin areas occurring in one or more of the following body areas: armpits, groin, under breasts, and other skin folds around the genitals and buttocks.

[0136] In another embodiment, the disease or disorder treated in accordance with the methods described herein is pustular psoriasis, symptoms of which include, but are not limited to, pus-filled blisters of various sizes and locations, but most often occurring on the hands and feet.

[0137] In another embodiment, the disease or disorder treated according to the methods described herein is erythrodermic psoriasis.Symptoms of erythrodermic psoriasis include, but are not limited to, periodic, widespread, intense reddening of the skin and shedding of scales in sheets rather than small flakes.The reddening and shedding of the skin is often accompanied by intense itching and pain, increased heart rate, and temperature fluctuations.

[0138] In another embodiment, the disease or disorder treated according to the methods described herein is rheumatoid arthritis. Symptoms of rheumatoid arthritis include, but are not limited to, fatigue, loss of appetite, low-grade fever, swollen glands, weakness, joint pain in the wrists, elbows, shoulders, hips, knees, ankles, toes, jaw, hands, feet, fingers, and / or neck, morning stiffness, chest pain when breathing (pleurisy), burning, itchy, and discharged eyes, skin nodules, numbness, tingling, or burning in the hands and feet.

[0139] In another embodiment, the disease or disorder treated according to the methods described herein is Crohn's disease. Symptoms of Crohn's disease include, but are not limited to, crampy abdominal (abdominal area) pain, fever, fatigue, loss of appetite, painful bowel movements (tenesmus), persistent watery diarrhea, unintentional weight loss, constipation, eye irritation, fistulas (usually around the rectum, which can cause the discharge of pus, mucus, or stool), joint pain, liver inflammation, mouth ulcers, rectal bleeding and blood in the stool, skin lumps or sores (ulcers), and swollen gums.

[0140] In another embodiment, the disease or disorder treated according to the methods described herein is ankylosing spondylitis. Symptoms of ankylosing spondylitis include, but are not limited to, frequent pain and stiffness in the lower back and buttocks, spine, and / or neck, and pain and tenderness radiating to the ribs, shoulder blades, buttocks, thighs, and heels, eye inflammation (iridocyclitis and uveitis) (causing redness, eye pain, loss of vision, floaters, and photophobia), fatigue, and nausea.

[0141] In another embodiment, the disease or disorder treated according to the methods described herein is diabetes. Symptoms of diabetes include, but are not limited to, weight loss, polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), cardiovascular disease, diabetic retinopathy, diabetic neuropathy, hyperosmolar nonketotic states, and diabetic ketoacidosis.

[0142] In some embodiments, an antibody or composition of the disclosure can be administered to an individual in combination with a JAK inhibitor prior to, concurrent with, and / or after transplantation.

[0143] As used herein, treatment of an individual in need of a transplant may refer to one or more of therapeutic treatment and prophylactic or preventative treatment (e.g., increasing the likelihood of a favorable treatment outcome (e.g., graft survival, graft function) or decreasing the likelihood of an unfavorable outcome (e.g., an unfavorable response to treatment, or a condition that reduces the likelihood of favorable treatment (e.g., transplant) occurring). Treatment may include, but is not limited to, alleviating or preventing conditions and symptoms associated with the disorder or condition, and / or problems or conditions that prevent or limit an individual's access to treatment options for the disorder or condition (e.g., sensitization, hypersensitization, high panel reactive antibody (PRA) levels, and / or the presence of pre-existing alloantibodies that limit the availability of grafts to individuals awaiting transplant). People in need of treatment include those in whom the disorder or condition is to be prevented, as well as those who already have the disorder or condition. Treating a disorder or condition can suppress immune-mediated events associated with the disorder or condition, ameliorate the symptoms of the disorder or condition, reduce the severity of the disorder or condition, alter the course of the progression of the disorder or condition, and / or ameliorate or cure the underlying disorder or condition.

[0144] For example, successful treatment of an individual awaiting a transplant includes, but is not limited to, reducing the level of alloantibodies, reducing panel reactive antibodies (PRA), enabling the individual to obtain a greater number of cross-match compatible donors, increasing the likelihood or probability that the individual will receive a graft, shortening the individual's expected waiting period for a graft, desensitizing the individual, reducing the risk of a transplant-related symptom or condition (e.g., an immune-mediated event as described below), or any combination thereof.

[0145] For example, successful treatment of an individual receiving a transplant includes, but is not limited to, protection and maintenance of the transplanted organ or tissue over time, including controlling, reversing, reducing, slowing, or preventing one or more symptoms or undesirable conditions associated with organ transplantation (e.g., immune-mediated events, including, but not limited to, donor-specific alloantibody (DSA) production, GVHD, antibody-mediated rejection (AMR), hyperacute transplant rejection, chronic transplant rejection, graft failure, and graft loss, as measured by functional or histological signs of the symptom or condition). A treatment capable of controlling a disorder or condition (e.g., transplant rejection) can include a treatment that slows the progression of the disease process if initiated after functional or histological signs of the disorder or condition (e.g., transplant rejection) are observed. Additionally, a treatment capable of reversing a disease or condition (e.g., transplant rejection) can include a treatment that reverses the disease process and returns functional and histological findings to a more normal state if initiated after functional or histological signs of the disease or condition (e.g., transplant rejection) appear. Treatments capable of "delaying the progression" of a disease or condition (e.g., transplant rejection) can include postponing, preventing, slowing, stabilizing, and / or postponing the onset of the disease or condition (e.g., transplant rejection). The length of time of this delay can vary depending on the history of the disease and / or the patient being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can actually encompass prevention, in that an individual (e.g., an individual at risk of developing the disorder or condition) does not develop the disorder or condition.

[0146] In some embodiments, the transplant of the present disclosure may be a transplant of one or more tissues or organs, including, but not limited to, bone marrow, kidney, heart, liver, neuronal tissue, lung, pancreas, skin, and intestine (e.g., the small intestine and / or large intestine, and any subtissues thereof).

[0147] In addition, combinations of antibodies and JAK inhibitors are useful in preventing and / or treating certain disorders and diseases associated with or caused (in whole or in part) by increased proliferation and / or number of activated T cells compared to proliferation and / or number of activated T cells observed in healthy individuals or individuals not suffering from a particular disorder or disease. Non-limiting examples of disorders and diseases that can be prevented and / or treated using the antibodies described herein in combination with a JAK inhibitor include graft-versus-host disease and cases of transplant rejection (including transplant rejection using allogeneic or xenogeneic tissue) (e.g., bone marrow transplants, liver transplants, kidney transplants, or any organ or tissue transplant).

[0148] In some embodiments, an antibody or composition of the disclosure can be administered to an individual in combination with a JAK inhibitor before, concomitantly with, and / or after a transfusion. For example, as described in more detail below, a tetravalent antibody or composition of the disclosure can be administered to increase the likelihood of a favorable therapeutic outcome, decrease the likelihood of an unfavorable outcome, and / or reduce or prevent symptoms that occur before, concomitantly with, or after a transfusion is completed.

[0149] As used herein, treatment of an individual in need of a transfusion may refer to one or more of therapeutic treatment and prophylactic or preventative treatment (e.g., increasing the likelihood of a favorable treatment outcome (e.g., replacement or replenishment of blood components / cells) or decreasing the likelihood of an unfavorable outcome (e.g., unfavorable response to treatment, ineffective treatment, or immunological reaction, or conditions that reduce the likelihood of favorable treatment (e.g., transfusion)). Treatment may include, but is not limited to, alleviating or preventing conditions and symptoms associated with the disorder or condition, and / or problems or conditions that prevent or limit an individual's access to treatment options for the disorder or condition. People in need of treatment include those who already have the disorder or condition, as well as those in whom the disorder or condition is to be prevented. Treatment of a disorder or condition may suppress immune-mediated events associated with the disorder or condition, ameliorate symptoms of the disorder or condition, reduce the severity of the disorder or condition, alter the course of progression of the disorder or condition, and / or improve or cure the underlying disorder or condition.

[0150] In some embodiments, the transfusion is a transfusion that includes one or more of white blood cells, red blood cells, and platelets, hi some embodiments, the transfusion includes whole blood or one or more blood products, including but not limited to white blood cells, red blood cells, platelets, fresh frozen plasma, cryoprecipitate, or blood clotting factors, antibodies, and / or blood substitutes. Exemplary conditions that can be treated with transfusions (e.g., transfusions of blood or blood products) include, but are not limited to, bleeding or blood loss, reduced hematocrit or hemoglobin (e.g., anemia), sickle cell disease, thalassemia, blood replacement during or after a surgical procedure, cardiac disease, traumatic injury, deficiency of one or more blood factors (e.g., hemophilia, von Willebrand disease, hypofibrinogenemia, or deficiency of Factor II, V, VII, IX, X, or XI), conditions requiring replacement of fibrinogen (e.g., liver disease, transfusions, etc.), bone marrow failure, platelet dysfunction, thrombocytopenia, immune deficiency (e.g., due to treatment or disease), etc. Explanations of the practice, administration, response, indications, and preparation for transfusions can be found, for example, in the American Red Cross Compendium of Transfusion Practice Guidelines.

[0151] In some embodiments, administration of an antibody of the present disclosure in combination with a JAK inhibitor of the present disclosure to a subject results in a reduction in one or more symptoms of a T cell mediated inflammatory disease of the present disclosure in the subject.

[0152] The dosage and frequency of administration of the antibody and / or JAK inhibitor or pharmaceutical composition thereof described herein is administered according to the method for prevention and / or treatment while minimizing side effects. The exact dosage of the antibody or pharmaceutical composition thereof described herein administered to a particular subject in combination with a JAK inhibitor can be determined by the practitioner in light of factors related to the subject in need of treatment. Factors that may be considered include the severity of the disease state, the general health of the subject, the age and weight of the subject, diet, time and frequency of administration, combination(s) with other therapeutic agents or drugs, reaction sensitivity, and tolerability / response to treatment. The dosage and frequency of administration of the antibody or pharmaceutical composition thereof described herein in combination with a JAK inhibitor can be adjusted over time to provide sufficient levels of the antibody or JAK inhibitor or to maintain the desired effect.

[0153] The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the inflammatory disorder or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0154] Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0155] In one embodiment, any antibody and / or JAK inhibitor described herein is formulated for administration by intraperitoneal, intravenous, subcutaneous, or intramuscular injection, or by other modes of administration (e.g., oral, transmucosal, inhalation, sublingual, etc.). In some embodiments, the antibody is administered by intravenous infusion and / or the JAK inhibitor is administered orally.

[0156] Parenteral administration is characterized in one embodiment by injection, and is contemplated herein to be either subcutaneous, intramuscular, or intravenous. Injections can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injections, solutions, and emulsions also contain one or more excipients. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents. Other routes of administration can include enteral administration, intracerebral administration, intranasal administration, intraarterial administration, intracardiac administration, intraosseous injection, intrathecal administration, intravenous injection, subcutaneous implantation or injection, intramuscular administration, intrarectal administration, intravaginal administration, intragastric administration, intratracheal administration, intrapulmonary administration, and intraperitoneal administration. Preparations for parenteral administration include sterile solutions that can be used for injection, sterile dry soluble products (e.g., lyophilized powders) that are combined with a solvent immediately before use, sterile dry insoluble preparations that are combined with a vehicle immediately before use, and sterile emulsions.The solutions can be aqueous or non-aqueous.For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS), water, and solutions containing thickening agents and solubilizers (e.g., glucose, polyethylene glycol, and polypropylene glycol), and mixtures thereof.

[0157] In another embodiment, the present disclosure also contemplates administration of compositions comprising the antibodies of the present disclosure conjugated to other molecules (e.g., detectable labels, or therapeutic or cytotoxic agents). The agents can include, but are not limited to, radioisotopes, toxins, toxoids, inflammatory agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents. Methods for conjugating antibodies to such molecules are well known to those of skill in the art. See, for example, PCT Publication Nos. WO 92 / 08495, WO 91 / 14438, WO 89 / 12624, U.S. Patent No. 5,314,995, and EP 396,387.

[0158] In one embodiment, the composition comprises an antibody or polypeptide conjugated with a cytotoxic agent. The cytotoxic agent may comprise any agent that is detrimental to cells. Exemplary classes of cytotoxic agents that can be conjugated with the antibody or fragment include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0159] V. Pharmaceutical Compositions The disclosure also provides a pharmaceutical composition comprising an antibody or JAK inhibitor described herein and a pharma- ceutical acceptable carrier or excipient, which can be used, for example, in the methods, uses, and / or kits of the disclosure.

[0160] Pharmaceutically acceptable carriers or excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, excipients can provide shape or viscosity or act as diluents. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, buffers, and skin penetration enhancers. In certain embodiments, the tetravalent antibody described herein is in a liquid pharmaceutical composition. Liquid pharma-ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing the antibody described herein in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain small amounts of non-toxic auxiliary substances (e.g., wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, etc.). Excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0161] The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms (e.g., sterile parenteral solutions or suspensions) containing a suitable amount of the tetravalent antibodies described herein. The tetravalent antibodies are in one embodiment formulated and administered in unit dosage forms or multiple dosage forms. As used herein, unit dosage forms refer to physically discrete units suitable for human and animal subjects, packaged individually as known in the art. Each unit dosage contains a predetermined amount of an antibody or an antigen-binding fragment derived from an antibody sufficient to produce a desired therapeutic effect, together with the required pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes. A unit dosage form can be administered in divided or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container for administration in separate unit dosage forms. Examples of multiple dosage forms include vials, or bottles of pints or gallons. Thus, a multiple dosage form is a plurality of unit doses that are not separated in packaging.

[0162] The concentration of the antibody in the pharmaceutical composition depends, for example, on the physicochemical properties of the antibody, the administration schedule and dosage, as well as other factors known to those skilled in the art.

[0163] In some embodiments, the disclosure provides antibodies and compositions (e.g., pharmaceutical compositions described herein) for use in any of the methods described herein, administered or formulated for administration with a JAK inhibitor of the disclosure, whether in the context of use as a medicament and / or for the manufacture of a medicament.

[0164] VI. Kit Certain aspects of the present disclosure relate to kits or articles of manufacture that include the anti-PSGL-1 antibodies of the present disclosure and / or the JAK inhibitors of the present disclosure. Optionally, the kits described herein can include one or more pharma- ceutically acceptable carriers (e.g., exemplary carriers described herein). In some embodiments, the kits of the present disclosure include the pharmaceutical compositions of the present disclosure. The kits described herein can be used, for example, in the methods or uses of the present disclosure.

[0165] The kit may optionally provide additional components (e.g., buffers and interpretive information). Typically, the kit includes a container and a label or package insert(s) on or associated with the container. The container may be a unit dose, bulk package (e.g., a multi-dose package), or sub-unit dose. The instructions provided with the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions embodied on a magnetic or optical storage disk) are also acceptable.

[0166] In some embodiments, the kit further comprises a package insert containing instructions for administering the antibody in combination with a JAK inhibitor to treat a T cell mediated disease (e.g., a T cell mediated inflammatory disease) or cancer, hi some embodiments, the kit further comprises a JAK inhibitor or a pharma- ceutical acceptable formulation thereof.

[0167] The kits of the present disclosure are suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages are also contemplated for use in combination with certain devices (e.g., inhalers, nasal administration devices (e.g., atomizers), or injection devices such as mini-pumps). The kits may have a sterile access port (e.g., the container may be an intravenous solution bag, or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag, or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a tetravalent antibody or polypeptide as described herein. The container may further comprise a second pharma- ceutically active agent. In some embodiments, the kits may further comprise any other material or device useful for treatment (e.g., blood transfusion or transplantation), including, but not limited to, one or more containers, tubes, sterilants or equipment, cannulas, syringes, and the like. EXAMPLES

[0168] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications and changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims.

[0169] Example 1: Cell apoptosis assay by combined treatment with anti-PSGL-1 antibody and JAK inhibitor. The Examples presented below describe preclinical studies using the Janus kinase (JAK) inhibitor ruxolitinib in combination with the anti-PSGL-1 antibody 15A7H to induce apoptosis of activated T cells.

[0170] method Preparation of human T cells T cell stimulation was achieved by phytohemagglutinin-L (PHA) treatment prior to the apoptosis assay described below. Briefly, human blood samples used in this study were obtained from healthy human donors. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from whole blood by Ficoll-Hypaque gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare; catalog number 17-1440-03) at room temperature for 15 min at 2400 revolutions per minute (rpm). The buffy coat layer containing the mononuclear cells was collected and washed three times with phosphate-buffered saline (PBS) to minimize platelet contamination. The collected PBMCs were used to generate activated T cells by incubating them with PHA (3 μg / mL, SIGMA; catalog number L2769-10mg) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) for 2 days, and then maintaining them in medium containing recombinant human interleukin-2 (5 ng / mL, R&D Systems; catalog number 202-IL-050) for an additional 4 to 6 days.

[0171] Cell apoptosis assay Fluorescence-activated cell sorting (FACS) analysis using Annexin V / propidium iodide (PI) staining was used to quantify the apoptotic rate in drug- and control-treated cells. Briefly, 1–1.5 × 10 5 Activated T cells (usually 5-7 days after PHA stimulation) were seeded and cultured with or without the addition of antibody 15A7H and / or JAK inhibitor. Aliquots of antibody 15A7H or ruxolitinib at the indicated concentrations, human IgG4 isotype control at only the highest concentration used for antibody 15A7H, and crosslinker (CL, mouse anti-human IgG antibody; Jackson Immuno Research; catalog no. 209-005-098) at half the concentration of antibody 15A7H were freshly prepared in complete RPMI-1640 medium (10% FBS, 1% penicillin / streptomycin, and 2 ng / mL interleukin-2) and added to test wells in a final volume of 100 μL. Plates were incubated at 37 °C for 48 h.

[0172] The cells were then washed with ice-cold annexin binding buffer and centrifuged at 300xg for 5 min. The cell pellet was resuspended in ice-cold annexin binding buffer and annexin-V-FITC and PI (Strong Biotech; AVK250) were added. The staining procedure was performed using the Annexin-V FITC Apoptosis Detection Kit (Strong Biotech; AVK250) according to the manufacturer's instructions.

[0173] Samples were run on a flow cytometer (BD LSR scanner; BD Biosciences, San Diego) and cell viability and apoptosis were calculated using quadrants of Annexin-V-FITC / PI histograms. Live cells were identified as those staining negatively with both fluorochromes, early apoptotic cells were identified as those staining only positive for Annexin-V-FITC, necrotic cells were identified as those staining only positive for PI, and late apoptotic cells were identified as those staining positively with both fluorochromes. Annexin-V-positive cells, PI-positive cells, and Annexin-V- and PI-positive cells were counted as apoptotic cells.

[0174] Fixed ratio combination cytotoxicity assay Activated T cells were cultured in a 96-well plate at 1–1.5 × 10 5 T cells were seeded at 1000x / mL and treated with ruxolitinib and antibody 15A7H alone or in combination at fixed concentration ratios. Combinations of ruxolitinib and antibody 15A7H at fixed ratios of 3.3:1 and 1.1:1 were tested in T cells from four donors. Additionally, a fixed ratio of ruxolitinib and antibody 15A7H at 0.37:1 was tested in T cells from two donors.

[0175] Cell apoptosis was measured using the Annexin-V FITC Apoptosis Detection Kit (Strong Biotech; AVK250) according to the manufacturer's instructions. Combinatorial drug effects were evaluated by calculating the combination index (CI) using Calcusyn software (version 2.0, Biosoft) based on the Chou-Talalay method (see, for example, Chou TC, (2010) Cancer Res., 70:440-446 and Bijnsdorp IV. et al., (2011) Methods Mol Biol., 731:421-434) (CI<1 indicates synergy, CI=1 indicates additivity, and CI>1 indicates antagonism).

[0176] reagent The JAK inhibitor ruxolitinib ("RUXO") was obtained from LC Laboratories (catalog number R-6688) and dissolved in dimethylsulfoxide (DMSO) according to the manufacturer's instructions. Ruxolitinib is part of the first generation of JAK inhibitors that have been found to inhibit multiple JAKs; ruxolitinib inhibits JAK1 and JAK2.

[0177] result Antibody 15A7H at concentrations of 0.03 μg / mL, 0.3 μg / mL, and 3 μg / mL and ruxolitinib at concentrations of 10 μM, 3.3 μM, 1.1 μM, 0.367 μM, and 0.122 μM, alone or in combination, were initially tested on activated T cells derived from PBMCs of three donors to quantify preliminary combination effects.

[0178] As shown in Figures 1A-1C, antibody 15A7H alone induced optimal apoptosis induction in activated T cells at a concentration of 3 μg / mL and in the presence of an antibody crosslinker 48 hours after treatment. Suboptimal antibody 15A7H concentrations of 0.03 μg / mL and 0.3 μg / mL generally induced minimal to moderate apoptosis in treated cells, depending on the donor. Ruxolitinib alone, at concentrations ranging from 0.122 to 10 μM, showed minimal or no increase in apoptosis over background levels after 48 hours of treatment. However, the combination of ruxolitinib and antibody 15A7H, especially at the suboptimal antibody concentrations of 0.03 μg / mL and 0.3 μg / mL, resulted in increased levels of cell apoptosis compared to cells treated with antibody 15A7H alone at the same concentrations or compared to cells treated with ruxolitinib and an isotype control antibody.

[0179] To further validate the combinatorial effect of antibody 15A7H and ruxolitinib on apoptosis induction in activated T cells, the combinatorial effect was evaluated using Chou-Talalay median effect analysis with a fixed ratio of antibody 15A7H and ruxolitinib (Chou TC. Cancer Res. 2010;70:440-446). Cells were treated with each drug alone and in combination, and cell apoptosis was measured using Annexin-V FITC Apoptosis Detection Kit (Strong Biotech; AVK250). Results were expressed as apoptotic fraction (affected fraction) based on the measured fluorescence counts of treated samples compared to untreated control samples. Combination index (CI) was measured for each combination using Calcusyn software (Biosoft) using seven diagonal lines representing various dose-effect curves for fixed drug ratios. Combinatorial effects were defined as "synergistic," "additive," and "antagonistic" when CIs were <1, 1, and >1, respectively.

[0180] As shown in Figure 2, the apoptosis assay of fixed ratio combinations confirmed that antibody 15A7H exhibited synergistic use with ruxolitinib at ratios of 3.3:1, 1.1:1, and 0.37:1, since most of the CI values ​​calculated for the 25% to 75% fraction of affected donor T cells remained below 0.9.

[0181] For drug combination evaluation, CI values ​​ranging from 0.1 to 0.3 are considered to indicate strong synergy, 0.3 to 0.7 to indicate synergy, and 0.7 to 0.85 to indicate moderate synergy. Table 1 shows the EC ratios and donors for all drugs tested. 50 The CI index at the CI level is shown. The CI index for the combination of antibody 15A7H and ruxolitinib is in the range of 0.3 to 0.6, which indicates the synergistic effect of the combination of antibody 15A7H and ruxolitinib (see, for example, Karagianni F. et al., (2021) PLoS ONE, 16(3): e0248298). [Table 1]

[0182] Example 2: Cell apoptosis assay using combined treatment with anti-PSGL-1 antibody and mTOR inhibitor. The following examples describe in vitro cytotoxicity studies using the mTOR inhibitor rapamycin alone or in combination with the anti-PSGL-1 antibody 15A7H. Rapamycin, a potent immunosuppressant, interferes with signaling pathways required for T cell activation and development. Rapamycin is clinically approved for the prevention of organ rejection and for several cancer indications.

[0183] method reagent Rapamycin was obtained from AdooQ Bioscience (catalog number A10782-5) and dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions.

[0184] Preparation of human T cells PBMCs were obtained from peripheral blood of healthy donors. Isolation was performed by density centrifugation of blood over Ficoll (GE Healthcare; catalog number 17-1440-03). Harvested PBMCs were used to generate activated T cells by incubating with PHA (3 μg / mL, SIGMA; catalog number L2769-10mg) in RPMI-1640 medium (10% FBS, 1% penicillin / streptomycin, and 2-mercaptoethanol) for 2 days and then maintaining them in medium containing recombinant human interleukin-2 (5 ng / mL, R&D Systems; catalog number 202-IL-050) for an additional 4–6 days.

[0185] Cell apoptosis assay Rapamycin is a macrolide that exhibits potent immunosuppressant activity. To evaluate the effects of drug combinations, 1–1.5 × 10 5 Activated T cells were plated in 96-well plates and treated with 15A7H (a cross-linking, mouse anti-human IgG antibody (Jackson; catalog no. 209-005-098)) and rapamycin, either alone or in combination, at the indicated concentrations for 48 h. Cytotoxicity was assessed by Annexin V / propidium iodide (PI) staining to quantify the apoptotic rate of drug-treated cells.

[0186] result Treatment of cells with rapamycin alone ranging from 0.122 to 10 μM did not result in any significant levels of cell apoptosis after 48 hours (Figures 3A-3C). Combined treatment with rapamycin and 15A7H did not increase the levels of apoptotic cells compared to cells treated with 15A7H alone at the same dose levels. These data suggested that treatment with 15A7H and rapamycin did not have any additive effect on activated T cells.

[0187] Example 3: Fixed ratio combination cytotoxicity assay with ruxolitinib and anti-PSGL-1 antibody. The Examples below demonstrate that in addition to 15A7H, other anti-PSGL-1 antibodies with cytotoxic function can synergize with ruxolitinib to induce activated T cell apoptosis.

[0188] method reagent The JAK inhibitor ruxolitinib ("RUXO") was obtained from LC Laboratories (catalog number R-6688) and dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions.

[0189] Preparation of human T cells PBMCs were obtained from peripheral blood of healthy donors. Isolation was performed by density centrifugation of blood over Ficoll (GE Healthcare; catalog number 17-1440-03). Harvested PBMCs were used to generate activated T cells by incubating with PHA (3 μg / mL, SIGMA; catalog number L2769-10mg) in RPMI-1640 medium (10% FBS, 1% penicillin / streptomycin, and 2-mercaptoethanol) for 2 days and then maintaining them in medium containing recombinant human interleukin-2 (5 ng / mL, R&D Systems; catalog number 202-IL-050) for an additional 4–6 days.

[0190] Fixed ratio combination cytotoxicity assay In addition to 15A7H, the anti-PSGL-1 antibodies LH10, ​​c43B6, and h9F9 were tested for their ability to induce cell death in activated T cells. The aim of this study was to determine whether apoptosis-inducing anti-PSGL-1 antibodies and ruxolitinib would have a better cytotoxic effect on activated T cells. Combinatorial effects were assessed using Chou-Talalay median effect analysis with a fixed ratio of antibodies and ruxolitinib in the cytotoxicity assay. Activated T cells were treated with 1–1.5 × 10 5Cells / mL were seeded into 96-well plates and treated for 48 hours with ruxolitinib and anti-PSGL1 antibodies alone or in combination at fixed concentration ratios of 3.3:1, 1.1:1, and 0.37:1. The anti-PSGL-1 antibodies LH10, ​​c43B6, and h9F9 were tested in 3-fold titrations from 0.004 μg / mL to 3 μg / mL (including a crosslinker, mouse anti-human IgG antibody (Jackson; catalog no. 209-005-098) at half the concentration of the anti-PSGL-1 antibodies), and ruxolitinib was tested in these assays alone or in combination at 3-fold titrations from 0.0015 μM to 10 μM.

[0191] result Cell apoptosis was determined as described in Example 1, and the combination index (CI) of combinatorial drug effects was analyzed using CalcuSyn (Biosoft, Ferguson, MO, USA), which allows for automated simulation of synergy or antagonism (CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism). As shown in Figures 4A (LH10), 4B (c43B6), and (h9F9), the combination of anti-PSGL-1 antibodies, LH10, ​​c43B6, and h9F9, which have cytotoxic potential in activated T cells, showed synergy with ruxolitinib at ratios of 3.3:1, 1.1:1, and 0.37:1, since most of the CI values ​​calculated for the 35%-85% fraction of affected donor T cells remained below 0.9.

[0192] Example 4: Fixed ratio combination cytotoxicity assay with tofacitinib and anti-PSGL-1 antibody. The Examples presented below describe studies using the Janus kinase (JAK) 1 / 3 inhibitor tofacitinib in combination with the anti-PSGL-1 antibodies 15A7H and LH10 to induce apoptosis in activated T cells.

[0193] method reagent The JAK inhibitor tofacitinib ("TOFA") was obtained from AdooQ Bioscience (A10241-10) and dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions.

[0194] Preparation of human T cells PBMCs were obtained from peripheral blood of healthy donors. Isolation was performed by density centrifugation of blood over Ficoll (GE Healthcare; catalog number 17-1440-03). Harvested PBMCs were used to generate activated T cells by incubating with PHA (3 μg / mL, SIGMA; catalog number L2769-10mg) in RPMI-1640 medium (10% FBS, 1% penicillin / streptomycin, and 2-mercaptoethanol) for 2 days and then maintaining them in medium containing recombinant human interleukin-2 (5 ng / mL, R&D Systems; catalog number 202-IL-050) for an additional 4–6 days.

[0195] Fixed ratio combination cytotoxicity assay Tofacitinib, a first-generation jakinib that inhibits JAK3, JAK1, and to a lesser extent JAK2, was the first JAK inhibitor developed for the treatment of inflammatory diseases, including rheumatoid arthritis (RA), psoriatic arthritis, and active ulcerative colitis. In this experiment, cytotoxicity assays were performed in activated T cells treated with a fixed 1:1 combination of tofacitinib (at concentrations of 0.01–3 μM) and anti-PSGL-1 antibodies 15A7H or LH10 (at concentrations of 0.01–3 μg / mL) to evaluate the combination effect. Briefly, 1–1.5 × 10 5Activated T cells were seeded and treated with antibody 15A7H or LH10 and tofacitinib. Aliquots of the indicated concentrations of antibodies or tofacitinib, and cross-linker (mouse anti-human IgG antibody; Jackson; catalog number 209-005-098) at half the concentration of anti-PSGL-1 antibody, were freshly prepared in complete RPMI-1640 medium (10% FBS, 1% penicillin / streptomycin, and 2 ng / mL interleukin-2) and added to the test wells in a final volume of 100 μL. Plates were incubated at 37° C. for 48 hours. Combinatorial effects were evaluated using Calcusyn software to calculate combination index (CI) based on the apoptosis assay results of single agent and combination treatments, as described in Example 1. Cell apoptosis was measured using Annexin-V FITC Apoptosis Detection Kit (Strong Biotech; AVK250) according to the manufacturer's instructions.

[0196] result As shown in Figure 5, the experimental results indicate that the combination of tofacitinib with 15A7H or LH10 produced a significant synergistic effect, as most of the CI values ​​calculated for the 25% to 75% fraction of affected donor T cells remained below 0.5.

[0197] Example 5: Combination treatment of ruxolitinib and anti-PSGL-1 antibody in xenograft-versus-host disease (GvHD) model mice The Examples presented below describe studies using a combination of ruxolitinib and the anti-PSGL-1 antibody 15A7H to improve survival in mice engrafted with human PBMCs and developing xenogeneic GvHD.

[0198] method reagent The JAK inhibitor ruxolitinib ("RUXO") was obtained from LC Laboratories (catalog number R-6688), dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions, and formulated in a 0.5% methylcellulose vehicle for oral gavage administration.

[0199] Preparation of human PBMCs PBMCs were obtained from peripheral blood of healthy donors. Isolation was performed by density centrifugation of blood over Ficoll (GE Healthcare; Cat. No. 17-1440-03). Harvested PBMCs were washed and split in PBS.

[0200] Mouse experiments Female severely immunodeficient (ASID) mice (NOD.Cg-Prkdc) aged 8–9 weeks were cultured. scid Il2rg tm1Wjl / YckNarl; NLAC NARLabs Taiwan) was administered a total body irradiation dose of 2.0 Gy, and 24 hours after irradiation, 3 × 10 6 Human PBMCs were transplanted. Mice were kept under pathogen-free conditions and housed under standardized conditions with a set temperature of 22±1°C, a 12-h light / dark cycle, and free access to food and water. Experimental animals were allowed a minimum of 3 days for acclimatization before being randomly assigned to treatment groups.

[0201] 3×10 6 Xenogeneic GvHD (xGvHD) was induced in ASID mice administered human PBMCs (n = 5–7 per group). After 72 h of human PBMC engraftment, 45 mg / kg ruxolitinib or vehicle was administered orally twice daily for 15 days, followed by 10 mg / kg anti-PSGL-1 15A7H antibody or vehicle intravenously every 3–4 days for a total of 5 doses. Mice were assessed for survival daily. The time when treatment was initiated was designated as day 1.

[0202] result As shown in Figure 6, treatment with ruxolitinib and anti-PSGL-1 antibody 15A7H alone or in combination ameliorated xenogeneic GVHD induced by human lymphocytes. Furthermore, mice treated with ruxolitinib (solid line, open circle) or antibody 15A7H (solid line, open triangle) alone showed delayed mortality, but none of the animals survived beyond day 18, whereas none of the animals in the vehicle-treated group (dashed line, open square) survived beyond day 16. The combination treatment group showed the best survival rate, with 43% (3 of 7) of the mice surviving until the end of the experiment (day 21).

[0203] Example 6: Clinical trial of anti-PSGL-1 antibodies in patients with steroid-resistant acute graft-versus-host disease (SR-aGVHD) or treatment-resistant acute graft-versus-host disease (TR-aGVHD). The example presented below describes a Phase 1, open-label study of the anti-PSGL-1 antibody 15A7H in patients with steroid-resistant acute graft-versus-host disease (SR-aGVHD) or treatment-resistant acute graft-versus-host disease (TR-aGVHD).

[0204] Test Design The anti-PSGL-1 antibody 15A7H was tested in patients with SR-aGVHD or TR-aGVHD using multiple doses of the antibody. Study participants received an initial dose of 6 mg / kg of antibody 15A7H, followed by weekly doses of 4 mg / kg of antibody 15A7H for three weeks (6-4-4-4 regimen).

[0205] The study included patients with SR-aGVHD who had not received any prior systemic treatment other than corticosteroids, and patients with TR-aGVHD who had received one prior systemic treatment for aGVHD, including a JAK inhibitor in addition to corticosteroids. Participants treated with the antibody 15A7H in combination with additional systemic treatment were also included in the study.

[0206] result Efficacy analyses were performed to determine the likelihood of reaching pre-established futility criteria after 24 patients were enrolled in the study. Of the 24 participants, 12 were classified as steroid-resistant (SR) and 12 were classified as treatment-resistant (TR).

[0207] Improved clinical responses were observed in participants with TR (8 / 12 = 67% overall response) compared with those with SR (4 / 12 = 33%). This finding was unexpected, as patients who had previously received systemic treatment are likely to have more severe or aggressive disease. Further analysis showed that clinical responses were improved in participants who received the combination of antibody 15A7H plus a JAK inhibitor therapy.

[0208] The observed improved patient outcomes treated with a combination of antibody 15A7H and JAK inhibition in patients with T cell mediated diseases is supported by preclinical studies showing synergistic effects when antibody 15A7H is combined with JAK inhibitors such as ruxolitinib, e.g., as described in Example 1 herein.

[0209] Although the foregoing embodiments have been described in some detail by way of illustration and example, for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present disclosure.

Claims

1. A pharmaceutical for treating or preventing a T cell-mediated inflammatory disease in a subject, the pharmaceutical comprising an antibody that specifically binds to human PSGL-1 and is administered in combination with a Janus kinase (JAK) inhibitor.

2. A pharmaceutical for treating or preventing cancer in a subject, the pharmaceutical comprising an antibody that specifically binds to human PSGL-1, and being administered in combination with a Janus kinase (JAK) inhibitor. (a) the JAK inhibitor inhibits JAK1 and / or JAK2; (b) the JAK inhibitor inhibits JAK1 and / or JAK3; (c) the JAK inhibitor is ruxolitinib, or (d) the JAK inhibitor is tofacitinib; The pharmaceutical composition according to claim 1 or claim 2.

4. The pharmaceutical composition of claim 1 or 2, wherein the antibody that specifically binds to human PSGL-1 comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO: 8), CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7), and the antibody is optionally a humanized antibody, and optionally the VH domain comprises the amino acid sequence of SEQ ID NO: 4 and / or the VL domain comprises the amino acid sequence of SEQ ID NO:

3.

5. The pharmaceutical composition of claim 1 or 2, wherein the antibody that specifically binds to human PSGL-1 comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence AYYIH (SEQ ID NO: 24), CDR-H2 comprising the amino acid sequence RVNPNTGGTSYNPKFKG (SEQ ID NO: 25), and CDR-H3 comprising the amino acid sequence SGSPYYRYDD (SEQ ID NO: 26), and the VL domain comprises CDR-L1 comprising the amino acid sequence RASSTVNSTYLH (SEQ ID NO: 28), CDR-L2 comprising the amino acid sequence GSSNLAS (SEQ ID NO: 29), and CDR-L3 comprising the amino acid sequence QQYSGYPLT (SEQ ID NO: 30), and wherein, optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 27 and / or the light chain comprises the amino acid sequence of SEQ ID NO:

31.

6. The pharmaceutical composition of claim 1 or 2, wherein the antibody that specifically binds to human PSGL-1 comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence TNAMN (SEQ ID NO: 32), CDR-H2 comprising the amino acid sequence RIRSKSNNYATYYADSVKD (SEQ ID NO: 33), and CDR-H3 comprising the amino acid sequence GGSYWYFDV (SEQ ID NO: 34), and the VL domain comprises CDR-L1 comprising the amino acid sequence RSSQSIVNSNGNTYLE (SEQ ID NO: 36), CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 37), and CDR-L3 comprising the amino acid sequence FQGSHVPWT (SEQ ID NO: 38), and wherein, optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 35 and / or the light chain comprises the amino acid sequence of SEQ ID NO:

39.

7. 3. The pharmaceutical of claim 1 or claim 2, wherein the heavy chain further comprises an antibody constant domain, optionally the constant domain is a human IgG4 constant domain, optionally the constant domain is a human IgG4 constant domain comprising a S228P amino acid substitution at position 228, wherein numbering is according to EU numbering.

8. The pharmaceutical composition of claim 1 or claim 2, wherein the light chain is a human kappa light chain. (a) the heavy chain comprises the amino acid sequence of SEQ ID NO:2, and / or the light chain comprises the amino acid sequence of SEQ ID NO:1, or (b) the antibody that specifically binds to human PSGL-1 comprises the amino acid sequence of SEQ ID NO: 23; The pharmaceutical composition according to claim 1 or claim 2.

10. The pharmaceutical composition according to claim 1, wherein the T cell-mediated inflammatory disease is graft-versus-host disease (GVHD) or transplant rejection, and optionally the T cell-mediated inflammatory disease is acute GVHD, steroid-resistant acute GVHD (SR-aGVHD), treatment-resistant acute GVHD (TR-aGVHD), or chronic GVHD.

11. 2. The pharmaceutical composition of claim 1, wherein the T cell-mediated inflammatory disease is selected from the group consisting of skin disorders, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type 1 diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, psoriasis, ankylosing spondylitis, arthritis, diabetes, systemic lupus erythematosus, dermatitis, Sjogren's syndrome, ulcerative colitis, asthma, allergic asthma, scleroderma, uveitis, Stevens-Johnson syndrome, vitiligo, alopecia areata, cytokine release syndrome, hidradenitis suppurativa, sarcoidosis, primary biliary cirrhosis, posterior uveitis, allergy, psoriatic arthritis, atopic dermatitis, cutaneous lupus erythematosus, and autoimmune thyroid disorders.

12. 3. The pharmaceutical composition of claim 2, wherein the cancer is T-cell leukemia or T-cell lymphoma, and optionally the T-cell leukemia or lymphoma is adult T-cell leukemia / lymphoma (ATLL), anaplastic large cell lymphoma (ALCL), or cutaneous T-cell lymphoma (CTCL).

13. The pharmaceutical composition of claim 1 or 2, wherein the human PSGL-1 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

22.

14. The pharmaceutical according to claim 1 or claim 2, wherein the subject is a human.

15. The pharmaceutical according to claim 1 or 2, wherein the antibody is a monoclonal antibody.

16. The method of claim 1 or claim 2, wherein the subject has been treated with a corticosteroid prior to administration of the antibody and the JAK inhibitor.

17. 3. The pharmaceutical of claim 1 or claim 2, wherein administration of the antibody and the JAK inhibitor results in a reduction of one or more symptoms of the T cell-mediated inflammatory disease or cancer in the subject.

18. A kit comprising: (a) an antibody that specifically binds human PSGL-1 to treat or prevent a T cell-mediated inflammatory disease in a subject; or (b) to treat or prevent cancer in a subject; and a package insert containing instructions for using the antibody in combination with a Janus kinase (JAK) inhibitor, wherein optionally the antibody comprises a heavy chain comprising a heavy chain variable (VH) domain and a light chain comprising a light chain variable (VL) domain, and the VH domain has the amino acid sequence SFGMH( a CDR-H1 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 8), a CDR-H2 comprising the amino acid sequence YINGGSSTIFYANAVKG (SEQ ID NO: 9), and a CDR-H3 comprising the amino acid sequence YASYGGGAMDY (SEQ ID NO: 10), wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence RSSQSIVHNDGNTYFE (SEQ ID NO: 5), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 6), and a CDR-L3 comprising the amino acid sequence FQGSYVPLT (SEQ ID NO: 7).

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