Novel PD-L2 variants

Abstract

An isolated polypeptide is described that is a variant of programmed cell death 1 ligand 2 ("PD-L2") and can activate or suppress immune cells through the activation or suppression of programmed cell death protein 1 ("PD-1"). A pharmaceutical composition comprising the polypeptide and a method of using the polypeptide are also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application 63 / 471,298, filed June 6, 2023, which is incorporated herein by reference in its entirety.

[0002] Embedding by citation Any patents, patent publications, journals, or other documents cited herein are incorporated herein by express citation in their entirety.

[0003] Field of Invention This invention generally relates to polypeptide modulators and immunomodulation. More specifically, the invention relates to ligands that specifically bind to or modulate programmed cell death protein 1 ("PD-1"), and methods of using the same. [Background technology]

[0004] Background of the Invention PD-1 is a protein on the surface of T and B cells that plays a role in regulating the immune system's response to cells in the human body and promoting autoimmune tolerance through T cell inflammatory activity by downregulating the immune system. This biological pathway prevents autoimmune diseases, but 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, suppressing the immune response. The PD-1 / PD-L1 pathway has shown promising clinical success as a target for cancer immunotherapy. Current antibodies targeting PD-1 or PD-L1 can block this interaction and boost the immune response against cancer cells. Successful clinical trials with PD-1 monoclonal antibodies and other immune checkpoint inhibitors have opened new avenues in cancer immunology. However, the failure of a large group of cancer patients to respond to novel immunotherapies has led to increased research into combination therapies and predictive biomarkers. See, for example, Iwai, Y, et al., Journal of Biomedical Science, 24:26 (2017), which is included in its entirety here by citation.

[0006] In addition to PD-L1, programmed cell death ligand 1-2 ("PD-L2") is another protein known to bind to PD-1. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, the object of one or more inventions disclosed herein is to provide compositions and methods for modulating PD-1 signaling and related biological pathways. [Means for solving the problem]

[0008] Summary of the Invention In one embodiment, an isolated polypeptide that binds to PD-1 is described. In one embodiment, the polypeptide can modulate an immune response in vitro or in vivo and / or can be used individually or in combination with other agents for the prevention or treatment of a variety of conditions. In another embodiment, the isolated polypeptide is a variant of PD-L2 in which the inhibitory or activating site is inactive. In yet another embodiment, pharmaceutical compositions containing the polypeptide and methods of using these compositions individually or in combination with other agents or compositions for the prevention or treatment of a variety of conditions are described.

[0009] In one embodiment, an isolated polypeptide is described that contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2.

[0010] In any of the embodiments disclosed herein, the polypeptide is a variant of programmed cell death ligand 1-2 ("PD-L2").

[0011] In any of the embodiments disclosed herein, an amino acid is fused with an immunoglobulin.

[0012] In any of the embodiments disclosed herein, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

[0013] In any 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0014] In any 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: 11 or SEQ ID NO: 12.

[0015] In any of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0016] In any of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.

[0017] In any of the embodiments disclosed herein, the polypeptide binds to programmed cell death protein 1 ("PD-1") and activates PD-1.

[0018] In any of the embodiments disclosed herein, the polypeptide activates immune cells.

[0019] In any 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0020] In any 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: 11.

[0021] In any of the embodiments disclosed herein, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0022] In any of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO: 11.

[0023] In any of the embodiments disclosed herein, the polypeptide binds to PD-1 and inhibits cells via PD-1.

[0024] In any of the embodiments disclosed herein, the polypeptide suppresses immune cells.

[0025] In any of the embodiments disclosed herein, the polypeptide induces central memory T cells ("Tcm").

[0026] In any of the embodiments disclosed herein, the polypeptide prevents T cell exhaustion.

[0027] In any 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: 12 or SEQ ID NO: 13.

[0028] In any 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: 12.

[0029] In any of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

[0030] In any of the embodiments disclosed herein, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

[0031] In yet another embodiment, a conjugate is described comprising a polypeptide of any of the embodiments disclosed herein and a detectable marker or carrier molecule, wherein the polypeptide is bound to the detectable marker or carrier molecule.

[0032] In any of the embodiments disclosed herein, the carrier molecule is selected from the group consisting of glycosaminoglycans, proteoglycans, albumin, and polyalkylene glycols.

[0033] In yet another embodiment, nucleic acids encoding any of the polypeptides of the embodiments disclosed herein are described.

[0034] In yet another embodiment, a pharmaceutical composition comprising any polypeptide of the embodiments disclosed herein is described.

[0035] In any of the embodiments disclosed herein, the polypeptide is encapsulated in a liposome.

[0036] In any of the embodiments disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent.

[0037] In any of the embodiments disclosed herein, the second therapeutic agent is a chemotherapeutic agent or an immunomodulator (e.g., an immunosuppressant or an immunoactivator).

[0038] In yet another embodiment, a method is described for inducing, promoting or enhancing an immune response in a subject requiring treatment, the method comprising administering to the subject an effective amount of any polypeptide of any embodiment disclosed herein, any conjugate of any embodiment disclosed herein, or any pharmaceutical composition of any embodiment disclosed herein.

[0039] In yet another embodiment, a method is provided for treating cancer or reducing tumor burden in a subject requiring treatment, the method comprising administering to the subject an effective amount of any polypeptide of any embodiment disclosed herein, any conjugate of any embodiment disclosed herein, or any pharmaceutical composition of any embodiment disclosed herein.

[0040] In any of the embodiments disclosed herein, cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder, brain, breast, cervix, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterus, ovarian, and testicular cancers.

[0041] In any of the embodiments disclosed herein, the method further includes upregulation of inducible T cell kinase ("ITK").

[0042] In yet another embodiment, a method for reducing, suppressing or preventing an immune response in a subject requiring treatment is described, the method for administering to the subject an effective amount of any polypeptide of any embodiment disclosed herein, any conjugate of any embodiment disclosed herein, or any pharmaceutical composition of any embodiment disclosed herein.

[0043] In yet another embodiment, a method for treating an autoimmune disease in a subject requiring treatment is described, comprising administering to the subject an effective amount of any polypeptide of any embodiment disclosed herein, any conjugate of any embodiment disclosed herein, or any pharmaceutical composition of any embodiment disclosed herein.

[0044] In any of the embodiments disclosed herein, the autoimmune disease is achalasia, Addison's disease, Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-tubular basement membrane antibody nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic dysphatitis, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neurogenic neuropathy, Barlow's disease, Behçet's disease, benign visceral dysplasia Membranous pemphigoid, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing polymyelitis, Churg-Strauss syndrome, eosinophilic granulomatosis, scarring pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, Crest syndrome, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosis Alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, bullous pemphigoid of pregnancy, hidradenitis suppurativa (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immunothrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, sclerosing atrophic lichen, woody lumps Euveitis, linear IgA disease, lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mohlen's ulcer, Mukka-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, childhood autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, ciliary uveitis (peripheral uveitis), Personage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis,Pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia syndrome type I, polymyalgia syndrome type II, polymyalgia syndrome type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone 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 The following conditions are selected from the group consisting of machistomas, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Suzac syndrome, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease, and combinations thereof.

[0045] In any of the embodiments disclosed herein, the method further includes downward control of the ITK.

[0046] In any of the embodiments disclosed herein, the polypeptide is used for ex vivo treatment of T cells for adoptive transfer of engineered T cells, CAR-T cells, and / or TCT-T cells.

[0047] Any embodiment disclosed herein may be combined with any other embodiment disclosed herein. Combinations of any embodiment disclosed herein with any other embodiment disclosed herein are expressly intended. Specifically, a selection of one or more embodiments of a given substituent may be appropriately combined with a selection of one or more specific embodiments of any other substituent. Such combinations may be made in any one or more embodiments of the applications described herein or in any formula described herein. [Brief explanation of the drawing]

[0048] [Figure 1] Figures 1A and 1B illustrate the design principles of PD-L2 mutants according to one or more embodiments described herein.

[0049] [Figure 2] Figures 2A-2C show that PD-L2 increases Tcm and prevents T cell exhaustion in one or more embodiments described herein.

[0050] [Figure 3] Figures 3A to 3C show that PD-L2 is necessary for Tcm generation and fatigue prevention in one or more embodiments described herein.

[0051] [Figure 4] Figures 4A to 4D show that PD-L2 is required to initiate the activation of both CD4 and CD8 T cells in one or more embodiments described herein.

[0052]

[0053] Figures 5A to 5C show the binding of PD-L2 mutants to PD-1 by flow cytometry assays in one or more embodiments described herein.

[0054] [Figure 6] Figures 6A-6B show the functionality of PD-L2 mutants as measured by luminescence assays in one or more embodiments described herein.

[0055] [Figure 7] Figures 7A-7B show the functionality of PD-L2 mutants as measured by IFNg production assays in one or more embodiments described herein.

[0056] [Figure 8] Figures 8A to 8C show the in vivo effects of PD-L2 variants on human T cell activation according to one or more embodiments described herein.

[0057] [Figure 9]Figures 9A-9B show the in vivo effects of PD-L2 variants on human T cell activation according to one or more embodiments described herein. [Modes for carrying out the invention]

[0058] Detailed description of the invention definition It should be recognized that the present invention is not limited to the compositions, methods, and experimental conditions described herein, as they may vary. It should also be understood that the terms used herein are for the purpose of describing certain embodiments only and are not intended to be limiting.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly recognized by those skilled in the art to which this invention pertains. Any composition, method, and substance similar or equivalent to those described herein may be used in carrying out or testing this invention.

[0060] In the context of the claimed invention (particularly in the context of the claims), singular expressions and similar references should be interpreted as covering both singular and plural, unless otherwise specifically stated or unless it is clearly contrary to the context.

[0061] The range of values ​​described herein is intended to serve as a shortened method for listing each individual value that falls within that range independently, unless otherwise specified, and each individual value is incorporated herein as if it were listed individually.

[0062] The use of the term "approximately" is intended to indicate values ​​within a range of approximately ±10% above or below the stated value. In some embodiments, the value may be within a range of approximately ±5% above or below the stated value. In some embodiments, the value may be within a range of approximately ±2% above or below the stated value. In some embodiments, the value may be within a range of approximately ±1% above or below the stated value. The aforementioned ranges are intended to be evident from the context and do not imply any further limitation. All methods described herein can be performed in any suitable order unless otherwise clearly indicated herein or otherwise clearly contradicted by the context. Any and all examples or illustrative terms provided herein (e.g., "exemplary," "etc.," "for example," "including but not limited to") are merely intended to better illustrate the invention and do not impose limitations on the scope of the invention unless otherwise indicated.

[0063] As used herein, the terms “cancer” and, equally, “tumor” refer to a condition in which abnormally replicating cells of host origin are present in a detectable amount in a subject. Cancer may be malignant or non-malignant. Cancer or tumors include, but are not limited to, adult T-cell leukemia / lymphoma (including those caused by human T-cell lymphophosphorus virus ("HTLV-1")), cholangiocarcinoma; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; stomach cancer; neoplasm in situ; 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 cancer of the lymphatic system or blood cancer that originates from lymphocytes. Cancer may be primary or metastatic. Non-cancerous diseases may be associated with mutational alterations of components of the Ras signaling pathway, and the polypeptides disclosed herein may be used to treat these non-cancerous diseases. Such non-cancerous diseases include, but are not limited to, neurofibromatosis; Leopard syndrome; Noonan syndrome; Regius syndrome; Costello syndrome; cardiac-facial-cutaneous syndromes; hereditary gingival fibromatosis type 1; autoimmune lymphoproliferative syndromes; and capillary malformations-arteriovenous malformations.

[0064] As used herein, "effective dose" refers to any quantity necessary or sufficient to achieve or promote a desired outcome. In some cases, the effective dose is the therapeutic effective dose. The therapeutic effective dose is any quantity necessary or sufficient to achieve or promote a desired biological response in a subject. The effective dose for any particular application may vary depending on factors such as the disease or condition being treated, the specific drug being administered, the size of the subject, or the severity of the disease or condition. A person skilled in the art can empirically determine the effective dose of a particular drug without requiring excessive experimentation.

[0065] The term "subject" as used herein refers to vertebrates. In some embodiments, the subject is a mammal or a 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, livestock, racehorses, domesticated animals, and undomesticated animals.

[0066] 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, and oligodendrocytes), monocytes, macrophages, dendritic cells, and lymphocytes (e.g., B cells, T cells, and natural killer ("NK") cells).

[0067] The “conventional T cells” used herein are T lymphocytes that express the αβ T cell receptor ("TCR") and co-receptors such as CD4 or CD8. Conventional T cells are found in peripheral blood, lymph nodes, and tissues. See, for example, Roberts and Girardi, “Conventional and Unconventional T Cells”, Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari and Tyring (ed.)), Springer London (2008), which is included here by whole word. The “unconventional T cells” used herein are lymphocytes that express the γδ TCR and can commonly be found in epithelial environments such as the skin, gastrointestinal tract, or urogenital tract. Another subset of unconventional T cells is invariant natural killer T ("NKT") cells, which possess the phenotype and functional capabilities of conventional T cells as well as the characteristics of natural killer cells (e.g., cytolytic activity). See, for example, ibid. The regulatory T cells ("Treg") used herein refer to a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, suppress autoimmune diseases, and otherwise suppress the immunostimulatory or activating responses of other cells. Tregs can take many forms, the most well understood being those expressing CD4, CD25, and Foxp3. The "native Treg" or "nTreg" used herein refers to Tregs or cells that develop in the thymus. The "inducible Treg" or "iTreg" used herein refers to Tregs or cells that develop outside the thymus from mature CD4+ conventional T cells.

[0068] As used herein, the terms "peptide" and "polypeptide" refer to amino acid chains of any length, regardless of modifications (e.g., phosphorylation or glucosylation). The terms "peptide" and "polypeptide" are used interchangeably. These terms include proteins and their fragments. Polypeptides may be "exogenous," meaning they are "heterogeneous," i.e., foreign to the host cell from which they are utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right, in the direction from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named using the following three-letter or one-letter abbreviations: 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).

[0069] The term “immune response” includes, for example, the development of beneficial humoral (antibody-mediated) and / or cellular (antigen-specific T cell-mediated or their secretion-mediated) responses to polypeptides in a recipient patient. Such responses may be active responses induced by immunogen administration or passive responses induced by antibody or primed T cell administration. Cellular immune responses are induced by the presentation of polypeptide epitopes that bind to class I or class II major histocompatibility complex ("MHC") molecules to activate antigen-specific CD4+ T helper cells and / or CD8+ cytotoxic T cells. Responses may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophil activation or recruitment, or other components of innate immunity. The presence of a cell-mediated immunological response may 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 immunized allograft animals and measuring their protective or therapeutic effects in a second target.

[0070] The term "immunogenic factor" or "immunogen" refers to a factor that, when administered to a mammal, preferably with an adjuvant, can induce an immunological response against its own body.

[0071] polypeptide In addition to PD-L1, PD-L2 is another protein that binds to PD-1. The applicant has surprisingly discovered that when PD-L2 binds to PD-1 via two main interaction sites on PD-L2, these sites have opposite functions: one site on PD-L2 activates PD-1 and the immune response, while the other inhibits PD-1 and the immune response. The site that activates PD-1 is referred to here as the "activating site," and the site that inhibits PD-1 is referred to here as the "inhibitory site." The two interaction sites of PD-L2 are shown in Figures 1A and 1B. In one embodiment, the activating site on PD-L2 contains one or more amino acids selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114. In one embodiment, the inhibitory site on PD-L2 contains one or more amino acids selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111.

[0072] In one embodiment, a polypeptide is described that blocks or inactivates the inhibitory site of PD-L2, which activates PD-1 and the immune response. In another embodiment, a polypeptide is described that blocks or inactivates the activating site of PD-L2, which inhibits PD-1 and suppresses the immune response. For example, as illustrated in Figure 1B, a PD-L2 mutant with an inactivated inhibitory site was able to activate the immune response via PD-1 signaling. Furthermore, as illustrated in Figure 1A, a PD-L2 mutant with an inactivated activating site was able to suppress the immune response via PD-1 signaling.

[0073] Sequence ID 1 is the amino acid sequence that defines human PD-L2: LFTVTVPKELYIIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHP.

[0074] Sequence ID 21 is the amino acid sequence that defines the activation site of human PD-L2: QXIXIXXXXWXYXY [Here, X is any amino acid.]

[0075] Sequence ID 22 is an amino acid sequence that defines the inhibitory site of human PD-L2: IXGVAWD [Here, X is any amino acid.]

[0076] Sequence ID 2 is an amino acid sequence that defines a variant of human PD-L2: LFTVTVPKELYIIIEHGSNVTLECNFDTGSHVNLGAITASLQKVEX1DTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCX2IX3YX4X5X6WDYKYLT LKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHP [Here, X1 is any amino acid; X2 is any amino acid; X3 is any amino acid; X4 is any amino acid; X5 is any amino acid; and X6 is any amino acid.

[0077] In one 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: 1; and the amino acid sequence is fused with an immunoglobulin or a fragment thereof. In one embodiment, the immunoglobulin is IgG, IgM, or IgA. In one embodiment, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the immunoglobulin is human immunoglobulin. In one embodiment, the isolated polypeptide has an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 described. In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, the isolated polypeptide is a variant of human PD-L2.

[0078] Sequence ID 3 is the amino acid sequence that defines the first variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYNVMWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0079] Sequence ID 4 is the amino acid sequence that defines the second variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVEDDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYNVMWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0080] Sequence ID 5 is the amino acid sequence that defines the third variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYFVHWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0081] Sequence ID 6 is the amino acid sequence that defines the fourth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIIEHGSNVTLECNFDTGSHVNLGAITASLQKVESDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYFVHWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0082] Sequence ID 7 is the amino acid sequence that defines the fifth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVESDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYNRMWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0083] Sequence ID 8 is the amino acid sequence that defines the sixth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVESDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0084] Sequence ID 9 is the amino acid sequence that defines the seventh variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVEDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0085] Sequence ID No. 10 is the amino acid sequence that defines the eighth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIMYGTAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0086] Sequence ID 11 is the amino acid sequence that defines the ninth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIMYGTAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0087] In one embodiment, the polypeptide is a variant of PD-L2 in which the inhibitory site is inactive. In one embodiment, the polypeptide contains 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In one embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0088] In one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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. In one embodiment, the polypeptide contains 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: 8. In one embodiment, the polypeptide contains 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: 9. In one embodiment, the polypeptide contains 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: 10.In one embodiment, the polypeptide contains 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: 11. In one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In one embodiment, the polypeptide contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In one embodiment, the polypeptide contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 9. In one embodiment, the polypeptide contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 3. In one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 4. In one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 5.In one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 6. In another embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 7. In another embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 8. In another embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 9. In another embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 10. In another embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 11. In another embodiment, the polypeptide contains one or more amino acids selected from the group consisting of Tyr 112, Trp 110, Ile 103, Ile 105, Gln 101, and Tyr 114.

[0089] In one embodiment, the polypeptide binds to PD-1. In one embodiment, the polypeptide binds to PD-1 only via a binding site similar to the activating site of PD-L2. In one embodiment, the polypeptide does not have a binding site similar to the inhibitory site of PD-L2. In one embodiment, the polypeptide activates PD-1. In one embodiment, the polypeptide activates immune cells via activated 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 one embodiment, the polypeptide can be used to induce, promote, or enhance an immune response. In one embodiment, the polypeptide can be used to treat cancer.

[0090] Sequence ID 12 is the amino acid sequence that defines the tenth variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCVIIYGVAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0091] Sequence ID 13 is the amino acid sequence that defines the eleventh variant of human PD-L2: MGWSCIILFLVATATGVHSLFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIVYGVAWDYK YLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPIEGRMDD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0092] In one embodiment, the polypeptide is a variant of PD-L2 in which the activating site is inactive. In one embodiment, the polypeptide contains 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: 12 or SEQ ID NO: 13. In one embodiment, the polypeptide contains an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 12 or SEQ ID NO: 13. In one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

[0093] In one embodiment, the polypeptide contains 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: 12. In one embodiment, the polypeptide contains 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: 13. In one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains 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 one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 12. In one embodiment, the polypeptide contains the amino acid sequence of SEQ ID NO: 13. In one embodiment, the polypeptide contains one or more amino acids selected from the group consisting of Ile 105, Val 108, Gly 107, Ala 109, Trp 110, and Asp 111.

[0094] In any one or more embodiments described herein, the polypeptide is a variant of programmed cell death 1 ligand 2 ("PD-L2"). In some embodiments, the polypeptide binds to PD-1. In some embodiments, the polypeptide binds to PD-1 only via 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 cells via PD-1. In some embodiments, the polypeptide suppresses immune cells via inhibition of 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 the immune response. In some embodiments, the polypeptide can be used to treat autoimmune diseases.

[0095] In one embodiment, the polypeptide can be modified in a manner known to those skilled in the art. For example, the polypeptide can be used as a peptide mimetic. The polypeptide can form dimers.

[0096] In some embodiments, polypeptides can be pegylated. Pegylation can slow the removal of polypeptides from the circulation through various mechanisms. In some embodiments, pegylation slows degradation by proteolytic enzymes and reduces the rate of renal filtration by increasing the apparent molecular size. Therefore, polyethylene glycol ("PEG")-based modifications may be useful for enhancing the circulation time and bioavailability of polypeptides. In some embodiments, polypeptides are pegylated with linear PEG molecules. In other embodiments, polypeptides are pegylated with branched PEG molecules. The present invention further provides amino-, carboxy-, and side-chain-pegylated polypeptides. The PEG moiety may be PEG molecules with a molecular weight greater than 5 kDa. For example, the molecular weight can be 5kDa to 100kDa (e.g., 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 50kDa, 55kDa, 60kDa, 65kDa, 70kDa, 75kDa, 80kDa, 85kDa, 90kDa, 95kDa, or 100kDa), and more preferably The molecular weight is between 10kDa and 50kDa (for example, 10kDa, 12kDa, 14kDa, 16kDa, 18kDa, 20kDa, 22kDa, 24kDa, 26kDa, 28kDa, 30kDa, 32kDa, 34kDa, 36kDa, 38kDa, 40kDa, 42kDa, 44kDa, 46kDa, 48kDa, or 50kDa). The synthesis method for pegylated polypeptides is well known in this field.

[0097] In one embodiment, a polypeptide is bound to a detectable marker. In another embodiment, the detectable marker is bound to the C-terminus of the polypeptide. In yet another embodiment, the detectable label is bound to the N-terminus. The detectable marker may be a chemical label, but not limited to, a radioisotope, a fluorescent group, a chemiluminescent label, a colorimetric label, an enzyme marker, and an affinity moiety (e.g., biotin), which facilitates the detection of the labeled polypeptide. The present invention also provides dye-labeled polypeptides, but not limited to, fluorescein and rhodamine conjugates. Other chemical labels and methods for binding chemical labels to polypeptides are well known in the art.

[0098] In one embodiment, a polypeptide is conjugated to a carrier molecule. The polypeptide may also be used as a conjugate of at least one polypeptide or polypeptide fragment conjugated to the carrier. The carrier may provide a solid support for the polypeptide of the present invention. The carrier may be a biological carrier, such as, but not limited to, glycosaminoglycans, proteoglycans, or albumin, or a synthetic polymer, such as, but not limited to, polyalkylene glycol or synthetic chromatography supports. Other carriers include, but are not limited to, ovalbumin and human serum albumin, other proteins, and polyethylene glycol.

[0099] In one embodiment, the nucleic acid encoding the polypeptide is described. In another embodiment, the polypeptide can be prepared using recombinant DNA technology, where the expression vector comprises a nucleic acid sequence encoding the polypeptide of the present invention, the nucleic acid sequence being operably ligated to a promoter. The expression vector is delivered by means of, for example, transformation, transfection, and a host cell that enables the expression of the appropriate polypeptide, including but not limited to these. 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 human cells. In another embodiment, the host cell is a bacterial, fungal, or insect cell. In one embodiment, the polypeptide is recovered from the culture, where the recovery may include a step leading to the purification of the polypeptide. Preparation of the polypeptide by recombinant technology is advantageous if the polypeptide can be post-translationally modified. Furthermore, amide and ester derivatives of the polypeptide can be produced using a combination of synthetic and recombinant DNA technology, and fragments of the desired polypeptide can be produced and then assembled in a manner well known to those skilled in the art.

[0100] Expression vectors suitable for nucleic acid sequence delivery and polypeptide expression in human cells are known in this field. Non-limiting examples include plasmids, viruses, or bacterial vectors.

[0101] Polypeptides can also be commercially prepared by companies that offer polypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pennsylvania; AnaSpec, Inc., San Jose, California). Automated polypeptide synthesis machines, such as those manufactured by Perkin-Elmer Applied Biosystems, are also available.

[0102] Polypeptides useful by the methods described herein are purified after being isolated or synthesized by chemical or recombinant techniques. 4-, C2- or C 18 Standard methods for purification may be used, including reversed-phase high-performance liquid chromatography ("HPLC") using alkylated silica columns, such as, but not limited to, silica. In this method, a gradient mobile phase that increases the organic content is commonly used to achieve purification (e.g., acetonitrile in aqueous buffer, optionally containing a small amount of acid, e.g., trifluoroacetic acid). Alternatively, polypeptide compounds can be separated based on charge using ion exchange chromatography. The purity of the polypeptide compound can be diagnosed by the number of peaks identified by HPLC. In one embodiment, a useful level of polypeptide purity results in one peak in the HPLC chromatogram. In one embodiment, the target polypeptide is at least 94.99% of the feed into the HPLC column. In another embodiment, the target polypeptide is at least 96.99% of the feed into the HPLC column. In one embodiment, the target polypeptide is 97% to 99.5% of the feed into the HPLC column.

[0103] In one embodiment, a method for determining whether a polypeptide exhibits an effect on the immune response is a luciferase assay. In one embodiment, Jurkat PD-1 cells are used in the luciferase assay.

[0104] Pharmaceutical composition In one embodiment, a pharmaceutical composition comprising one or more polypeptides disclosed herein is described. In one embodiment, the polypeptides are encapsulated in liposomes in the pharmaceutical composition.

[0105] In some embodiments, carriers may be used in pharmaceutical compositions, comprising, but not limited to, ion exchangers, alumina, aluminum stearate, lecithin, non-albumin serum proteins, buffers (e.g., phosphoric acid, 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, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid, wax, polyethylene-polyoxypropylene block polymer, and wool fat). Such modifications can result in both increased apparent affinity and altered polypeptide stability. The number of polypeptide fragments that bind to each carrier may vary, but typically about 4 to 8 polypeptide fragments per carrier molecule can bind under standard coupling conditions.

[0106] In some embodiments, polypeptides can be prepared and stored in salt form. Various salt forms of polypeptides can be formed or exchanged by any of the various methods known in the art, for example, by the use of various ion exchange chromatography methods. Cationic counterions that can be used in compositions include, but are not limited to, amines (e.g., ammonium ions); monovalent, divalent, or trivalent ions of metal ions, particularly 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 can be used in compositions described herein include, but are not limited to, chlorides, fluorides, acetates, trifluoroacetates, phosphates, sulfates, carbonates, citrates, ascorbetes, sorbates, glutarates, ketoglutarates, and possible combinations thereof. The trifluoroacetate salts of the polypeptide compounds described herein are typically formed during purification in trifluoroacetate buffer using HPLC. While not always suitable for in vivo applications, the trifluoroacetate forms of the polypeptides described herein can be conveniently used in various in vitro cell culture tests, assays, or tests of the activity or efficacy of the target polypeptide compound. The polypeptides can then be converted from trifluoroacetate to a more acceptable salt form for pharmaceutical or nutritional supplement compositions by ion exchange or synthesis.

[0107] In some embodiments, pharmaceutical compositions can be delivered by a variety of routes or in various forms. These include, but are not limited to, non-enteral, oral, intratracheal, sublingual, pulmonary, topical, rectal, nasal, buccal, sublingual, vaginal, or via implanted reservoirs. Implanted reservoirs may function mechanically, osmotically, or otherwise. As used herein, “non-enteral” includes, but is not limited to, intravenous, intracranial, intraperitoneal, paravertebral, periarticular, periosteal, subcutaneous, intradermal, intraarterial, intramuscular, intraarticular, synovial, intrasternal, intrathecal, and intrafocal injection or infusion techniques. Such compositions are formulated for non-enteral administration, and preferably for intravenous, intracranial, or intraarterial administration. Generally, when administration is intravenous or intraarterial, pharmaceutical compositions may be given as a bolus, in divided doses, or in small amounts.

[0108] In one embodiment, the pharmaceutical composition may be in a form for sterile injection, for example, as an aqueous suspension for sterile injection. This suspension may be formulated by art known techniques using a suitable dispersant or suspending agent. The sterile injection form may also be a sterile injection solution or suspension in a non-toxic, non-enterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable solvents that may be used include, but are not limited to, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, fixative oils can be conveniently used as solvents or suspension media. For this purpose, any non-irritating fixative oil, including, but not limited to, synthetic mono or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the formulation of injections, as are naturally pharmaceutically acceptable oils such as olive oil or castor oil.

[0109] In some embodiments, the pharmaceutical composition may be administered orally in the form of capsules, tablets, caplets, pills, aqueous suspensions and solutions, or syrups. For tablets for oral use, carriers may be used, including but not limited to lactose and corn starch. Lubricants, including but not limited to magnesium stearate, may also be added. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. Capsules, tablets, pills, and caplets may be formulated for delayed or sustained release when long-term action is required. Alternatively, when administering an oral aqueous suspension, the polypeptide is advantageously mixed with an emulsifier and / or suspending agent. Certain sweeteners and / or flavorings and / or colorings may be added as desired. In some embodiments, the oral formulation provides the polypeptide described herein in a mixture that prevents or inhibits hydrolysis of the polypeptide compound by the digestive system, thereby enabling absorption into the bloodstream.

[0110] In some embodiments, the pharmaceutical composition may be administered to a mucous membrane (e.g., vagina or rectum). These dosage forms may be prepared by mixing the polypeptides described herein with a suitable non-irritating additive that is solid at room temperature but liquid at body temperature, and therefore changes state to a liquid form in a suitable body space, releasing the active compound. Examples of these solvents include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol. Furthermore, for other mucosal sites such as nasal or pulmonary delivery, absorption may occur via the nasal mucosa or be inhaled into the lungs. These administration methods typically require providing the composition in the form of a solution, liquid suspension, or powder, and then mixing it with a gas such as air, oxygen, or nitrogen, or a combination thereof, to produce an aerosol or suspension of droplets or particles. These formulations can be carried out by well-known techniques in the pharmaceutical art. These formulations may be prepared as a solution in saline solution using benzyl alcohol or other suitable preservatives known in the art, absorption enhancers to increase bioavailability, carbon fluoride, and solubilizers or dispersants.

[0111] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0112] Further examples of therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antivirals (e.g., protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic agents (e.g., parasitic worms or protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (e.g., humanized, single-stranded, and chimeric antibodies), antigens and vaccine preparations (including adjuvants), polypeptide drugs, anti-inflammatory agents, ligands that bind to Toll-like receptors for activating the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, NK cells and helper T cells, and other molecules that inactivate or downregulate suppressor or regulatory T cells.

[0113] Further therapeutic agents are selected based on the condition, disorder, or disease being treated. For example, the polypeptides described herein may be co-administered with one or more additional agents that function to enhance or promote the immune response or to reduce or inhibit the immune response.

[0114] Chemotherapy agents In one embodiment, the polypeptides described herein may be combined with one or more chemotherapeutic agents or apoptosis promoters. Typical chemotherapeutic agents include amsacrin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysanthaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, Liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, larcitrexed, satoraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof are included, but are not limited to these. Representative apoptosis promoters include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0115] Immunosuppressants In some embodiments, the polypeptides disclosed herein are used in combination with other immunotherapies, immune modulators, costimulatory activating agonists, other cytokines and chemokines and factors, vaccines, adjuvants, oncolytic viruses, cell therapies, small molecule and targeted therapies, chemotherapy, and radiotherapy. In some embodiments, the immune modulator includes, but is not limited to, checkpoint inhibitors such as anti-PD1, anti-CTLA4, anti-TIM3, and anti-LAG3. In some embodiments, the costimulatory activating agonist includes, but is not limited to, anti-OX40, anti-GITR, etc. In some embodiments, the cell therapy includes engineered T cells, CAR-T cells, TCR-T cells, and others. In some embodiments, the vaccine includes a pan-DR-epitope ("PADRE") peptide. In some embodiments, the adjuvant includes QuilA. In some embodiments, the pan-DR-epitope peptide and QuilA together constitute the vaccine.

[0116] In some embodiments, the polypeptides disclosed herein are used in combination with other immunotherapies, immunomodulators, biologics (e.g., antibodies), vaccines, small molecule and targeted therapies, anti-inflammatory therapies, cell therapies (e.g., engineered Tregs and other types of cells), chemotherapy, and radiotherapy.

[0117] In one embodiment, the polypeptides disclosed herein are administered in vivo to a patient by intravenous, intramuscular, or other non-enteral means, either alone or in combination with other agents. They may also be administered intranasally, by inhalation, rectally, vaginally, topically, orally, or by implant.

[0118] In one embodiment, a further therapeutic agent is an immunosuppressant. The immunosuppressant is another lymphocyte surface marker (e.g., CD40 and alpha-4 integrin) or cytokine, fusion protein (e.g., CTLA-4-Ig(Orencia)). (登録商標) ), and TNFR-Ig (Embrel (登録商標) Antibodies against )); TNF-α blockers, e.g., Enbrel(登録商標) , Remicade (登録商標) , Simulect (登録商標) , and Humira (登録商標) , cyclophosphamide (「CTX」) (e.g., Endoxan (登録商標) , Cytoxan (登録商標) , Neosar (登録商標) , Procytox (登録商標) , and Revimmune TM ), methotrexate (「MTX」) (e.g., Rheumatrex (登録商標) and Trexall (登録商標) ), and belimumab (e.g., Benlysta (登録商標) ); other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds, and steroids); antiproliferatives; cytotoxic agents; and other compounds that may assist with immunosuppression, but are not limited thereto.

[0119] In certain embodiments, the additional therapeutic agent can be a checkpoint inhibitor. In certain embodiments, the additional therapeutic agent can be a CTLA-4 fusion protein such as CTLA-4-Ig (abatacept). The CTLA-4-Ig fusion protein can compete with the costimulatory receptor, CD28, for the T cell to bind to CD80 / CD86 (B7-1 / B7-2) on the antigen-presenting cell, and thus functions to inhibit T cell activation. In other embodiments, the additional therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y), which can significantly increase its avidity for CD-86 in vivo. In other embodiments, the additional therapeutic agent is Maxy-4.

[0120] In other embodiments, the additional therapeutic agent is CTX. CTX (Endoxan (登録商標) , Cytoxan (登録商標) , Neosar (登録商標) , Procytox (登録商標) , and Revimmune TMCTX (generic name), also known as cytophosphan, is a nitrogen mustard alkylating agent from the oxazofolin group. It can be used to treat various types of cancer and some autoimmune disorders. CTX is the first-line drug used for generalized proliferative glomerulonephritis in patients with renal lupus.

[0121] In one embodiment, further therapeutic agents may be administered in an effective amount to reduce blood or serum levels of anti-double-stranded DNA ("anti-ds DNA") autoantibodies and / or proteinuria in patients requiring treatment.

[0122] In other embodiments, further therapeutic agents may increase the amount of adenosine in the serum (see, for example, WO08 / 147482, which is incorporated herein by reference). For example, the second therapeutic agent may be CD73-Ig, recombinant CD73, or other agents that increase the expression of CD73 (e.g., cytokines, monoclonal antibodies, or small molecules) (see, for example, WO04 / 084933, which is incorporated herein by reference). In other embodiments, further therapeutic agents may be interferon-beta.

[0123] In one embodiment, further therapeutic agents may be small molecules that inhibit or reduce differentiation, proliferation, activity, cytokine production, and / or cytokine secretion by other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to Th1, Th17, Th22, and / or IL-1β, TNF-α, TGF-beta, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment, further therapeutic agents may be small molecules that interact with Tregs, enhance Treg activity, promote or enhance IL-10 secretion by Tregs, increase Treg numbers, increase Treg inhibitory ability, or a combination thereof.

[0124] In one embodiment, further therapeutic agents include antibodies against pro-inflammatory molecules such as IL-6, IL-23, IL-22, or IL-21, for example, inhibitory antibodies.

[0125] In one embodiment, the further therapeutic agent comprises nucleic acid. In another embodiment, the further therapeutic agent comprises ribonucleic acid.

[0126] Methods for treating diseases In another embodiment, a method for treating a disease in a subject requiring treatment includes administering an effective amount of the polypeptide described herein to the subject.

[0127] In one embodiment, the disease is cancer or an autoimmune disease.

[0128] In one embodiment, 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 one embodiment, the immune cell is a Treg. In one embodiment, the polypeptide activates PD-1 signaling. In another embodiment, the polypeptide inhibits PD-1 signaling. Surprisingly, the inventors have found that in one embodiment, the polypeptide activates the immune response, while in another embodiment, the polypeptide suppresses the immune response. In one embodiment, the polypeptide is used in the ex vivo treatment of T cells for adoptive transfer of engineered T cells, CAR-T cells, and / or TCT-T cells.

[0129] The inventors have surprisingly discovered that in some embodiments, the polypeptide induces T cell activation, while in other embodiments, it induces T cell suppression. For example, in one embodiment, the polypeptide containing SEQ ID NO: 12, optionally combined with a vaccine, significantly reduces IFNg in the serum of treated animals, compared to the vaccine alone and / or wild-type PD-L1 (when present). In another embodiment, the polypeptide containing SEQ ID NO: 12, optionally combined with a vaccine, significantly reduces the number of vaccine-specific T cells in vivo, compared to the vaccine alone and / or wild-type PD-L1. The inventors have surprisingly discovered that in another embodiment, the polypeptide containing SEQ ID NO: 11, optionally combined with a vaccine, significantly increases the total IFNg-producing T cells, compared to the vaccine alone and / or wild-type PD-L2 (when present). In yet another embodiment, the polypeptide containing SEQ ID NO: 11, optionally combined with a vaccine, significantly increases the number of vaccine-specific T cells compared to the vaccine alone and / or wild-type PD-L2. In one embodiment, the vaccine is a PADRE, optionally containing an adjuvant (e.g., QuilA).

[0130] cancer In one embodiment, a method is provided for treating or preventing cancer in a subject requiring treatment, comprising regulating PD-1 signaling by administering an effective amount of the polypeptide described herein to the subject.

[0131] In one embodiment, 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, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, adult T-cell leukemia / lymphoma, and combinations thereof.

[0132] In some embodiments, the polypeptides and compositions disclosed herein are useful for the treatment of leukemia. In some embodiments, the polypeptides and compositions disclosed herein that activate PD-1 are useful for the treatment of 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 agents. The ability to activate PD-1 enables a more robust immune response. In some embodiments, the polypeptides and compositions disclosed herein are also useful for stimulating or enhancing T cell-involved immune stimuli or activating responses. In some embodiments, the polypeptides and compositions disclosed herein are useful for stimulating or enhancing the immune response in the host for the treatment of leukemia by selective activation of PD-1. In these embodiments, the polypeptides and compositions disclosed herein may be administered to a subject in an amount effective for stimulating T cells in the subject. The types of leukemia that can be treated with the polypeptides and compositions disclosed herein include, but are not limited to, acute myeloid leukemia ("AML"), chronic myeloid leukemia ("CML"), acute lymphoblastic leukemia ("ALL"), chronic lymphoblastic leukemia ("CLL"), adult T-cell leukemia / lymphoma ("ATLL"), and chronic myelomonocytic leukemia ("CMML").

[0133] In one embodiment, ATLL is diagnosed almost exclusively in adults, with a median age of mid-60s. In one embodiment, there are four types of ATLL: (1) acute, (2) chronic, (3) smoldering, and (4) lymphoma. In one embodiment, acute ATLL is the most common form and is characterized by high white blood cell count, hypercalcemia, organ enlargement, and high lactose dehydrogenase levels. In one embodiment, lymphoma ATLL manifests in the lymph nodes, with circulating lymphocytes less than 1%. In one embodiment, chronic and smoldering ATLL are characterized by a low-aggression clinical course and allow for long-term survival. In one embodiment, the 4-year survival rate for acute and lymphoma ATLL is less than 5%. In one embodiment, the 4-year survival rates for chronic and smoldering forms of ATLL are 26.9% and 62%, respectively. In one embodiment, adult T-cell leukemia / lymphoma is caused by HTLV-1.

[0134] In one embodiment, the polypeptides and compositions disclosed herein are useful for treating ATLL. In one embodiment, the polypeptides and compositions disclosed herein that activate PD-1 are useful for treating ATLL. In one embodiment, ATLL cells exhibit an activated helper / inducer T cell phenotype but show strong immunosuppressive activity. In one embodiment, 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 immune-stimulating response to overcome the strong immunosuppressive activity of ATLL cells.

[0135] autoimmune diseases In one embodiment, a method is provided for treating or preventing an autoimmune disease in a subject requiring treatment, comprising regulating PD-1 signaling by administering an effective amount of the polypeptide described herein to the subject.

[0136] Non-limited examples of autoimmune diseases include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-tubular basement membrane antibody nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic ataxia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neurogenic neuropathy, Barlow's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, and catfish disease. Sulman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuritis, chronic relapsing polymyelitis, Churg-Strauss syndrome, eosinophilic granulomatosis, pemphigoid scarring, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, Crest syndrome, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrous alveolitis, giant cell arteritis (temporal). Arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, bullous pemphigoid of pregnancy, hidradenitis suppurativa (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immunothrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, sclerosing atrophic lichen, woody conjunctivitis, linear IgA disease, loop Chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mohren's ulcer, Mukka-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, childhood autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, ciliary uveitis (peripheral uveitis), Personage-Turner syndrome, pemphigus, peripheral neuropathy, perivenosis encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, Syndrome of Polyglandular Disease Type I, Syndrome of Polyglandular Disease Type II, Syndrome of Polyglandular Disease Type III, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrene, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless syndrome of the lower extremities, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis This includes sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Suzac syndrome, sympathetic ophthalmitis, Takayasu arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease, and combinations thereof.

[0137] Equal parts The following representative examples are intended to aid in the explanation of the present invention and are not intended to limit the scope of the invention, nor should they be construed as such. Indeed, various modifications of the invention and many embodiments thereof will be apparent to those skilled in the art from the entirety of this description, including the following examples and references to scientific and patent documents cited herein, in addition to those shown and described herein. It should be further recognized that the contents of these cited references are included herein by reference to aid in explaining common technical knowledge. The following examples contain important further information, examples, and guidance which may be applied in the implementation of the invention, its various embodiments and equivalents. [Examples]

[0138] Example 1. Polypeptide design Figures 1A and 1B illustrate the design of the polypeptide described herein. Figures 1A and 1B show that when bound to PD-1, PD-L2 has two interaction sites: one is an inhibitory site and the other is an activating site. As illustrated in Figure 1A, a mutant of PD-L2 with an inactive activating site binds to PD-1 only via the inhibitory site. This binding to PD-1 suppressed the immune response via PD-1 signaling. Furthermore, as illustrated in Figure 1B, a mutant of PD-L2 with an inactive inhibitory site binds to PD-1 only via the activating site and therefore activates the immune response via PD-1 signaling.

[0139] Example 2. PD-L2 increases central memory T cells ("Tcm") and prevents T cell exhaustion. As illustrated in Figure 2A, human CD4 T cells (BioIVT) sorted by flow cytometry 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, the cells were treated with PD-L2-IgG at a concentration of 25 μg / mL. The control well was left untreated. 48 hours after treatment, the cells were collected, washed, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Tcm was identified as CD45RO 高 / CD62L 高 / CD45RA 低 This was defined as exhausted T cells or terminal effector T cells ("Tte") being treated with CD45RO 低 / CD62L 低 / CD45RA 高 This was defined as follows. As shown in Figures 2B-2C, PD-L2-IgG treatment resulted in a significant increase in Tcm (*p<0.05, Figure 2B) and a significant decrease in Tte (*p<0.05, Figure 2C).

[0140] Example 3. PD-L2 is necessary for Tcm production and prevention of exhaustion. As illustrated in Figure 3A, human CD4 T cells (BioIVT) sorted by flow cytometry 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 100 μg / mL anti-PD-L2 antibody or left untreated. After 48 hours, the cells were collected, washed, stained with fluorophore-labeled antibody, and analyzed by flow cytometry. Tcm was CD45RO 高 / CD62L 高 / CD45RA 低 Defined as follows: Tte is CD45RO 低 / CD62L 低 / CD45RA 高 This was defined as follows. As shown in Figures 3B-3C, blocking PD-L2 binding to PD-1 with an anti-PD-L2 antibody prevented Tcm generation (****p<0.0001, Figure 3B) and significantly increased Tte (Figure 3C).

[0141] Example 4. PD-L2 is required for the initiation of activation in both CD4 and CD8 T cells. Human CD4 and CD8 T cells (BioIVT) sorted by flow cytometry were treated 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 either treated with 100 μg / mL anti-PD-L2 antibody or left untreated. After 48 hours, cells and supernatant were collected. The supernatant was analyzed for IFNg and TNF using a CBA assay (BD Biosciences). Cells were washed, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. CD25 expression in CD4 T cells and granzyme B and IFNg in CD8 T cells were used as indicators of activation and functionality. As shown in Figure 4A, anti-PD-L2 treatment inhibited cytokine production in CD4 T cells and similarly reduced CD4 T cell activation (shown in Figure 4B). Similarly, PD-L2 blockade reduced CD25 expression in CD8 T cells (shown in Figure 4C) and decreased intracellular granzyme B and IFNg (shown in Figure 4D).

[0142] Example 5. Flow cytometry assay to evaluate PD-L2 mutant binding The binding of mutants to PD-1 was evaluated using a flow cytometry assay. Briefly, PD-1-expressing Jurkat cells were treated with wild-type PD-L2 or various mutants at a concentration of 10 μg / ml for 30 minutes. After washing, anti-human IgG AlexaFluor647 was added to the samples for 20 minutes. As a negative control, cells were incubated with anti-human IgG AlexaFluor647 alone. After washing, stained cells were incubated in 200 μL of PBS and acquired using an Attune flow cytometer (Thermo). Data were analyzed using FlowJo software (BD Biosciences), and the results are shown in Figures 5A–5C.

[0143] Example 6. Luminescence assay to evaluate the functionality of PD-L2 mutants The functionality of the mutants was evaluated using luminescence assays. PD-1 expression reporter Jurkat-luciferase cells (Invivogen) were incubated with mutants at the concentrations described and in plates coated with anti-human CD3 antibody (0.4 μg / mL - 100 μL for 3 hours). After overnight incubation, the supernatant was collected and luminescence was measured using a VarioSkan luminometer. Relative luminescence units (RLU) were evaluated, and the results are shown in Figures 6A-6B. In Figure 6A, mutants with wild-type PD-L2 and SEQ ID NO: 10 were tested at concentrations of 0, 6.25 μg / mL, and 12.5 μg / mL (from left to right). Mutants with SEQ ID NO: 11 were tested at concentrations of 0, 6.25 μg / mL, 12.5 μg / mL, and 25 μg / mL (from left to right).

[0144] Example 7. IFNg production assay to evaluate the functionality of PD-L2 mutants. The activating function was tested by evaluating IFNg production from human CD4 T cells using an ELISA assay (Invitrogen). For the activating mutants, purified human CD4 T cells (BioIVT) were pre-stimulated with anti-CD3 / anti-CD28 Dyna beads (0.5 μL / 80,000 cells) for 24 hours. After stimulation, cells were treated with SEQ ID NOs. 10 and 11 mutants at concentrations indicating cell activity. IFNg was evaluated 24 hours after treatment using an ELISA assay in the supernatant according to the manufacturer's specifications (Invitrogen), and the results are shown in Figure 7A. For the inhibitory mutants, 96-well plates were coated with SEQ ID NOs. 12 and 13 mutants for 3 hours (10 μg / mL, 100 μL in PBS). After washing with PBS, purified human CD4 T cells (BioIVT) were added with anti-CD3 / anti-CD28 Dyna beads (0.5 μL / 80,000 cells) and incubated for 48 hours. IFNg was evaluated using an ELISA assay in the supernatant 48 hours after processing, according to the manufacturer's specifications (Invitrogen), and the results are shown in Figure 7B.

[0145] Example 8. Ligand-based activator in vivo human T cell activation As shown in Figure 8A, NSG mice transplanted with human PBMCs were treated with PADRE (pan-DR epitope) peptide and QuilA adjuvant as a vaccine on days 0, 7, and 14. Recombinant wild-type ("WT") human PD-L2 or SEQ ID NO: 11 mutant intravenously was administered twice weekly (see Figure 8A). Five days after the final immunization (day 19), the mice were sacrificed and the spleen was isolated (see Figure 8A). Total and antigen (PADRE)-specific IFNg-producing cell counts were assessed using a standard ELISPOT assay. As shown in Figure 8B (total T cell response), the addition of SEQ ID NO: 11 to the vaccine treatment significantly increased total IFNg-producing T cells compared to the all-encompassing group including vaccine + WT PD-L2 (*p<0.05, **p<0.01). As shown in Figure 8C (antigen-specific T cell response), the SEQ ID NO: 11+ vaccine also significantly increased the number of PADRE-specific T cells compared to the vaccine alone (*p<0.05).

[0146] Example 9. Ligand-based inhibitor in vivo human T cell activation NSG mice transplanted with human PBMCs were treated with PADRE (pan-DR epitope) peptide and QuilA adjuvant as vaccines on days 0, 7, and 14. Recombinant WT human PD-L1 or SEQ ID NO: 12 mutant intravenously were administered twice weekly. Five days after the final immunization (day 19), mice were collected, sacrificed, and spleens isolated. A standard ELISA assay was used to test serum IFNg levels, and an ELISPOT assay was used to evaluate the number of antigen (PADRE)-specific IFNg-producing cells. As shown in Figure 9A (total T cell response in serum), the addition of SEQ ID NO: 12 to the vaccine treatment significantly reduced serum IFNg in treated mice compared to the vaccine alone group (*p<0.05). As shown in Figure 9B (antigen-specific T cell response), and similar to the PD-L1+ vaccine, the SEQ ID NO: 12+ vaccine also significantly reduced the number of PADRE-specific T cells compared to the vaccine alone group (*p<0.05).

Claims

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

2.

2. The polypeptide according to claim 1, wherein the polypeptide is a variant of programmed cell death ligand 1 ("PD-L2").

3. The polypeptide according to claim 1 or 2, wherein an amino acid is fused with an immunoglobulin.

4. The polypeptide according to claim 3, wherein the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

5. The polypeptide according to any one of claims 1 to 4, wherein 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13.

6. The polypeptide according to any one of claims 1 to 5, wherein 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 or SEQ ID NO:

12.

7. The polypeptide according to claim 5, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

8. The polypeptide according to claim 6, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO:

12.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide binds to programmed cell death protein 1 ("PD-1") and activates PD-1.

10. The polypeptide according to claim 9, which activates polypeptide immune cells.

11. The polypeptide according to claim 9 or 10, wherein 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

12. The polypeptide according to any one of claims 9 to 11, wherein 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.

13. The polypeptide according to claim 11, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

11.

14. The polypeptide according to claim 12, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

11.

15. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide binds to PD-1 and inhibits cells via PD-1.

16. The polypeptide according to any one of claims 1 to 8 and 15, wherein the polypeptide suppresses immune cells.

17. The polypeptide according to any one of claims 1 to 8 and 15 to 16, wherein the polypeptide induces central memory T cells ("Tcm").

18. The polypeptide according to any one of claims 1 to 8 and 15 to 17, wherein the polypeptide prevents T cell exhaustion.

19. The polypeptide according to any one of claims 15 to 17, wherein 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 or SEQ ID NO:

13.

20. The polypeptide according to claims 15 to 17 and 19, wherein 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.

21. The polypeptide according to claim 19, wherein the polypeptide comprises an amino acid sequence that is sequence number 12 or sequence number 13.

22. The polypeptide according to claim 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

12.

23. A conjugate comprising a polypeptide according to any one of claims 1 to 22 and a detectable marker or carrier molecule, wherein the polypeptide is bound to the detectable marker or carrier molecule.

24. The conjugate of claim 23, wherein the carrier molecule is selected from the group consisting of glycosaminoglycans, proteoglycans, albumin, and polyalkylene glycols.

25. A nucleic acid encoding a polypeptide according to any one of claims 1 to 22.

26. A pharmaceutical composition comprising the polypeptide described in any one of claims 1 to 22.

27. The pharmaceutical composition according to claim 26, wherein the polypeptide is encapsulated in liposomes.

28. The pharmaceutical composition according to claim 26 or 27, further comprising a second therapeutic agent.

29. The pharmaceutical composition according to claim 28, wherein the second therapeutic agent is a chemotherapeutic agent or an immunomodulator.

30. A method for inducing, promoting, or enhancing an immune response in a subject requiring treatment, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 14, a conjugate according to claim 23 or 24, or a pharmaceutical composition according to any one of claims 26 to 29.

31. A method for treating cancer or reducing tumor burden in a subject requiring treatment, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 14, a conjugate according to claim 23 or 24, or a pharmaceutical composition according to any one of claims 26 to 29.

32. The method according to claim 31, wherein the cancer is selected from the group consisting of adult T-cell leukemia / lymphoma, bladder, brain, breast, cervix, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterus, ovarian, and testicular cancer.

33. The method according to any one of claims 30 to 32, wherein the method further includes upregulation of inducible T cell kinase ("ITK").

34. A method for reducing, suppressing, or preventing an immune response in a subject requiring treatment, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 8 and 15 to 22, a conjugate according to claim 23 or 24, or a pharmaceutical composition according to any one of claims 26 to 29.

35. A method for treating an autoimmune disease in a subject requiring treatment, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 6 and 15 to 22, a conjugate according to claim 23 or 24, or a pharmaceutical composition according to any one of claims 26 to 29.

36. Autoimmune diseases include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-tubular basement membrane antibody nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic dysphagia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neurogenic neuropathy, Barlow's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, and Castleman disease. Celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuritis, chronic relapsing polymyelitis, Churg-Strauss syndrome, eosinophilic granulomatosis, pemphigoid scarring, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, Crest syndrome, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrous alveolitis, giant cell arteritis (temporal arteritis) ), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, bullous pemphigoid of pregnancy, hidradenitis suppurativa (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immunothrombocytopenic purpura, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, sclerosing atrophic lichen, woody conjunctivitis, linear IgA disease, lupus Chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mohlen's ulcer, Mukka-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, childhood autoimmune neuropsychiatric disorders, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, ciliary body squamous cellulitis (peripheral uveitis), Personage-Turner syndrome, pemphigus, peripheral neuropathy, perivenosis encephalomyelitis, pernicious anemia, POEMS syndrome,Polyarteritis nodosa, Syndrome of Polyglandular Disease Type I, Syndrome of Polyglandular Disease Type II, Syndrome of Polyglandular Disease Type III, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless syndrome of the lower extremities, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, S The method according to claim 35, selected from the group consisting of Eummidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Suzac syndrome, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease, and combinations thereof.

37. The method according to any one of claims 34 to 36, wherein the method further includes downward control of ITK.

38. The method according to any one of claims 30 to 37, for use in ex vivo treatment of T cells for adoptive transfer of polypeptide-modified T cells, CAR-T cells, and / or TCT-T cells.

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