Polypeptide Modulators

Abstract

Described are isolated polypeptides that are mutants of programmed cell death 1 ligand 2 ("PD-L2") and that can either activate or inhibit immune cells by activating or inhibiting programmed cell death protein 1 ("PD-1"). Also described are isolated polypeptides that block activating or inhibitory sites on PD-L2, thereby inhibiting or activating PD-1. Additionally, described are pharmaceutical compositions comprising the polypeptides, and methods of using the polypeptides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 288,330, filed December 10, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by Reference All patents, patent publications, journal publications, or other documents cited herein are expressly incorporated herein by reference in their entirety.

[0003] FIELD OF THE PRESENT ART The present invention relates generally to polypeptide modulators and immune regulation. More specifically, the present invention relates to ligands that specifically bind to or modulate programmed cell death protein 1 ("PD-1"), and methods of use thereof. [Background technology]

[0004] PD-1 is a protein on the surface of T and B cells that plays a role in controlling the immune system's response to the body's own cells by suppressing the inflammatory activity of T cells, downregulating the immune system and promoting self-tolerance. Although this biological pathway prevents autoimmune diseases, it can also prevent the immune system from killing cancer cells.

[0005] Programmed cell death ligand 1 ("PD-L1") binds to PD-1 and transmits an inhibitory signal to suppress immune responses. The PD-1 / PD-L1 pathway has shown promising clinical success as a target for cancer immunotherapy. Current antibodies targeting either PD-1 or PD-L1 can block this interaction and enhance immune responses against cancer cells. Successful clinical trials with PD-1 monoclonal antibodies and other immune checkpoint inhibitors have opened new avenues in cancer immunology. However, a large proportion of cancer patients do not respond favorably to new immunotherapies, stimulating research into combination therapies and predictive biomarkers. See, for example, Iwai, Y, et al., Journal of Biomedical Science, 24:26 (2017).

[0006] In addition to PD-L1, programmed cell death 1 ligand 2 ("PD-L2") is a protein known to bind to PD-1.

[0007] It is therefore an object of one or more of the inventions disclosed herein to provide compositions and methods for modulating PD-1 signaling and associated biological pathways. Summary of the Invention

[0008] In one aspect, isolated polypeptides that bind to PD-1 are described. In one embodiment, the polypeptides can modulate immune responses in vitro or in vivo and / or can be used alone or in combination with other agents to prevent or treat a variety of conditions. In another aspect, isolated polypeptides that block inhibitory or activating sites on PD-L2 are described. In yet another aspect, pharmaceutical compositions comprising the polypeptides and methods of using these compositions, alone or in combination with other agents or compositions, to prevent or treat a variety of conditions are described. In yet another aspect, inducible T cell kinase ("ITK") activators or inhibitors are described and / or can be used alone or in combination with other agents to prevent or treat a variety of conditions.

[0009] In one aspect, an isolated polypeptide is described that comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1.

[0010] In another aspect, an isolated polypeptide is described that comprises the amino acid sequence of SEQ ID NO:21.

[0011] In yet another aspect, an isolated polypeptide is described that comprises the amino acid sequence of SEQ ID NO:22.

[0012] In any one of the embodiments disclosed herein, the amino acid is fused to an immunoglobulin.

[0013] In any one of the embodiments disclosed herein, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

[0014] In any one of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0015] In any one of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0016] In any one of the embodiments disclosed herein, the polypeptide comprises one or more amino acids each selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114.

[0017] In any one of the embodiments disclosed herein, the polypeptide binds to and activates PD-1.

[0018] In any one of the embodiments disclosed herein, the polypeptide activates an immune cell.

[0019] In any one of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or SEQ ID NO:6.

[0020] In any one of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0021] In any one of the embodiments disclosed herein, the polypeptide comprises one or more amino acids each selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111.

[0022] In any one of the embodiments disclosed herein, the polypeptide binds to and inhibits PD-1.

[0023] In any one of the embodiments disclosed herein, the polypeptide inhibits an immune cell.

[0024] In any one of the embodiments disclosed herein, the polypeptide induces central memory T cells ("Tcm").

[0025] In any one of the embodiments disclosed herein, the polypeptide prevents T cell exhaustion.

[0026] In yet another aspect, a conjugate is described comprising a polypeptide of any one of the embodiments disclosed herein, wherein the polypeptide is linked to a detectable marker or carrier molecule.

[0027] In any one of the embodiments disclosed herein, the carrier molecule is selected from the group consisting of glycosaminoglycans, proteoglycans, albumin, and polyalkylene glycols.

[0028] In yet another aspect, there is provided a nucleic acid encoding a polypeptide of any one of the embodiments disclosed herein.

[0029] Yet another aspect includes a pharmaceutical composition comprising a polypeptide of any one of the embodiments disclosed herein.

[0030] In any one of the embodiments disclosed herein, the polypeptide is encapsulated within a liposome.

[0031] In any one of the embodiments disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent.

[0032] In any one of the embodiments disclosed herein, the second therapeutic agent is a chemotherapeutic agent or an immunosuppressant agent.

[0033] In yet another aspect, a method of inducing, promoting or enhancing an immune response in a subject in need thereof is described, comprising administering to the subject an effective amount of a polypeptide according to any one of the embodiments disclosed herein, a conjugate according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0034] In yet another aspect, a method of treating cancer or reducing tumor burden in a subject in need thereof is described, comprising administering to the subject an effective amount of a polypeptide according to any one of the embodiments disclosed herein, a conjugate according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0035] In any one of the embodiments disclosed herein, the cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, renal cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, and testicular cancer.

[0036] In any one of the embodiments disclosed herein, the method further comprises upregulating ITK.

[0037] In yet another aspect, a method of reducing, suppressing or preventing an immune response in a subject in need thereof is described, comprising administering to the subject an effective amount of a polypeptide according to any one of the embodiments disclosed herein, a conjugate according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0038] In yet another aspect, a method of treating an autoimmune disease in a subject in need thereof is described, comprising administering to the subject an effective amount of a polypeptide according to any one of the embodiments disclosed herein, a conjugate according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0039] In any one of the embodiments disclosed herein, the autoimmune disease is selected from the group consisting of achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane disease, antitubular basement membrane antibody nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy, Baro's disease, Behcet's disease, Benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing polymyelitis, Churg-Strauss syndrome, eosinophilic granulomatosis, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans' syndrome, fibromyalgia, fibrous alveolar pulmonary Inflammation, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational pemphigoid, hidradenitis suppurativa (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, Linear IgA disease, lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, pediatric autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis,Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Polyendocrine syndrome type I, Polyendocrine syndrome type II, Polyendocrine syndrome type III, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progestational dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm and testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome syndrome), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and combinations thereof.

[0040] In any one of the embodiments disclosed herein, the method further comprises downregulating ITK.

[0041] In yet another embodiment, a method of inducing, promoting or enhancing an immune response is described that comprises administering an effective amount of an ITK activator.

[0042] In yet another embodiment, a method of treating cancer or reducing tumor burden is described that comprises administering an effective amount of an ITK activator.

[0043] In any one of the embodiments disclosed herein, the ITK activator is a small molecule compound, a polypeptide, or a nucleic acid.

[0044] In yet another aspect, a method of reducing, suppressing, or preventing an immune response in a subject in need thereof is described, comprising administering to the subject an effective amount of an ITK inhibitor.

[0045] In yet another aspect, a method of treating an autoimmune disease in a subject in need thereof is described, comprising administering to the subject an effective amount of an ITK inhibitor.

[0046] In any one of the embodiments disclosed herein, the IKT inhibitor is a small molecule compound, a polypeptide, or a nucleic acid.

[0047] In yet another embodiment, the isolated polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO:11 to SEQ ID NO:13.

[0048] In any one of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 to SEQ ID NO:13.

[0049] In any one of the embodiments disclosed herein, the polypeptide binds to PD-L2.

[0050] In any one of the embodiments disclosed herein, the polypeptide blocks the inhibitory site of PD-L2.

[0051] In yet another embodiment, the isolated polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:14.

[0052] In any one of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO:14.

[0053] In any one of the embodiments disclosed herein, the polypeptide blocks the activation site of PD-L2.

[0054] In any one of the embodiments disclosed herein, the polypeptide inhibits an immune cell.

[0055] In yet another aspect, a method of reducing, suppressing, or preventing an immune response in a subject in need thereof is described, comprising administering to the subject an effective amount of a small molecule that binds to the inhibitory site of PD-1.

[0056] In yet another embodiment, a method of treating an autoimmune disease is described that includes administering to a subject an effective amount of a small molecule that binds to the inhibitory site of PD-1.

[0057] Any embodiment disclosed herein can be appropriately combined with any other embodiment disclosed herein.Any one of the embodiments disclosed herein and any other embodiment disclosed herein are expressly contemplated for combination.Specifically, the selection of one or more embodiments for one substituent can be appropriately combined with the selection of one or more specific embodiments for any other substituent.Such combinations are possible in any one or more embodiments of the application described herein or in any formulation described herein. [Brief description of the drawings]

[0058] [Figure 1] FIG. 1 shows the relative light units ("RLU") of PD-1 reporter cell populations treated with or without a polypeptide according to one or more embodiments described herein.

[0059] [Diagram 2] 2A-2C show the design rationale for a polypeptide that binds to PD-L2, according to one or more embodiments described herein.

[0060] [Diagram 3] 3A-3B show the design rationale for PD-L2 variants according to one or more embodiments described herein.

[0061] [Figure 4]FIG. 4 illustrates that, according to one or more embodiments described herein, the type of PD-1 signaling depends on the balance between ITK and Src homology domain 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2).

[0062] [Diagram 5] 5A-5C show immune responses that depend on the balance between ITK and SHP2 and upstream pathways that affect the balance between ITK and SHP2, according to one or more embodiments described herein.

[0063] [Figure 6] 6A-6C show the differences in levels of PD-1-bound ITK and SHP2 following treatment of primary T cells with PD-L1 and PD-L2, according to one or more embodiments described herein.

[0064] [Figure 7] 7A-7C show that PD-L2 increases Tcm and prevents T cell exhaustion, according to one or more embodiments described herein.

[0065] [Figure 8] 8A-8C show that PD-L2 is required for the generation and prevention of exhaustion of Tcm, in accordance with one or more embodiments described herein.

[0066] [Figure 9] Figures 9A-9D show that PD-L2 is required to initiate activation of both CD4 and CD8 T cells, in accordance with one or more embodiments described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Detailed Description of the Invention definition It is understood that the disclosure is not limited to the compositions and methods described herein and the experimental conditions described, as these may vary, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All compositions, methods, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0069] The use of the singular and similar terms in the context of describing the presently claimed invention (particularly in the context of the claims) are to be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0070] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein.

[0071] The use of the term "about" is intended to describe values ​​above or below the stated value, within a range of about ±10%. In some embodiments, values ​​may be above or below the stated value, within a range of about ±5%. In some embodiments, values ​​may be above or below the stated value, within a range of about ±2%. In some embodiments, values ​​may be above or below the stated value, within a range of about ±1%. The foregoing ranges are intended to be made clear by the context, and no further limitations are implied. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. Any examples provided herein, or the use of exemplary language (e.g., "exemplary," "such as," "for example," "including but not limited to") are intended merely to make the invention more clear, and do not limit the scope of the invention, unless otherwise indicated.

[0072] The term "cancer" and equivalently "tumor" as used herein refers to a condition in which abnormally replicating cells derived from the host are present in a detectable amount in a subject. Cancer can be malignant or non-malignant. Cancer or tumor includes, but is not limited to, adult T-cell leukemia / lymphoma (including those caused by human T-cell lymphotropic virus ("HTLV-1")), biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasia, leukemia, lymphoma, liver cancer, lung cancer (e.g., small cell and non-small cell), melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and other carcinomas and sarcomas. As used herein, the term "lymphoma" refers to a cancer of the lymphatic system or a blood cancer that arises from lymphocytes. Cancer can be primary or metastatic. Diseases other than cancer may be related to the mutational alteration of components of the Ras signaling pathway, and the polypeptides disclosed herein may be used to treat these non-cancer diseases. Such non-cancer diseases include, but are not limited to, neurofibromatosis, Leopard syndrome, Noonan syndrome, Regius syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, hereditary gingival fibromatosis type 1, autoimmune lymphoproliferative syndrome, and capillary malformation-arteriovenous malformation.

[0073] As used herein, "effective amount" refers to any amount necessary or sufficient to achieve or promote a desired result. In some instances, the effective amount is a therapeutically effective amount. A therapeutically effective amount is an amount necessary or sufficient to promote or achieve a desired biological response in a subject. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular drug being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular drug without undue experimentation.

[0074] The term "subject" as used herein refers to a vertebrate. In some embodiments, the subject is a mammal or mammalian species. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human vertebrate, including, but not limited to, non-human primates, laboratory animals, farm animals, race horses, pets, and non-domesticated animals.

[0075] As used herein, the term "immune cells" refers to cells of the innate and adaptive immune systems, including, but not limited to, neutrophils, eosinophils, basophils, glial cells (e.g., astrocytes, microglia, oligodendrocytes), monocytes, macrophages, dendritic cells, lymphocytes (e.g., B cells, T cells, natural killer ("NK") cells).

[0076] As used herein, a "conventional T cell" is a T lymphocyte that expresses an αβ T cell receptor ("TCR") and a co-receptor such as CD4 or CD8. Conventional T cells are present in peripheral blood, lymph nodes and tissues. See Roberts and Girardi, "Conventional and Unconventional T Cells", Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari and Tyring (ed.)), Springer London (2008), which is incorporated herein by reference in its entirety. As used herein, a "non-conventional T cell" is a lymphocyte that expresses a γδ TCR and may be commonly present in epithelial environments such as the skin, gastrointestinal tract, or urogenital tract. Another subset of non-conventional T cells is the invariant natural killer T ("NKT") cell, which has the phenotype and functional capabilities of conventional T cells as well as the characteristics of natural killer cells (e.g., cytolytic activity). See id. As used herein, regulatory T cells ("Tregs") are a subpopulation of T cells that regulate the immune system, maintain immune tolerance to self-antigens, prevent autoimmune disease, and otherwise suppress the immune stimulatory or activation responses of other cells. There are various types of Tregs, but the most well-known are those that express CD4, CD25, and Foxp3. As used herein, "natural Tregs" or "nTregs" refers to Tregs or cells that develop in the thymus. As used herein, "inducible Tregs" or "iTregs" refers to Tregs or cells that develop outside the thymus from mature CD4+ conventional T cells.

[0077] The term "peptide" or "polypeptide" as used herein refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The terms "peptide" and "polypeptide" are used interchangeably. The term includes proteins and fragments thereof. A polypeptide is "foreign," meaning "heterologous," i.e., foreign to the host cell in which it is utilized, and can be, for example, a human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right from the amino terminus to the carboxy terminus. According to standard nomenclature, amino acid residue sequences are designated by either three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0078] The term "immune response" includes, for example, the development of beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) responses induced against a polypeptide in a recipient patient. Such responses can be active responses induced by administration of an immunogen, or passive responses induced by administration of antibodies or primed T cells. A cellular immune response is triggered by the presentation of a polypeptide epitope in association with class I or class II major histocompatibility complex ("MHC") molecules to activate antigen-specific CD4+ T helper cells and / or CD8+ cytotoxic T cells. This response can also involve the activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, neutrophils, or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+ T cells) or cytotoxic T lymphocyte ("CTL") assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized allogeneic animal and measuring the protective or therapeutic effect in a second subject.

[0079] The term "immunogenic agent" or "immunogen" refers to a substance that is capable of inducing an immunological response against itself when administered to a mammal, optionally in combination with an adjuvant.

[0080] Polypeptides In addition to PD-L1, PD-L2 is another protein that binds to PD-1. Applicants have surprisingly discovered that when PD-L2 binds to PD-1 through two major interaction sites on PD-L2, these sites have opposite functions, i.e., one site on PD-L2 activates PD-1 and immune responses, while the other site inhibits PD-1 and immune responses. The site that activates PD-1 is referred to herein as the "activation site," and the site that inhibits PD-1 is referred to herein as the "inhibition site." The two interaction sites of PD-L2 are shown in FIG. 2A. In some embodiments, the activation site on PD-L2 comprises one or more amino acids selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114, respectively. In some embodiments, the inhibitory site on PD-L2 comprises one or more amino acids selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111.

[0081] In some embodiments, polypeptides are described that block or disable the inhibitory site of PD-L2, which activates PD-1 and the immune response. In another embodiment, polypeptides are described that block or disable the activating site of PD-L2, which inhibits PD-1 and suppresses the immune response. For example, as shown in Figure 3B, a mutant PD-L2 with an inhibiting site disabled can activate the immune response through PD-1 signaling. Furthermore, as shown in Figure 3A, a mutant PD-L2 with an inhibiting site disabled can suppress the immune response through PD-1 signaling.

[0082] SEQ ID NO:1 is the amino acid sequence defining human PD-L2: LFTVVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHP.

[0083] SEQ ID NO: 21 is the amino acid sequence defining the activation site of human PD-L2: QXIXIXXXXWXYXY (wherein X is any amino acid).

[0084] SEQ ID NO: 22 is an amino acid sequence defining the inhibitory site of human PD-L2: IXGVAWD (wherein X is any amino acid).

[0085] SEQ ID NO:2 is the amino acid sequence defining human PD-L2 fused to hIgG1: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0086] In some embodiments, an isolated polypeptide or variant thereof is described that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polypeptide is a variant of SEQ ID NO: 1. In some embodiments, the polypeptide blocks or neutralizes the activating site of PD-L2, resulting in inhibition of PD-1 and suppression of the immune response. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 22.

[0087] In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 21, and 22, wherein the amino acid sequence is fused to an immunoglobulin or a fragment thereof. In some embodiments, the immunoglobulin is IgG, IgM, or IgA. In some embodiments, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin is a human immunoglobulin. In some embodiments, the polypeptide is described as having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2. In some embodiments, the polypeptide is a variant of SEQ ID NO: 2.

[0088] SEQ ID NO:3 is an amino acid sequence defining the first variant of PD-L2: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYDDDWLYKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0089] SEQ ID NO:4 is an amino acid sequence defining a second variant of PD-L2: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIDYDDDDLYKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0090] In some embodiments, the polypeptide is a mutant of PD-L2 with a disabled inhibitory site. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the polypeptide comprises one or more amino acids each selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114.

[0091] In some embodiments, the polypeptide binds to PD-1. In some embodiments, the polypeptide binds to PD-1 only through a binding site similar to the activation site of PD-L2. In some embodiments, the polypeptide does not have a binding site similar to the inhibitory site of PD-L2. In some embodiments, the polypeptide activates PD-1. In some embodiments, the polypeptide activates immune cells by activating PD-1. Non-limiting examples of immune cells include T cells (e.g., Tregs), B cells, macrophages, and glial cells (e.g., astrocytes, microglia, or oligodendrocytes). In some embodiments, the polypeptide can be used to induce, promote, or enhance an immune response. In some embodiments, the polypeptide can be used to treat cancer.

[0092] SEQ ID NO:5 is an amino acid sequence defining a third variant of PD-L2: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYLCDIIYGVAWDDKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0093] SEQ ID NO:6 is an amino acid sequence defining a fourth variant of PD-L2: LFTTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYLCDIDYGVADDKDLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0094] In some embodiments, the polypeptide is a mutant of PD-L2 with a disabled activation site. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:5. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:6. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the polypeptide comprises one or more amino acids each selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111.

[0095] In some embodiments, the polypeptide binds to PD-1. In some embodiments, the polypeptide binds to PD-1 only through a binding site similar to the inhibitory site of PD-L2. In some embodiments, the polypeptide does not have a binding site similar to the activating site of PD-L2. In some embodiments, the polypeptide inhibits PD-1. In some embodiments, the polypeptide suppresses immune cells by inhibiting PD-1. Non-limiting examples of immune cells include T cells (e.g., Tregs), B cells, macrophages, and glial cells (e.g., astrocytes, microglia, or oligodendrocytes). In some embodiments, the polypeptide can be used to reduce, suppress, or prevent an immune response. In some embodiments, the polypeptide can be used to treat an autoimmune disease.

[0096] SEQ ID NO:7 is an amino acid sequence defining a fifth variant of PD-L2 on the linker region: IDTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVDWDYKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0097] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% identical to SEQ ID NO:7.

[0098] In some embodiments, a polypeptide is described that blocks or neutralizes the inhibitory site of PD-L2, which activates PD-1 and the immune response. For example, as shown in Figure 2B, a polypeptide that blocks the activating site of PD-L2 can induce an inhibitory signal from PD-1. Furthermore, as shown in Figure 2C, a polypeptide that blocks the inhibitory site of PD-L2 can induce an activating signal from PD-1.

[0099] SEQ ID NO:11 is an amino acid sequence defining a first polypeptide that binds PD-L2: TSESFVLNWYRMSPS.

[0100] SEQ ID NO:12 is an amino acid sequence defining a second polypeptide that binds to PD-L2: FSNTSESFVLNWYRM.

[0101] SEQ ID NO:13 is an amino acid sequence defining a third polypeptide that binds to PD-L2: ESFVLNWYRMSP.

[0102] In some embodiments, a polypeptide that blocks the inhibitory site of PD-L2 is described. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 13.

[0103] In some embodiments, the polypeptide binds to PD-L2. In some embodiments, the polypeptide activates PD-1 by blocking the inhibitory site of PD-L2. In some embodiments, the polypeptide activates immune cells by activating PD-1. Non-limiting examples of immune cells include T cells (e.g., Tregs), B cells, macrophages, and glial cells (e.g., astrocytes, microglia, or oligodendrocytes). In some embodiments, the polypeptide can be used to induce, promote, or enhance an immune response. In some embodiments, the polypeptide can be used to treat cancer.

[0104] SEQ ID NO:14 is an amino acid sequence defining a fourth polypeptide that binds to PD-L2: TYLCGAISLAPKAQI.

[0105] In some embodiments, polypeptides that block the activation site of PD-L2 are described. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 14.

[0106] In some embodiments, the polypeptide binds to PD-L2. In some embodiments, the polypeptide inhibits PD-1 by blocking the activation site of PD-L2. In some embodiments, the polypeptide suppresses immune cells by inhibiting PD-1. Non-limiting examples of immune cells include T cells (e.g., Tregs), B cells, macrophages, and glial cells (e.g., astrocytes, microglia, or oligodendrocytes). In some embodiments, the polypeptide can be used to reduce, suppress, or prevent an immune response. In some embodiments, the polypeptide can be used to treat an autoimmune disease.

[0107] In some embodiments, the polypeptides may be modified in a manner known to those of skill in the art. For example, the polypeptides may be used as peptidomimetics. The polypeptides may form dimers.

[0108] In some embodiments, the polypeptide can be PEGylated. PEGylation can delay the clearance of the polypeptide from the circulation by various mechanisms. In some embodiments, PEGylation inhibits proteolytic enzyme degradation and decreases the rate of renal filtration by increasing the apparent molecular size. Thus, PEG-based modifications can be useful for extending the circulation time of the polypeptide and for increasing bioavailability. In some embodiments, the polypeptide is PEGylated with a linear PEG molecule. In another embodiment, the polypeptide is PEGylated with a branched PEG molecule. The present invention further provides amino-, carboxy- and side-chain-PEGylated polypeptides. The PEG moiety can be a PEG molecule with a molecular weight greater than 5 kDa. For example, the molecular weight can be between 5 kDa and 100 kDa (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 kDa), and more preferably, the molecular weight is between 10 kDa and 50 kDa (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 kDa). Methods for the synthesis of pegylated polypeptides are well known in the art.

[0109] In some embodiments, the polypeptide is conjugated to a detectable marker. In some embodiments, the detectable marker is conjugated to the C-terminus of the polypeptide. In other embodiments, the detectable label is conjugated to the N-terminus. The detectable marker can be, but is not limited to, a chemical label, such as a radioisotope, a fluorescent group, a chemiluminescent label, a colorimetric label, an enzyme marker, and an affinity moiety (e.g., biotin) that facilitates detection of the labeled polypeptide. The present invention also provides dye-labeled polypeptides, including, but not limited to, fluorescein and rhodamine conjugates. Other chemical labels and methods of chemically labeling polypeptides are well known in the art.

[0110] In some embodiments, the polypeptide is bound to a carrier molecule. The polypeptide can also be used as a conjugate of at least one polypeptide or polypeptide fragment bound to a carrier. The carrier can provide a solid phase support for the polypeptide of the present invention. The carrier can be a biological carrier, including but not limited to glycosaminoglycans, proteoglycans, or albumin, or a synthetic polymer, including but not limited to polyalkylene glycol or synthetic chromatographic carrier. Other carriers include but are not limited to ovalbumin, human serum albumin, other proteins, and polyethylene glycol.

[0111] In one embodiment, a nucleic acid encoding a polypeptide is described. In another embodiment, the polypeptide can be prepared using recombinant DNA technology methods, where an expression vector contains a nucleic acid sequence encoding a polypeptide of the invention, and the nucleic acid sequence is operably linked to a promoter. The expression vector can be delivered by methods such as, but not limited to, transformation, transfection, and a suitable host cell that allows expression of the polypeptide. The host cell containing the expression vector is cultured under appropriate conditions to express the polypeptide. In one embodiment, the host cell is a mammalian cell, including but not limited to a human cell. In another embodiment, the host cell is a bacterial, fungal or insect cell. In one embodiment, the polypeptide is recovered from the culture, where recovery can include a step leading to purification of the polypeptide. Preparation of a polypeptide by recombinant techniques is advantageous when the polypeptide can be post-translationally modified. Still further, methods combining synthetic and recombinant DNA techniques can be used to prepare amide and ester derivatives of the polypeptide, as well as to prepare fragments of a desired polypeptide that are subsequently assembled by methods well known to those skilled in the art.

[0112] Suitable expression vectors for the delivery of nucleic acid sequences and for the expression of polypeptides in human cells are known in the art. Non-limiting examples are plasmid, viral or bacterial vectors.

[0113] Polypeptides can also be commercially prepared by companies that provide polypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pennsylvania; AnaSpec, Inc., San Jose, California). Automated polypeptide synthesizers, such as those manufactured by Perkin-Elmer Applied Biosystems, are also available.

[0114] The polypeptides useful in the methods described herein are isolated or synthesized, either by chemical or recombinant techniques, and then purified. 4 -, C 2 - or C 18 Standard methods for purification purposes can be used, including reversed-phase high pressure liquid chromatography ("HPLC") using alkylated silica columns, including but not limited to cyclohexane-silica. In this method, a gradient mobile phase of increasing organic content is generally used to achieve purification (e.g., acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid). Alternatively, ion exchange chromatography can be used to separate polypeptide compounds based on their charge. The purity of a polypeptide compound can be determined by the number of peaks identified by HPLC. In some embodiments, a useful level of polypeptide purity can result in a single peak on an HPLC chromatogram. In some embodiments, the polypeptide of interest is at least 94.99% of the input material on the HPLC column. In other embodiments, the polypeptide of interest is at least 96.99% of the input material on the HPLC column. In some embodiments, the polypeptide of interest is between 97% and 99.5% of the input material on the HPLC column.

[0115] In some embodiments, the method for determining whether a polypeptide has an effect on an immune response is a luciferase assay. In some embodiments, Jurkat PD-1 cells are used in the luciferase assay.

[0116] Pharmaceutical Compositions In one aspect, a pharmaceutical composition is described that comprises one or more of the polypeptides disclosed herein. In some embodiments, the polypeptide is encapsulated in a liposome in the pharmaceutical composition.

[0117] In some embodiments, carriers including, but not limited to, ion exchangers, alumina, aluminum stearate, lecithin, non-albumin serum proteins, buffer substances (e.g., phosphates, glycine, sorbic acid, potassium sorbate), and partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, and wool fat) may be used in the pharmaceutical compositions. Such modifications may increase the apparent affinity as well as alter the stability of the polypeptide. The number of polypeptide fragments bound to each carrier may vary, but typically about 4-8 polypeptide fragments are bound per carrier molecule under standard coupling conditions.

[0118] In some embodiments, the polypeptides may also be prepared and stored in salt form. Various salt forms of the polypeptides may also be formed or exchanged by any of a variety of methods known in the art, for example, by using various ion exchange chromatography methods. Cationic counterions that may be used in the compositions include, but are not limited to, amines (e.g., ammonium ions), metal ions, particularly monovalent, divalent, or trivalent ions of alkali metals (e.g., sodium, potassium, lithium, and cesium), alkaline earth metals (e.g., calcium, magnesium, and barium), transition metals (e.g., iron, manganese, zinc, cadmium, and molybdenum), other metals such as aluminum, and possible combinations thereof. Anionic counterions that may be used in the compositions described herein include, but are not limited to, chloride, fluoride, acetate, trifluoroacetate, phosphate, sulfate, carbonate, citrate, ascorbate, sorbate, glutarate, ketoglutarate, and possible combinations thereof. The trifluoroacetate salt of the polypeptide compounds described herein is typically formed during purification in trifluoroacetate buffer using HPLC.Although not usually suitable for in vivo use, the trifluoroacetate salt form of the polypeptides described herein can be conveniently used in various in vitro cell culture studies, assays, or testing the activity or effectiveness of the polypeptide compounds of interest.The polypeptides can then be converted from trifluoroacetate salt by ion exchange methods, or synthesized as acceptable salt forms for pharmaceutical or nutraceutical compositions.

[0119] In some embodiments, the pharmaceutical composition can be delivered by various routes or modes, including, but not limited to, parenteral, oral, intratracheal, sublingual, pulmonary, topical (external), rectal, nasal, buccal, sublingual, vaginal, or via an implanted reservoir. The implanted reservoir may function by mechanical, osmotic, or other means. The term "parenteral" as used herein includes, but is not limited to, intravenous, intracranial, intraperitoneal, paraspinal, periarticular, periosteal, subcutaneous, intradermal, intraarterial, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, and localized injection or infusion techniques. Such compositions are formulated for parenteral administration, and are often formulated for intravenous, intracranial, or intraarterial administration. In general, when administration is intravenous or intraarterial, the pharmaceutical composition can be administered as a bolus, as a separate dose.

[0120] In some embodiments, the pharmaceutical composition may be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Solvents that may be used include, but are not limited to, mannitol, water, Ringer's solution, and isotonic sodium chloride solution (saline). In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be used, including but not limited to synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharma-ceutically acceptable oils, such as olive oil or castor oil.

[0121] In some embodiments, the pharmaceutical composition may be administered orally via capsules, tablets, caplets, pills, aqueous suspensions, and solutions, or syrups. For oral tablets, carriers including, but not limited to, lactose and cornstarch may be used. Lubricants including, but not limited to, magnesium stearate and the like may also be added. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried cornstarch. Capsules, tablets, pills, and caplets may be formulated for delayed or sustained release if extended expression is required. Alternatively, when oral aqueous suspensions are administered, the polypeptide is advantageously combined with emulsifying and / or suspending agents. Optionally, certain sweeteners and / or flavorings and / or colorings may be added. In some embodiments, preparations for oral administration provide the polypeptides described herein in a mixture that prevents or inhibits hydrolysis of the polypeptide compound by the digestive system, thereby allowing absorption into the bloodstream.

[0122] In some embodiments, the pharmaceutical compositions may be administered mucosally (e.g., vaginally or rectally). These dosages may be prepared by mixing the polypeptides described herein with suitable non-irritating excipients that are solid at room temperature but liquid at body temperature, and thus undergo a state change to liquid form in the relevant body space to release the active compound. Examples of these solvents include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols. They may also be absorbed via inhalation into the nasal mucosa or lungs for other mucosal sites, such as nasal or pulmonary delivery. These modes of administration typically require the composition to be provided in the form of a solution, liquid suspension, or powder, which is then mixed with a gas, such as air, oxygen, nitrogen, or a combination thereof, to generate an aerosol or suspension of droplets or particles. These preparations are made according to well-known techniques in the pharmaceutical formulation art. These formulations may be prepared as solutions in saline with benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and solubilizers or dispersants known in the art.

[0123] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0124] Exemplary additional therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral agents (e.g., protease inhibitors alone or in combination with nucleosides for the treatment of HIV or Hepatitis B or C), antiparasitic agents (e.g., helminths or protozoans), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (e.g., humanized antibodies, single chain antibodies, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), polypeptide formulations, anti-inflammatory agents, ligands that bind to Toll-like receptors and activate the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that recruit and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, NK cells, helper T cells, and other molecules that inactivate or downregulate suppressor or regulatory T cells.

[0125] The additional therapeutic agent is selected based on the condition, disorder, or disease to be treated. For example, the polypeptide described herein can be co-administered with one or more additional agents that function to enhance or promote immune response, or to reduce or inhibit immune response.

[0126] Chemotherapeutic agents In some embodiments, the polypeptides described herein can be combined with one or more chemotherapeutic or pro-apoptotic agents. Representative chemotherapeutic agents include amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, and cyclophosphamide. Representative pro-apoptotic agents include, but are not limited to, sorbicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0127] Immunosuppressants In some embodiments, the polypeptides disclosed herein are used in combination with other immunotherapeutic agents, immunomodulatory agents, costimulatory activation agonists, other cytokines and chemokines and factors, vaccines, oncolytic viruses, cell therapy, small molecule and targeted therapy, chemotherapy, and radiation therapy. In some embodiments, immunomodulatory agents include, but are not limited to, checkpoint inhibitors such as anti-PD1, anti-CTLA4, anti-TIM3, and anti-LAG3. In some embodiments, costimulatory activation agonists include anti-OX40, anti-GITR, and the like. In some embodiments, cell therapy includes engineered T cells, CAR-T, TCR-T cells, and the like.

[0128] In some embodiments, the polypeptides disclosed herein are used in combination with other immunotherapeutic agents, immunomodulatory agents, biologics (e.g., antibodies), vaccines, small molecule and targeted therapies, anti-inflammatory agents, cell therapies (e.g., engineered Tregs and other types of cells), chemotherapy, and radiation therapy.

[0129] In some embodiments, the polypeptides disclosed herein are used alone or in combination with other agents and are administered to a patient in vivo by intravenous, intramuscular, or other parenteral means, and may also be administered nasally, by inhalation, rectally, vaginally, topically, orally, or as an implant.

[0130] In some embodiments, the additional therapeutic agent is an immunosuppressant. Immunosuppressants include, but are not limited to, antibodies against other lymphocyte surface markers (e.g., CD40 and alpha-4 integrin) or against cytokines, fusion proteins (e.g., CTLA-4-Ig (Orencia®) and TNFR-Ig (Enbrel®)), TNF-α blockers such as Enbrel, Remicade, Cimzia, and Humira, cyclophosphamide ("CTX") (e.g., Endoxan®, Cytoxan®, Neosar®, Procytox®, and Revimmune™), methotrexate (MTX) (e.g., Rheumatrex® and Trexall®), belimumab (e.g., Benlysta®), other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, and steroids), antiproliferative agents, cytotoxic agents, and other compounds that may aid in immunosuppression.

[0131] In some embodiments, the additional therapeutic agent can be a checkpoint inhibitor. In some embodiments, the additional therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion protein can compete with CD28, a costimulatory receptor on T cells, for binding to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, thus functioning to inhibit T cell activation. In another embodiment, the additional therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that can significantly increase activity against CD86 in vivo. In another embodiment, the additional therapeutic agent is Maxy-4.

[0132] In another embodiment, the additional therapeutic agent is CTX. CTX (the generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, and Revimmune™), also known as cytophosphane, is a nitrogen mustard alkylating agent of the oxazophorines group. CTX is used to treat various types of cancer and some autoimmune diseases. CTX is the primary drug used for diffuse proliferative glomerulonephritis in patients with nephrogenic lupus.

[0133] In some embodiments, the additional therapeutic agent can be administered in an effective amount to reduce blood or serum levels of anti-double stranded DNA ("anti-ds DNA") autoantibodies and / or to reduce proteinuria in a patient in need thereof.

[0134] In another embodiment, the additional therapeutic agent can increase the amount of adenosine in serum (see, e.g., WO 08 / 147482). For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent (e.g., a cytokine, monoclonal antibody, or small molecule) that increases expression of CD73 (see, e.g., WO 04 / 084933). In another embodiment, the additional therapeutic agent is interferon beta.

[0135] In some embodiments, the additional therapeutic agent may be a small molecule that inhibits or reduces the differentiation, proliferation, activity, cytokine production and / or cytokine secretion of Th1, Th17, Th22, and / or other cells that secrete, or cause other cells to secrete, proinflammatory molecules including, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment, the additional therapeutic agent is a small molecule that interacts with Tregs, enhances the activity of Tregs, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or a combination thereof.

[0136] In some embodiments, the additional therapeutic agent is an antibody, for example a function-blocking antibody against a pro-inflammatory molecule such as IL-6, IL-23, IL-22, or IL-21.

[0137] In some embodiments, the additional therapeutic agent comprises a nucleic acid. In some embodiments, the additional therapeutic agent comprises a ribonucleic acid.

[0138] Methods for Treating Disease In another aspect, a method of treating a disease in a subject in need thereof comprises administering to the subject an effective amount of a polypeptide described herein.

[0139] ITK plays an important role in immune response. The type of PD-1 signal depends on the balance between ITK and SHP2. In some embodiments, the type of PD-1 signal depends on the ITK-SHP2 ratio, as shown in FIG. 4. In some embodiments, when SHP2 is more than ITK, an inhibitory signal is generated, as shown in FIG. 4. In some embodiments, when ITK is more than SHP2, a stimulatory signal is generated. In some embodiments, activators of ITK are described that are useful for treating cancer. In another embodiment, inhibitors of ITK are described that are useful for treating autoimmune diseases. In some embodiments, the activators of ITK are small molecule compounds, polypeptides, or nucleic acids. In some embodiments, the inhibitors of ITK are small molecule compounds, polypeptides, or nucleic acids.

[0140] In another embodiment, a method of treating a disease in a subject in need thereof comprises administering to the subject an effective amount of an activator or inhibitor of ITK described herein.

[0141] In some embodiments, the disease is cancer or an autoimmune disease.

[0142] In some embodiments, the polypeptide modulates PD-1 in immune cells. Non-limiting examples of immune cells include T cells (e.g., Tregs), B cells, macrophages, and glial cells (e.g., astrocytes, microglia, or oligodendrocytes). In some embodiments, the immune cells are Tregs. In some embodiments, the polypeptide activates PD-1 signaling. In other embodiments, the polypeptide inhibits PD-1 signaling. The inventors have surprisingly found that in some embodiments, the polypeptide activates an immune response, while in other embodiments, the polypeptide suppresses an immune response.

[0143] In some embodiments, an activator of ITK can induce, promote, or enhance an immune response in a subject in need thereof. In another embodiment, an inhibitor of ITK can reduce, suppress, or prevent an immune response in a subject in need thereof. In some embodiments, an activator of ITK is a small molecule compound, a polypeptide, or a nucleic acid. In some embodiments, an inhibitor of ITK is a small molecule compound, a polypeptide, or a nucleic acid.

[0144] In some embodiments, small molecules that bind to the inhibitory site of PD-1 are also described. In some embodiments, small molecules that bind to the inhibitory site of PD-1 can reduce, suppress, or prevent an immune response in a subject in need thereof. In some embodiments, small molecules that bind to the inhibitory site of PD-1 can be useful in treating autoimmune diseases.

[0145] cancer In some embodiments, there is provided a method of treating or preventing cancer in a subject in need thereof, comprising modulating PD-1 signaling by administering to the subject an effective amount of a polypeptide described herein. In some embodiments, there is provided a method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject an effective amount of an activator of ITK.

[0146] In some embodiments, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, adult T-cell leukemia / lymphoma, and combinations thereof.

[0147] In some embodiments, the polypeptides and compositions disclosed herein are useful for treating leukemia. In some embodiments, the polypeptides and compositions disclosed herein that activate PD-1 are useful for treating leukemia. In these embodiments, the polypeptides and compositions disclosed herein that activate PD-1 are useful in vivo and ex vivo as immune response stimulating therapies. The ability to activate PD-1 allows for a more robust immune response. In some embodiments, the polypeptides and compositions disclosed herein are also useful for stimulating or enhancing immune stimulatory or immune activation responses involving T cells. In some embodiments, the polypeptides and compositions disclosed herein are useful for stimulating or enhancing immune responses in a host for treating leukemia by selectively activating PD-1. In these embodiments, the polypeptides and compositions disclosed herein can be administered to a subject in an amount effective to stimulate T cells in the subject. Types of leukemia that may be treated with the polypeptides and compositions disclosed herein include, but are not limited to, acute myeloid leukemia ("AML"), chronic myeloid leukemia ("CML"), acute lymphocytic leukemia ("ALL"), chronic lymphocytic leukemia ("CLL"), adult T-cell leukemia / lymphoma ("ATLL"), and chronic myelomonocytic leukemia ("CMML").

[0148] In some embodiments, ATLL is diagnosed almost exclusively in adults, with a median age in the mid-60s. In some embodiments, there are four types of ATLL: (1) acute, (2) chronic, (3) smoldering, and (4) lymphomatous. In some embodiments, acute ATLL is the most common type and is characterized by high white blood cell counts, hypercalcemia, visceromegaly, and high lactose dehydrogenase. In some embodiments, lymphomatous ATLL is expressed in lymph nodes and has less than 1% circulating lymphocytes. In some embodiments, chronic and smoldering ATLL are characterized by a less aggressive clinical course and are capable of long-term survival. In some embodiments, acute and lymphomatous ATLL have a 4-year survival rate of less than 5%. In some embodiments, chronic and smoldering ATLL have a 4-year survival rate of 26.9% and 62%, respectively. In some embodiments, adult T-cell leukemia / lymphoma is caused by HTLV-1.

[0149] In some embodiments, the polypeptides and compositions disclosed herein are useful for treating ATLL. In some embodiments, the polypeptides and compositions disclosed herein that activate PD-1 are useful for treating ATLL. In some embodiments, ATLL cells exhibit an activated helper / inducer T cell phenotype but exhibit strong immunosuppressive activity. In some embodiments, the polypeptides and compositions disclosed herein that activate PD-1 reduce the immunosuppressive response of ATLL cells. In another embodiment, the polypeptides and compositions disclosed herein that activate PD-1 increase the immunostimulatory response to overcome the strong immunosuppressive activity of ATLL cells.

[0150] autoimmune disease In some embodiments, there is provided a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising modulating PD-1 signaling by administering to the subject an effective amount of a polypeptide described herein. In some embodiments, there is provided a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of ITK.

[0151] Non-limiting examples of autoimmune diseases include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane disease, antitubular basement membrane antibody nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy, Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease, Celiac disease, Chagas' disease, Chronic inflammatory demyelinating polyneuropathy, Chronic relapsing multiple myelitis, Churg-Strauss syndrome, Eosinophilic granulomatosis, Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (Neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans' syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (Temporal arteritis) , giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational pemphigoid, hidradenitis suppurativa (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus, chronic Lyme disease Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, childhood autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, Polyendocrine syndrome type I, Polyendocrine syndrome type II, Polyendocrine syndrome type III, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progestational dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm and testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome syndrome), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and combinations of these.

[0152] Equivalent The representative examples that follow are intended to be illustrative of the present invention and are not intended to, and should not be construed to, limit the scope of the present invention. Indeed, in addition to those shown and described herein, various modifications of the present invention and many further embodiments thereof will become apparent to those skilled in the art from the entire contents of this specification, including the examples that follow and references to the scientific and patent literature cited herein. Furthermore, it is understood that the contents of these cited documents are incorporated herein by reference to describe the state of the art. The following examples contain important additional information, exemplification, and guidance that can be adapted to the practice of the present invention in its various embodiments and equivalents thereof. EXAMPLES

[0153] Example 1. Biological Assays Jurkat-PD-1-NFAT reporter cell line (BPS Bioscience) ("PD-1 reporter cells") were cultured according to the vendor's specifications. PD-1 reporter cells were plated at 20,000 cells / well in 384-well plates. Group 1 cells were untreated. Groups 2 and 3 cells were cultured according to the vendor's specifications. TM Dynabeads TM Activation was performed with Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (100:1 V / V cell:bead ratio). Prior to activation, cells from group 3 were preincubated for 15 min with a 5-fold excess of a polypeptide having the amino acid sequence of SEQ ID NO: 14. All cells were incubated at 37 °C for 15 min in 5% CO 2 , and incubated at 37°C for 24 hours.

[0154] After 24 h of culture, luciferase reagent (One-Step, Thermo Fisher Scientific) was added at a 1:1 V / V ratio and incubated for 15 min at room temperature. Luminescence was measured using a Varioscan Instrument (Thermo Fisher Scientific) at a 100 ms setting. The RLUs for the three groups of cells are shown in Figure 1.

[0155] Example 2. Polypeptide design Figure 2 and Figure 3 show the design of the polypeptides described herein. Figure 2A shows that PD-L2 has two interaction sites when binding to PD-1, one is an inhibitory site and the other is an activating site. Figure 2B shows that when a polypeptide blocks the activating site of PD-L2, PD-L2 only binds to PD-1 through its inhibitory site, thereby inducing an inhibitory signal from PD-1. Furthermore, as shown in Figure 2C, in a polypeptide that blocks the inhibitory site of PD-L2, PD-L2 only binds to PD-1 through its activating site, thereby inducing an activating signal from PD-1.

[0156] Figure 3 shows the design of PD-L2 mutants. As shown in Figure 3A, the PD-L2 mutant with the disabled activation site only bound to PD-1 through its inhibitory site. This binding to PD-1 could suppress immune responses through PD-1 signaling. Furthermore, as shown in Figure 3B, the PD-L2 mutant with the disabled inhibitory site only bound to PD-1 through its activation site, thus activating immune responses through PD-1 signaling.

[0157] Example 3. Regulation of ITK Figure 4 and Figure 5 show the regulation of ITK and its effect on immune responses. As shown in Figure 4, when SHP2 was more abundant than ITK, an inhibitory signal was generated. However, when ITK was more abundant than SHP2, a stimulatory signal was generated. When ITK and SHP2 were present in equal amounts, the immune response was either inhibitory or activating. Figure 5 shows that SHP2 is controlled by the upstream PD-1 pathway, and ITK is influenced by the upstream T cell receptor (TCR) pathway. Thus, activators of ITK may enhance immune responses, while inhibitors of ITK may have the effect of suppressing immune responses. Figure 6 (T cells, 48 ​​h activation, IP with Ab / ligand) further shows the levels of SHP2 and ITK bound to PD-1. Preactivated primary mouse CD4 T cells were treated with PD-L1-IgG, PD-L2-IgG, or anti-PD-1 mouse blocking antibody (clone RMP1-14) for 5 min before immunoprecipitation of PD-1 with beads. Western blot ("WB") assays were used to measure the levels of PD-1-bound ITK and SHP2 after different treatments, and showed that the ITK / SHP2 ratio increased after PD-L2 treatment and slightly decreased after PD-L1 treatment compared to the Ab control group.

[0158] Example 4. PD-L2 Increases Central Memory T Cells ("Tcm") and Prevents T Cell Exhaustion As shown in Figure 7A, flow cytometry sorted human CD4 T cells (BioIVT) were stimulated with Dyna beads (Gibco) (coated with anti-CD3 and anti-CD28 antibodies) and IL-2 (100 U / mL - R&D Systems) for 72 hours. After 72 hours, cells were treated with PD-L2-IgG (concentration 25 μg / mL). Control wells were left untreated. After 48 hours of treatment, cells were harvested, washed, stained with fluorescently labeled antibodies, and analyzed by flow cytometry. Tcm are CD45RO high / CD62L high / CD45RA low Exhausted T cells or terminal effector T cells ("Tte") are CD45RO low / CD62L low / CD45RA high As shown in Figures 7B-7C, PD-L2-IgG treatment resulted in a significant increase in Tcm (*p<0.05, Figure 7B) and a significant decrease in Tte (*p<0.05, Figure 7C).

[0159] Example 5. PD-L2 is required for the generation and prevention of exhaustion of Tcm As shown in Figure 8A, flow cytometry sorted human CD4 T cells (BioIVT) were stimulated with Dyna beads (Gibco) (coated with anti-CD3 and anti-CD28 antibodies) and IL-2 (100 U / mL - R&D Systems) for 24 hours. After 24 hours, the cells were either treated with anti-PD-L2 antibody (concentration 100 μg / mL) or left untreated. After 48 hours of treatment, the cells were harvested, washed, stained with fluorescently labeled antibodies, and analyzed by flow cytometry. Tcms are CD45RO high / CD62L high / CD45RA low Tte was defined as CD45RO low / CD62L low / CD45RA highAs shown in Figures 8B-8C, blocking the binding between PD-L2 and PD-2 with anti-PD-L2 antibody prevented the development of Tcm (****p<0.0001, Figure 8B), and significantly increased Tte (Figure 8C).

[0160] Example 6. PD-L2 is required to initiate activation of CD4 and CD8 T cells Flow cytometrically sorted human CD4 and CD8 T cells (BioIVT) were stimulated with Dyna beads (Gibco) (coated with anti-CD3 and anti-CD28 antibodies) and IL-2 (100 U / mL - R&D Systems) for 24 hours. After 24 hours, cells were treated with anti-PD-L2 antibody (concentration 100 μg / mL) or left untreated. After 48 hours of treatment, cells and supernatants were harvested. Supernatants were analyzed for IFNg and TNF using a CBA assay (BD Biosciences). Cells were washed, stained with fluorescently labeled antibodies, and analyzed by flow cytometry. CD25 expression on CD4 T cells, granzyme B and IFNg in CD8 T cells were used as indicators of activation and function. Anti-PD-L2 treatment reduced CD4 T cell activation (shown in Figure 9B) and inhibited CD4 T cell cytokine production as shown in Figure 9A. Similarly, PD-L2 blockade reduced CD25 expression on CD8 T cells (shown in Figure 9C) and reduced intracellular granzyme B and IFNg (shown in Figure 9D).

Claims

1. An isolated polypeptide comprising the following amino acid sequences (a) to (e): (a) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1; (b) the amino acid sequence of SEQ ID NO: 21; (c) the amino acid sequence of SEQ ID NO: 22; (d) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 11-13; optionally, wherein: (i) the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-13; (ii) the polypeptide binds to programmed cell death 1 ligand 2 (“PD-L2”); (iii) the polypeptide blocks the inhibitory site of PD-L2; and / or (iv) the polypeptide activates immune cells; or (e) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 14; optionally, wherein: (i) comprising the amino acid sequence of SEQ ID NO: 14; (ii) the polypeptide binds to PD-L2; (iii) the polypeptide blocks the activation site of PD-L2; and / or (iv) the polypeptide inhibits immune cells; Isolated polypeptide.

2. An isolated polypeptide described in claim 1, comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

1.

3. An isolated polypeptide described in claim 1, comprising the amino acid sequence of SEQ ID NO:

21.

4. An isolated polypeptide described in claim 1, comprising the amino acid sequence of SEQ ID NO:

22.

5. 2. The isolated polypeptide of claim 1, wherein the amino acid is fused to an immunoglobulin, optionally wherein the immunoglobulin is IgG1, IgG2, IgG3, or IgG4. (a) the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:4; optionally, wherein the polypeptide comprises an amino acid sequence that is SEQ ID NO:3 or SEQ ID NO:4; (b) the polypeptide comprises one or more amino acids each selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114; (c) the polypeptide binds to programmed cell death protein 1 (“PD-1”) and activates PD-1; and / or (d) the polypeptide activates immune cells; The isolated polypeptide of claim 1. (a) the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or SEQ ID NO:6; (b) comprises an amino acid sequence that is at least 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:7; optionally, wherein the polypeptide comprises an amino acid sequence that is SEQ ID NO:7; (c) the polypeptide comprises one or more amino acids each selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111; (d) the polypeptide binds to and inhibits PD-1; (e) the polypeptide suppresses immune cells; (f) the polypeptide induces central memory T cells (“Tcm”); and / or (g) the polypeptide prevents T cell exhaustion; The isolated polypeptide of claim 1.

8. A conjugate comprising the isolated polypeptide of any one of claims 1 to 6, wherein the polypeptide is conjugated to a detectable marker or carrier molecule; optionally, wherein the carrier molecule is selected from the group consisting of glycosaminoglycans, proteoglycans, albumin, and polyalkylene glycols. Conjugates.

9. A conjugate comprising the isolated polypeptide of claim 7, wherein the polypeptide is conjugated to a detectable marker or carrier molecule; optionally, wherein the carrier molecule is selected from the group consisting of glycosaminoglycans, proteoglycans, albumin, and polyalkylene glycols. Conjugates.

10. A nucleic acid encoding the isolated polypeptide of any one of claims 1 to 5.

11. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 5; optionally comprising: (a) the polypeptide is encapsulated in a liposome; (b) further comprising a second therapeutic agent; optionally, wherein the second therapeutic agent is a chemotherapeutic agent or an immunosuppressant; Pharmaceutical compositions.

12. A pharmaceutical composition for inducing, promoting, or enhancing an immune response or treating cancer or reducing tumor burden, wherein the pharmaceutical composition comprises: (a) a polypeptide according to any one of claims 1, 2, 3, 5 and 6; and (b) activators of inducible T-cell kinase (“ITK”) optionally, wherein the activator of ITK is a small molecule compound, a polypeptide, or a nucleic acid; (c) optionally for use in a method comprising upregulating ITK; (d) optionally, the cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, and testicular cancer; Pharmaceutical compositions.

13. A pharmaceutical composition for inducing, promoting, or enhancing an immune response or treating cancer or reducing tumor burden, wherein the pharmaceutical composition comprises: (a) the conjugate of claim 8; or (b) the pharmaceutical composition of claim 10; and (c) activators of inducible T-cell kinase (“ITK”) optionally, wherein the activator of ITK is a small molecule compound, a polypeptide, or a nucleic acid; (d) optionally for use in a method comprising upregulating ITK; (e) optionally, the cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, and testicular cancer; Pharmaceutical compositions.

14. A pharmaceutical composition for inducing, promoting, or enhancing an immune response or treating cancer or reducing tumor burden, wherein the pharmaceutical composition comprises: (a) the conjugate of claim 9; and (b) activators of inducible T-cell kinase (“ITK”) optionally, wherein the activator of ITK is a small molecule compound, a polypeptide, or a nucleic acid; (c) optionally for use in a method comprising upregulating ITK; (d) optionally, the cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, and testicular cancer; Pharmaceutical compositions.

15. A pharmaceutical composition for reducing, suppressing, or preventing an immune response or treating an autoimmune disease, wherein the pharmaceutical composition comprises: (a) a polypeptide according to any one of claims 1, 2, 4, 5 and 7; and (b) an inhibitor of ITK, optionally wherein the inhibitor of ITK is a small molecule compound, a polypeptide, or a nucleic acid; or (c) Small molecule compounds that bind to the inhibitory site of PD-1 Including; (d) optionally for use in a method of downregulating ITK; (e) Optionally, the autoimmune disease is selected from the group consisting of achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane disease, antitubular basement membrane antibody nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neural neuropathy, Barrow's disease, Behçet's disease, benign mucous membrane pemphigoid, and bullous pemphigoid. , Castleman's disease, Celiac disease, Chagas' disease, Chronic inflammatory demyelinating polyneuropathy, Chronic relapsing multiple myelitis, Churg-Strauss syndrome, Eosinophilic granulomatosis, Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, Crest syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (Neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans' syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis) ), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational pemphigoid, hidradenitis suppurativa (acne suppurativa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus, chronic Lyme disease Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Much-Habermann's disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, pediatric autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, polyendocrine syndrome type I, polyendocrine syndrome type II, polyendocrine syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome syndrome), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and combinations thereof; Pharmaceutical compositions.

16. A pharmaceutical composition for reducing, suppressing, or preventing an immune response or treating an autoimmune disease, wherein the pharmaceutical composition comprises: (a) the conjugate of claim 8; or (b) the pharmaceutical composition of claim 10; and (c) an inhibitor of ITK, optionally wherein the inhibitor of ITK is a small molecule compound, a polypeptide, or a nucleic acid; or (d) Small molecule compounds that bind to the inhibitory site of PD-1 Including; (e) optionally for use in a method of downregulating ITK; (f) Optionally, the autoimmune disease is achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane disease, antitubular basement membrane antibody nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neural neuropathy, Barrow's disease, Behçet's disease, benign mucous membrane pemphigoid, or bullous pemphigoid. , Castleman's disease, Celiac disease, Chagas' disease, Chronic inflammatory demyelinating polyneuropathy, Chronic relapsing multiple myelitis, Churg-Strauss syndrome, Eosinophilic granulomatosis, Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, Crest syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (Neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans' syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis) ), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational pemphigoid, hidradenitis suppurativa (acne suppurativa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus, chronic Lyme disease Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Much-Habermann's disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, pediatric autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, polyendocrine syndrome type I, polyendocrine syndrome type II, polyendocrine syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome syndrome), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and combinations thereof; Pharmaceutical compositions.

17. A pharmaceutical composition for reducing, suppressing, or preventing an immune response or treating an autoimmune disease, wherein the pharmaceutical composition comprises: (a) the conjugate of claim 9; and (b) an inhibitor of ITK, optionally wherein the inhibitor of ITK is a small molecule compound, a polypeptide, or a nucleic acid; or (c) Small molecule compounds that bind to the inhibitory site of PD-1 Including; (d) optionally for use in a method of downregulating ITK; (e) Optionally, the autoimmune disease is selected from the group consisting of achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, antiglomerular basement membrane disease, antitubular basement membrane antibody nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neural neuropathy, Barrow's disease, Behçet's disease, benign mucous membrane pemphigoid, and bullous pemphigoid. , Castleman's disease, Celiac disease, Chagas' disease, Chronic inflammatory demyelinating polyneuropathy, Chronic relapsing multiple myelitis, Churg-Strauss syndrome, Eosinophilic granulomatosis, Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, Crest syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (Neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans' syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis) ), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational pemphigoid, hidradenitis suppurativa (acne suppurativa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus, chronic Lyme disease Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Much-Habermann's disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, pediatric autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, polyendocrine syndrome type I, polyendocrine syndrome type II, polyendocrine syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome syndrome), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and combinations thereof; Pharmaceutical compositions.

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