Anti-HVEM antibodies and uses thereof

Abstract

To provide medicaments that bind to HVEM and inhibit HVEM-BTLA interaction.SOLUTION: Disclosed is an antibody or antigen binding fragment thereof that binds to a herpesvirus entry mediator (HVEM) to inhibit the interaction between the HVEM and B- and T-lymphocyte attenuator (BTLA), comprising three heavy chain CDRs comprising amino acid sequences as set forth in SEQ ID NOs: 1 to 3, and three light chain CDRs comprising amino acid sequences as set forth in SEQ ID NOs: 4 to 6.SELECTED DRAWING: Figure 9B

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 965,921, filed on January 26, 2020, and U.S. Provisional Patent Application No. 62 / 839,841, filed on April 29, 2019, the entire contents of which are hereby incorporated by reference in their entirety.

[0002] The present invention is in the field of immunotherapy.

Background Art

[0003] Immunotherapies designed to enhance the immune response are thought to activate immunotherapy and are at the forefront of cancer treatment. Currently, immune checkpoint inhibition therapies have been successfully used in the treatment of advanced non - small cell lung cancer (NSCLC), metastatic melanoma, and advanced renal cell carcinoma (RCC). Some such agents developed and manufactured by various companies have shown great promise in various types of cancer and have received FDA approval, such as antibodies against the programmed cell death protein 1 receptor (PD1), the programmed cell death protein 1 ligand (PDL1), and cytotoxic T - lymphocyte - associated protein 4 (CTLA - 4). However, only usually 10% - 40% of the treated patients benefit, and at the same time patients may suffer from the side effects of the treatment after immunotherapy, so the patient response rate is not yet optimal. Furthermore, treatment with these agents may induce resistance through up - regulation of additional immune checkpoints. The combination of anti - PD1 therapy and anti - CTLA - 4 therapy in melanoma patients showed a higher response rate (60%) compared to single agents, but this combination therapy also includes severe side effects related to the treatment. Therefore, new anti - tumor immune activators are clearly needed.

[0004] Herpes virus entry mediator (HVEM) is a protein found on the surface of various cell types, including hematopoietic and non-hematopoietic cells. Since HVEM functions as a receptor for standard TNF-related ligands such as LIGHT and LTα, it functions as a signaling receptor. However, it also functions as a ligand for immunoglobulin (Ig) superfamily molecules such as the inhibitory receptors BTLA and CD160. Therefore, bidirectional signaling is possible in the signaling network via HVEM and may be involved in positive or negative immune responses under various circumstances. Dysregulation of this network is involved in the etiology of autoimmune diseases, inflammatory diseases, and cancer, and HVEM is targeted for immunotherapy. SUMMARY OF THE INVENTION

[0005] The present invention provides agents that bind to HVEM, inhibit HVEM-BTLA interaction, inhibit downstream BTLA signaling, and activate downstream HVEM signaling. Methods of treating diseases using those agents and kits containing those agents are also provided.

[0006] According to a first aspect, there is provided an agent that specifically binds to herpes virus entry mediator (HVEM) on a cell, a. inhibits the interaction between HVEM and B and T lymphocyte attenuator (BTLA), b. activates downstream signaling via HVEM within the cell.

[0007] According to another aspect, the antibody or its antigen-binding fragment comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 (QQYGSSPPYT).

[0008] According to another aspect, there is provided a pharmaceutical composition comprising a drug of the present invention, an antibody or an antibody-binding fragment of the present invention, and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0009] According to another aspect, there is provided a method for treating a disease or condition characterized by HVEM-positive cells, the method comprising administering a pharmaceutical composition of the present invention, thereby treating the disease or condition.

[0010] According to another aspect, there is provided a method for treating an HVEM-positive disease or condition, the method comprising: a. inhibiting the interaction between HVEM and BTLA; and b. activating HVEM signaling in immune cells, diseased cells, or both, thereby treating the HVEM-positive disease or condition. Thereby treating the HVEM-positive disease or condition.

[0011] According to another aspect, there is provided a method for determining the suitability of a subject to be treated by the method of the present invention, the method comprising obtaining a disease sample from the subject and determining the level of HVEM in the sample, wherein positive expression of HVEM indicates that the subject is suitable for the method of treatment of the present invention.

[0012] According to another aspect, a method for detecting HVEM in a sample is provided, the method comprising contacting the sample with the agent of the present invention, or an antibody of the present invention or an antigen-binding fragment thereof, thereby detecting HVEM.

[0013] According to another aspect, the pharmaceutical composition of the present invention, and a. an anti-PD-1 / PD-L1-based immunotherapy, b. a label indicating that the pharmaceutical composition of the present invention is for use in an anti-PD-1 / PD-L1-based immunotherapy, and c. a kit is provided that includes at least one of the secondary detection molecules for detecting the agent of the present invention, or an antibody of the present invention or an antigen-binding fragment thereof.

[0014] According to some embodiments, the agent does not substantially inhibit the interaction between HVEM and tumor necrosis factor superfamily member 14 (TNFSF14).

[0015] According to some embodiments, the agent inhibits the interaction between HVEM and CD160, lymphotoxin alpha (LTα), or both.

[0016] According to some embodiments, the agent does not directly induce apoptosis of cells expressing HVEM when binding to HVEM expressed by the cells.

[0017] According to some embodiments, the agent is an antibody or an antigen-binding fragment thereof.

[0018] According to some embodiments, the agent of the present invention comprises IgG2, IgG4 or modified IgG1 or IgG3 that is not cytotoxic to cells.

[0019] According to some embodiments, the agent is a single-chain antibody (scFv).

[0020] According to some embodiments, downstream signaling via HVEM includes the nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB)-mediated transcription.

[0021] According to some embodiments, downstream signaling via HVEM includes an increase in the cytotoxicity of immune cells expressing HVEM.

[0022] According to some embodiments, the increase in cytotoxicity includes an increase in the secretion of pro-inflammatory cytokines.

[0023] According to some embodiments, the cell is an immune cell and the signaling induces immune activation.

[0024] According to some embodiments, the immune cell is a tumor-infiltrating lymphocyte (TIL) or a peripheral blood mononuclear cell (PBMC).

[0025] According to some embodiments, the cell is a cancer cell and the signaling includes an anti-tumor effect.

[0026] According to some embodiments, the agent of the present invention is for use in the treatment of a disease characterized by disease cells expressing HVEM.

[0027] According to some embodiments, the disease is an HVEM-positive cancer or pre-cancerous lesion.

[0028] According to some embodiments, the HVEM-positive cancer is selected from melanoma, renal cancer, cervical cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, and head and neck cancer.

[0029] According to some embodiments, the disease is an infectious disease and the infected cells include HVEM expression.

[0030] According to some embodiments, the antibody or its antigen-binding fragment comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 (QQYGSSPPYT).

[0031] According to some embodiments, the agent of the present invention comprises a heavy chain comprising the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7).

[0032] According to some embodiments, the agent of the present invention comprises a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8).

[0033] According to some embodiments, the agent of the present invention comprises a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9), and a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0034] According to some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain comprising the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7).

[0035] According to some embodiments, the antibody or antigen-binding fragment of the present invention comprises a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8).

[0036] According to some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9), and a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0037] According to some embodiments, the method of the present invention further comprises administering an anti-PD-1 / PD-L1 based immunotherapy.

[0038] According to some embodiments, the method of the present invention further comprises administering adoptive cell therapy.

[0039] According to some embodiments, the adoptive cell therapy includes adoptive TIL therapy.

[0040] According to some embodiments, the adoptive cell therapy includes administering a chimeric antigen receptor (CAR) that expresses immune cells, wherein the CAR targets a non-HVEM protein on the surface of HVEM-expressing cells.

[0041] According to some embodiments, the method of the present invention further comprises inhibiting PD-1 and PD-L1 interaction.

[0042] According to some embodiments, the method of the present invention further comprises not substantially inhibiting the interaction between HVEM and TNFSF14.

[0043] According to some embodiments, the disease or condition is an HVEM-positive cancer or precancerous lesion.

[0044] According to some embodiments, the HVEM-positive cancer is selected from melanoma and renal cell carcinoma.

[0045] According to some embodiments, the disease or condition is an infectious disease, and the infected cells include HVEM expression.

[0046] According to some embodiments, the immune cells are TIL or PBMC.

[0047] According to some embodiments, the immune cells are CAR-T cells.

[0048] According to some embodiments, the method of the present invention comprises administering a pharmaceutical composition of the present invention.

[0049] According to some embodiments, positive expression of HVEM includes elevated HVEM levels as compared to a healthy sample or a predetermined threshold.

[0050] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0051]

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BRIEF DESCRIPTION OF THE INVENTION

[0052] In some embodiments, the present invention provides an agent that binds to HVEM, inhibits HVEM-BTLA interaction, and is also an agonist of HVEM. The present invention further relates to a method for treating HVEM-positive diseases and a kit containing these agents.

[0053] It is well known that cancers that are positive for HVEM can evade immune surveillance by binding BTLA to the surface of immune cells. The involvement of BTLA generates inhibitory signals within immune cells, reduces the proliferation of T cells, and increases cancer survival rates. It is known to inhibit this signaling with an antibody that binds to HVEM or an antibody that binds to BTLA. The present invention is based on the surprising discovery that an antibody that specifically binds to HVEM and activates HVEM signaling provides another benefit (increased immune cell activation) in the fight against cancer. Although HVEM is also expressed on the surface of immune cells, its role is not well understood. The antibodies of the present invention not only block the HVEM-BTLA interaction by binding HVEM to pathogenic cells and liberate immune cells from inhibition, but also bind to HVEM on immune cells and induce NF-kB signaling in those immune cells, enhancing their activation properties.

[0054] According to a first aspect, there is provided an agent that binds to herpesvirus entry mediator (HVEM) and inhibits the interaction between HVEM and B and T lymphocyte attenuator (BTLA).

[0055] In some embodiments, the agent activates downstream signaling via HVEM. In some embodiments, the agent inhibits downstream signaling via BTLA. In some embodiments, the agent activates downstream signaling via HVEM and inhibits downstream signaling via BTLA. In some embodiments, inhibiting the interaction between HVEM and BTLA inhibits downstream signaling via BTLA.

[0056] In some embodiments, the HVEM is mammalian HVEM. In some embodiments, the HVEM is rodent HVEM. In some embodiments, the HVEM is monkey HVEM. In some embodiments, the HVEM is human HVEM. In some embodiments, the HVEM is any one of mouse, monkey, and human HVEM. In some embodiments, the HVEM is membrane-bound HVEM. In some embodiments, the HVEM is cell surface HVEM. In some embodiments, the HVEM is cell-surface HVEM. In some embodiments, the HVEM is soluble HVEM.

[0057] In some embodiments, the cell is a pathogenic cell. In some embodiments, the cell is a cancerous cell. In some embodiments, the cell is a pathogen cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a eukaryotic cell infected with a pathogen. In some embodiments, the cell is a cell infected with a bacterium. In some embodiments, the cell is a cell infected with a virus. In some embodiments, the pathogen is selected from bacteria, viruses, and fungi.

[0058] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8-positive T cell. In some embodiments, the T cell is a cytotoxic CD8-positive T cell. In some embodiments, the T cell is a CD4-positive T cell. In some embodiments, the T cell is a CD4-positive helper T cell. In some embodiments, the T cell is selected from CD8-positive and CD4-positive T cells. In some embodiments, the T cell is a CD8-positive T cell, a CD4-positive T cell, or both. In some embodiments, the immune cell is a tumor-infiltrating lymphocyte (TIL). In some embodiments, the immune cell is not a peripheral blood immune cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a myeloid-derived suppressor cell (MDSC). In some embodiments, the cell is selected from T cells, B cells, NK cells, neutrophils, dendritic cells, MDSCs, and macrophages. In some embodiments, the cell is selected from T cells, B cells, NK cells, neutrophils, dendritic cells, and macrophages.

[0059] In some embodiments, the immune cell is a chimeric antigen receptor (CAR) that expresses the immune cell. In some embodiments, the CAR is a CAR-T cell. In some embodiments, the CAR is a CAR-NK cell. As used herein, the term "CAR" refers to an engineered receptor that has specificity for at least one target protein (e.g., a protein expressed by HVEM-expressing cells) and is grafted onto an immune effector cell (e.g., a T cell or an NK cell). In some embodiments, the CAR-T cell has the specificity of a monoclonal antibody grafted onto a T cell. In some embodiments, the CAR-NK cell has the specificity of a monoclonal antibody grafted onto an NK cell. In some embodiments, the T cell is selected from cytotoxic T lymphocytes and regulatory T cells. MART1 is an example of a target protein that co-expresses with HVEM on target cells. In some embodiments, the CAR targets a protein expressed by HVEM-expressing cells. In some embodiments, the protein is not HVEM. In some embodiments, the CAR targets a protein on the surface of HVEM-expressing cells. In some embodiments, the protein is an antibody. In some embodiments, the CAR targets an antibody. In some embodiments, the CAR targets an antibody of the present invention. In some embodiments, the CAR targets a cytotoxic antibody. In some embodiments, the CAR targets an antibody constant domain. In some embodiments, the CAR targets an Fc domain. In some embodiments, the CAR therapy further comprises administering an antibody that targets HVEM-expressing cells. In some embodiments, the antibody targeted by the CAR is not an antibody of the present invention.

[0060] CAR-T and CAR-NK cells, as well as their vectors, are well known in the art. Such cells target proteins to which the receptor binds and exhibit cytotoxicity. In some embodiments, the CAR-T or CAR-NK cells target at least one cancer protein. In some embodiments, the CAR-T or CAR-NK cells target multiple cancer proteins.

[0061] The construction of CAR-T cells is well known in the art. In one non-limiting example, a monoclonal antibody against a cancer protein can be produced, and then a vector encoding the antibody can be constructed. The vector also includes a co-stimulatory signal region. In some embodiments, the co-stimulatory signal region includes the intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of the ligands that specifically bind to CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the vector also includes a CD3Z signaling domain. This vector is then transfected into T cells, for example, by lentiviral infection.

[0062] In some embodiments, HVEM is Tumor Necrosis Factor Receptor Superfamily Member 14 (TNFRSF14). In some embodiments, HVEM is CD270. In some embodiments, HVEM is the receptor for BTLA. In some embodiments, HVEM is the ligand for BTLA. In some embodiments, BTLA is CD272. In some embodiments, HVEM is the receptor for Tumor Necrosis Factor Superfamily Member 14 (TNFSF14). In some embodiments, HVEM is the ligand for TNFSF14. In some embodiments, TNFSF14 is LIGHT. In some embodiments, TNFSF14 is CD258. In some embodiments, HVEM is the receptor for CD160. In some embodiments, HVEM is the ligand for CD160. In some embodiments, HVEM is the receptor for Lymphotoxin alpha (LTα). In some embodiments, HVEM is the ligand for LTα. In some embodiments, LTα is TNF-β.

[0063] In some embodiments, the agent specifically binds to HVEM. In some embodiments, the agent does not bind to other proteins other than HVEM. In some embodiments, the agent binds to the extracellular domain of HVEM. In some embodiments, the agent binds to the ligand-binding domain of HVEM. In some embodiments, the agent binds to the BTLA-binding domain of HVEM. In some embodiments, the agent occludes the BTLA-binding domain of HVEM. In some embodiments, the agent inhibits the interaction between HVEM and BTLA. In some embodiments, the agent blocks the interaction between HVEM and BTLA. In some embodiments, the agent inhibits HVEM-mediated BTLA-induced immunosuppression. In some embodiments, the agent binds to HVEM and prohibits the bound HVEM from further binding to BTLA. In some embodiments, the agent inhibits downstream signaling via BTLA. In some embodiments, the agent reduces downstream signaling via BTLA.

[0064] In some embodiments, the agent does not inhibit the interaction between HVEM and TNFSF14. In some embodiments, the agent inhibits the interaction between HVEM and TNFSF14. In some embodiments, TNFSF14 is membrane-bound TNFSF14 (mTNFS14). In some embodiments, TNFSF14 is soluble TNFSF14 (sTNFS14). In some embodiments, the agent does not inhibit the interaction between HVEM and one of mTNFS14 and sTNFS14, but inhibits the interaction with the other. In some embodiments, the agent does not inhibit the interaction between both mTNFS14 and sTNFS14 and HVEM. In some embodiments, the inhibition is substantial inhibition. In some embodiments, the inhibition is a reduction of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent does not block the interaction between HVEM and TNFSF14. In some embodiments, the agent does not substantially block the interaction between HVEM and TNFSF14. In some embodiments, the agent does not block / inhibit TNFSF14-mediated signaling. In some embodiments, the agent does not block / inhibit TNFSF14-mediated cell survival.

[0065] In some embodiments, the agent activates signaling via HVEM. In some embodiments, the agent is an HVEM agonist. In some embodiments, the agent induces signaling via HVEM. In some embodiments, signaling via HVEM is downstream signaling of HVEM. In some embodiments, the agent binds to HVEM on the cell and activates / induces HVEM signaling intracellularly. In some embodiments, HVEM signaling includes activation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) signaling. In some embodiments, NF-kB signaling includes regulation of genes having NF-kB response elements in their promoters. In some embodiments, the signaling includes an increase in the cytotoxicity of the cell. In some embodiments, the signaling includes an increase in the cytotoxicity of cells expressing HVEM contacted by the agent. In some embodiments, the increase in cytotoxicity includes an increase in the secretion of inflammatory cytokines. In some embodiments, the inflammatory cytokine is selected from IL-1, IL-1B, IL-4, IL-6, TNFα, IFNγ, MCP1, IL-12, IL-18, IL-23, and CM-CSF. In some embodiments, the inflammatory cytokine is IFNγ. In some embodiments, the increase is when compared to cells not contacted by the agent of the present invention. In some embodiments, the increase is an increase of at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500%. Each possibility represents a separate embodiment of the present invention.

[0066] In some embodiments, the cell is an immune cell and the signaling includes immune activation. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a CD8+ T cell. In some embodiments, the immune cell is a CD4+ T cell. In some embodiments, the immune cell is not a gamma / delta T cell. In some embodiments, the immune cell is a gamma / delta T cell. In some embodiments, the immune cell is an NK cell. In some embodiments, activating HVEM signaling includes activating immune cells. In some embodiments, immune activation includes an increase in proliferation. In some embodiments, immune activation includes an increase in cytotoxicity. In some embodiments, immune activation includes an increase in migration. In some embodiments, immune activation includes increased homing. In some embodiments, immune activation includes increased cell clustering. In some embodiments, immune activation is T cell activation. In some embodiments, immune activation includes an increase in the number of Th1 T cells. In some embodiments, the increase is a relative increase compared to Th2 T cells. In some embodiments, immune activation includes an increase in CD8+ T cells. In some embodiments, immune activation includes a decrease in regulatory T cells. In some embodiments, immune activation includes an increase in the expression of a marker selected from the secretion of 41BB, CD69, CD25, CD107a, HLA-DR, and cytokines. In some embodiments, the cytokine is an inflammation-inducing cytokine. In some embodiments, T cell activation includes an increase in T cell clustering.

[0067] In some embodiments, the cell is a cancerous cell and the signaling includes an anti-tumor effect. In some embodiments, the anti-tumor effect includes an increase in apoptosis. In some embodiments, the anti-tumor effect includes a decrease in proliferation. In some embodiments, the anti-tumor effect includes an increase in chemosensitivity. In some embodiments, the anti-tumor effect includes a decrease in motility. In some embodiments, the anti-tumor effect includes a decrease in invasion. In some embodiments, the anti-tumor effect includes a decrease in metastasis. In some embodiments, the anti-tumor effect includes a decrease in self-renewal. In some embodiments, the effect is compared to cancer cells that have not been contacted by the agent of the invention. In some embodiments, the decrease is a decrease of at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention.

[0068] In some embodiments, the agent does not induce apoptosis. In some embodiments, inducing apoptosis is directly inducing apoptosis. In some embodiments, the agent does not directly induce apoptosis. As used herein, "directly induce" refers to a result that occurs as an immediate result of the binding of the agent and does not characterize downstream signaling within the bound cell. In some embodiments, the agent is not cytotoxic. In some embodiments, the agent itself is not cytotoxic. In some embodiments, the agent does not induce antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the agent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the agent does not contain a cytotoxic moiety. In some embodiments, the agent does not induce apoptosis of cells expressing HVEM upon binding of HVEM expressed by the cells. In some embodiments, the agent does not induce apoptosis through interaction with another cell. In some embodiments, the agent does not directly induce the death of cells expressing HVEM. In some embodiments, the agent does not target cells for death. In some embodiments, the agent induces indirect apoptosis. In some embodiments, indirect apoptosis is apoptosis induced by downstream signaling via the HVEM receptor that results in apoptosis. In some embodiments, indirect apoptosis is apoptosis that requires signaling. In some embodiments, indirect apoptosis is apoptosis that does not require the involvement of a second cell. In some embodiments, the agent does not induce apoptosis in immune cells. In some embodiments, the agent induces apoptosis in cancer cells.

[0069] In some embodiments, the agent inhibits the interaction between HVEM and CD160. In some embodiments, the agent inhibits the interaction between HVEM and LTα. In some embodiments, the agent inhibits the interaction between HVEM and herpes simplex virus type 1 glycoprotein D (HSV1-gD). In some embodiments, the agent inhibits the interaction between HVEM and at least two of CD160, HSV1-gD and LTα. In some embodiments, the agent blocks the interaction between HVEM and CD160. In some embodiments, the agent blocks the interaction between HVEM and LTα. In some embodiments, the agent blocks the interaction between HVEM and HSV1-gD. In some embodiments, the agent blocks the interaction between HVEM and at least two of CD160, HSV1-gD and LTα. In some embodiments, the agent inhibits / blocks the interaction between HVEM and all of CD160, HSV1-gD and LTα. In some embodiments, the agent does not inhibit or block the interaction between HVEM and at least one of CD160, HSV1-gD and LTα.

[0070] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or fragment thereof is a fab fragment. In some embodiments, the antibody or fragment thereof is a single-chain antibody (scFv). In some embodiments, the antibody or fragment thereof is a single-domain antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a blocking antibody.

[0071] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one binding domain formed from the folding of a polypeptide chain having a three-dimensional binding space with a charge distribution complementary to the internal surface shape and the characteristics of the antigenic determinant of an antigen. An antibody typically has a tetrameric form comprising two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable regions of each light chain / heavy chain pair form the antibody binding site. Antibodies include, but are not limited to, oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and soluble or conjugatable and labelable antibodies, alone or in combination with other amino acid sequences, as well as epitope-binding fragments, variants or derivatives thereof. Antibodies may be from any species. The term "antibody" also includes binding fragments including, but not limited to, Fv, Fab, Fab’, F(ab’)2 single-chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-bonded variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including, but not limited to, the Fc region or fragments thereof. It is further understood by those skilled in the art that other fusion products may be generated including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions and scFv-scFv-Fc fusions.

[0072] The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody comprises IgG2 or IgG4. In some aspects, the antibody comprises IgG2. In some aspects, the antibody comprises IgG4. In some aspects, the antibody comprises IgG1. In some aspects, the antibody comprises IgG3. In some embodiments, the antibody comprises a modified IgG1 or IgG3 with reduced toxicity.

[0073] The basic unit of the naturally occurring antibody structure is a heterotetrameric glycoprotein complex of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains, which are linked by both non-covalent and disulfide bonds. Each heavy and light chain also has regularly spaced intra-chain disulfide cross-bridges. There are five human antibody classes (IgG, IgA, IgM, IgD, IgE), and the various subclasses within these classes are recognized based on differences in structure, such as the number of immunoglobulin units within a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0074] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxy-terminal regions and are thus called variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen-binding site, and thus there are two antigen-binding sites in the basic immunoglobulin unit. Certain amino acid residues are thought to form the interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985), Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0075] The carboxy-terminal portions of the heavy and light chains form the constant domains, namely CH1, CH2, CH3, CL. The diversity of these domains is much lower, but there are differences among animal species, and there are also several different antibody isotypes with different functions within the same individual.

[0076] The term "framework region" or "FR" refers to the amino acid residues within the variable domain of an antibody other than the hypervariable region amino acid residues as defined herein. The term "hypervariable region" as used herein refers to the amino acid residues within the variable domain of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from "complementary determining regions" or "CDRs". CDRs are mainly involved in binding to the epitope of an antigen. The ranges of FR and CDR are precisely defined (see Kabat et al.).

[0077] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments containing CDRs. See, for example, Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0078] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to enhance its similarity to a human antibody. A humanized antibody can be produced by generating recombinant DNA encoding the CDRs of a non-human antibody surrounded by sequences similar to those of a human antibody. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanization includes inserting the CDRs of the present invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art, and any method for generating them that retains the CDRs of the present invention can be used.

[0079] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible variants that may arise during the production of the monoclonal antibody, and / or bind to the same epitope, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as being produced by any particular method of preparation. Monoclonal antibodies used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991).

[0080] The mAbs of the present invention may be of any immunoglobulin class including IgG, IgM, IgD, IgE or IgA. The hybridomas producing the mAbs may be cultured in vitro or in vivo. High titer mAbs can be obtained by in vivo production by injecting cells from individual hybridomas intraperitoneally into the original primed Balb / c mice to generate ascites containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG can be purified from such ascites or from culture supernatants using column chromatography methods well known to those of skill in the art.

[0081] An "antibody fragment" includes a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2, Zapata et al, Protein Eng. 8(10):1057-1062 (1995)), single-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv), and Bispecific T cell engagers (BiTE), but are not limited thereto.

[0082] Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site and the residue "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab’)2 fragment that has two antigen-binding sites but can cross-link antigens.

[0083] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are tightly non-covalently associated. Three surfaces of the VH-VL dimer are in this configuration. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind an antigen, although with a lower affinity than the entire binding site.

[0084] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab’-SH is herein the designation for Fab’ in which the cysteine residue(s) of the constant domain retain at least one free thiol group. F(ab’)2 antibody fragments were originally generated as pairs of Fab’ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0085] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinguishable types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0086] Antibodies can be assigned to different classes according to the amino acid sequences of their heavy chain constant domains. Intact antibodies have five major classes, IgA, IgD, IgE, IgG, IgM, and some of these can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known.

[0087] The "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding. For a discussion of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0088] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) of the same polypeptide chain (VH-VL). By using a linker that is too short to permit pairing between the two domains of the same chain, the domains pair with the complementary domains of another chain to form two antigen-binding sites. The generation of diabodies is known in the art and is described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0089] The monoclonal antibodies of the present invention can be prepared using methods well known in the art. Examples include various techniques such as those described in Kohler, G. and Milstein, C, Nature 256:495-497 (1975), Kozbor et al, Immunology Today 4:72 (1983), Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0090] In addition to conventional methods of producing antibodies in vivo, antibodies can be generated in vitro using phage display technology. The generation of such recombinant antibodies is much faster compared to conventional antibody generation, and they can be generated against a vast number of antigens. Furthermore, when using conventional methods, many antigens have been found to be non-immunogenic or highly toxic and thus cannot be used for antibody generation in animals. Additionally, affinity maturation of recombinant antibodies (i.e., increase in affinity and specificity) is very simple and relatively fast. Finally, a large number of different antibodies against a particular antigen can be generated in a single selection procedure. To generate recombinant monoclonal antibodies, large pools of antibodies with different antigen recognition sites can be generated using various methods all based on display libraries. Such libraries can be created in several ways: a synthetic repertoire can be generated by cloning synthetic CDR3 regions into a pool of heavy chain germline genes, and a large antibody repertoire can be generated from which recombinant antibody fragments with various specificities can be selected. A pool of human lymphocytes can be used as starting material for antibody library construction. It is possible to construct a naive repertoire of human IgM antibodies and thus create a highly diverse human library. This method has been widely and successfully used for selecting a large number of antibodies against various antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are described in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992 - 2000), Chapter 17, Section 17.1.

[0091] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementarity-determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0092] In some embodiments, antibodies and portions thereof include, but are not limited to, antibodies, antibody fragments, Fab and F(ab’)2, single domain antigen-binding recombinant fragments, and natural nanobodies. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab’)2, scFv, scFv2, or scFv4 fragments.

[0093] In some embodiments, the invention provides a nucleic acid sequence encoding an antibody or antigen-binding portion of the invention.

[0094] For example, a polynucleotide may encode an entire immunoglobulin molecular chain, such as a light or heavy chain. A complete heavy chain includes not only the heavy chain variable region (VH), but also the heavy chain constant region (CH), typically three constant regions, CH1, CH2, and CH3, as well as a “hinge” region. In some situations, the presence of the constant region is desirable.

[0095] Other polypeptides that can be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fvs, scFvs, Fab', and CHIs, in which the CK or CL domain has been excised. Minibodies are smaller than conventional antibodies and should therefore have improved tissue penetration for use in clinical / diagnostic applications, but being bivalent, should retain a higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only the whole antibody molecule but also antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide can include at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, the framework region can include a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region. Given the properties of the individual amino acids that make up the disclosed protein products, some reasonable substitutions will be recognized by those skilled in the art. Amino acid substitutions, i.e., "conservative substitutions," can be made, for example, based on the similarity of the residues involved in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity.

[0096] Suitably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0097] As used herein, the term "non-naturally occurring" substances, compositions, entities, and / or any combination of substances, compositions or entities, or grammatical variations thereof, expressly excludes those forms of substances, compositions, entities, and / or any combination of substances, compositions or entities that are well understood by those skilled in the art to be "naturally occurring" or that can be determined or construed to be "naturally occurring" by a judge or administrative or judicial body at any time, but is a qualifying term only for the purpose of exclusion.

[0098] In some embodiments, the antibody comprises IgG4. In some embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG2 or IgG4. In some embodiments, the antibody comprises IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody does not comprise IgG1. In some embodiments, the antibody does not comprise IgG3. In some embodiments, the antibody does not comprise IgG1 or IgG3. In some embodiments, the antibody comprises mutated IgG1 and / or IgG3, where the mutation inhibits the induction of ADCC, CDC, or both. In some embodiments, the mutation is in the FcR gamma binding motif.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6 (QQYGSSPPYT). In some embodiments, the CDRs follow the Kabat numbering system.

[0100] According to another aspect, an antibody or an antigen-binding fragment thereof is provided, which comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 6 (QQYGSSPPYT). In some embodiments, the CDRs follow the Kabat numbering system.

[0101] In some embodiments, the antibody or an antigen-binding fragment thereof comprises three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 11 (GFTFSSYA), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 12 (ISGSGGST), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 13 (AKAPGDYTAYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 14 (QSVSSY), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 15 (GAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 16 (QQYGSSPPYT). In some embodiments, the CDRs are according to the IMGT numbering system.

[0102] According to another aspect, an antibody or an antigen-binding portion thereof comprising three heavy-chain CDRs (CDR-H) and three light-chain CDRs (CDR-L) is provided, wherein CDR-H1 comprises the amino acid sequence shown in SEQ ID NO: 11 (GFTFSSYA), CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 12 (ISGSGGST), CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 13 (AKAPGDYTAYFDY), CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 14 (QSVSSY), CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 15 (GAS), and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 16 (QQYGSSPPYT). In some embodiments, the CDRs are according to the IMGT numbering system.

[0103] In some embodiments, the antibody comprises a heavy chain comprising the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO: 7. In some aspects, the antibody comprises a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO: 9.

[0104] In some embodiments, the antibody comprises a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8). In some embodiments, the light chain consists of SEQ ID NO: 8. In some aspects, the antibody comprises a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). In some embodiments, the light chain consists of the sequence of SEQ ID NO: 10.

[0105] In some embodiments, the heavy chain and the light chain are linked by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24 or 25 amino acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24 or 25 amino acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is between 1 and 25, 5 and 25, 10 and 25, 15 and 25, 20 and 25, 1 and 20, 5 and 20, 10 and 20, 15 and 20, 1 and 15, 5 and 15, 10 and 15, 1 and 10, 5 and 10 or 1 and 5 amino acids in length. Each possibility represents a separate embodiment of the invention.

[0106] According to another aspect, there is provided a pharmaceutical composition comprising the agent of the invention.

[0107] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant. In some embodiments, the pharmaceutical composition is a therapeutically effective amount of the agent of the present invention.

[0108] As used herein, the terms "carrier", "excipient", or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium such as physiological saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can serve as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic physiological saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art and are described, for example, in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001), the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004), and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, normal saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al., Eds., Pergamon Press (1990), Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), the entire contents of each of which are hereby incorporated by reference in their entirety.The compositions described herein may also be included in artificially produced structures such as liposomes, ISCOMs, sustained release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids that generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally determined by considerations such as the size of the liposome and its stability in the blood. For example, as discussed by Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, various methods are available for preparing liposomes, see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0109] The carrier may generally constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0110] The term "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount effective over the dosage and time period required to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen will be determined by the physician in accordance with the condition of the patient.

[0111] In some embodiments, the agent is for use in treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In some embodiments, the disease or condition is characterized by cells that express HVEM. In some embodiments, the cells are diseased cells characterized by expressing HVEM. In some embodiments, the cells are characterized by overexpressing HVEM. In some embodiments, the overexpression is compared to healthy cells. In some embodiments, the healthy cells are non-afflicted cells. In some embodiments, the overexpression is an increase in expression. In some embodiments, the disease or condition is an HVEM-positive disease or condition. In some embodiments, the disease or condition is a BTLA-positive disease or condition.

[0112] In some embodiments, the disease or condition is an HVEM-positive cancer. In some embodiments, the disease or condition is a BTLA-positive cancer. In some embodiments, the disease or condition is an HVEM-positive pre-cancerous lesion. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is selected from melanoma, kidney cancer, cervical cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, and head and neck cancer. In some embodiments, the kidney cancer is renal cell carcinoma. One of ordinary skill in the art will understand that any cancer expressing HVEM can be a therapeutic target. In some embodiments, the disease is an inflammatory disease. In some embodiments, the infected cells comprise HVEM expression. In some embodiments, the infected cells comprise HVEM expression. In some embodiments, the infection is bacterial and the bacterial cells comprise HVEM expression. In some embodiments, the infection is a fungal infection and the fungal cells comprise HVEM expression. In some embodiments, the infection is viral and the target cells infected with the virus comprise HVEM expression. In some embodiments, the virus is a herpes virus. In some embodiments, the HVEM expression is HVEM overexpression. In some embodiments, the HVEM expression is an increase in HVEM expression.

[0113] In another aspect, a method of treating an HVEM-positive disease or condition is provided, the method comprising inhibiting HVEM and BTLA interaction and activating HVEM signaling in immune cells, diseased cells, or both.

[0114] In some embodiments, the method comprises administering an agent of the invention. In some embodiments, the method comprises administering a pharmaceutical composition of the invention.

[0115] In another aspect, a method of treating an HVEM-positive disease or condition is provided, the method comprising administering an agent of the invention.

[0116] In another aspect, a method of treating an HVEM-positive disease or condition is provided, the method comprising administering a pharmaceutical composition of the invention.

[0117] In some embodiments, the method further comprises not inhibiting the HVEM-TNFSF14 interaction. In some embodiments, the method further comprises substantially not inhibiting the HVEM-TNFSF14 interaction.

[0118] In some embodiments, the method further comprises inhibiting the interaction between PD-1 and one of its ligands. In some embodiments, the PD-1 ligand is selected from PD-L1 and PD-L2. In some embodiments, the method further comprises inhibiting PD-1, PD-L1 or PD-L2. In some embodiments, the method further comprises inhibiting the interaction from PD-1 to PD-L1. In some embodiments, the method further comprises inhibiting the interaction from PD-1 to PD-L2. In some embodiments, the method further comprises inhibiting the interaction from PD-1 to PD-L1 or PD-L2. In some embodiments, the inhibition of the interaction comprises administering an agent that inhibits the interaction from PD-1 to PD-L1 or the interaction from PD-1 to PD-L2. In some embodiments, inhibiting the interaction comprises administering an agent that inhibits the interaction between PD-1 and at least one of its ligands. In some embodiments, the at least one ligand is two ligands. In some embodiments, the inhibition of the interaction comprises PD-1 / PD-L1 blockade. In some embodiments, the inhibition of the interaction comprises PD-1 / PD-L1 or PD-L2 blockade. In some embodiments, the inhibition of the interaction comprises anti-PD-1 / PD-L1 therapy. In some embodiments, the inhibition of the interaction comprises anti-PD-1 / PD-L1 or PD-L2 therapy. In some embodiments, the therapy is immunotherapy. In some embodiments, the agent that inhibits the interaction is an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the agent that inhibits the interaction is an anti-PD-1, anti-PD-L1 or anti-PD-L2 antibody. In some embodiments, the agent that inhibits the interaction is a PD-1 / PD-L1 inhibitor. In some embodiments, the agent that inhibits the interaction is a PD-1 / PD-L1 / PD-L2 inhibitor. In some embodiments, the antibody is a blocking antibody.PD-L1 / L2 and PD-1 therapies are well known in the art and include, but are not limited to, nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab, avelumab, durvalumab, cemiplimab (Libtayo), pidilizumab, AMP-224, AMP-514, and PDR001.

[0119] In some embodiments, the method of treatment further comprises administering another therapeutic agent to HVEM-expressing cells. In some embodiments, the other therapeutic agent is an anti-cancer therapeutic agent. In some embodiments, the therapeutic agent is a non-autologous immune cell. In some embodiments, the other therapy is an autologous immune cell. In some embodiments, the immune cell is a CAR-expressing immune cell. In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the autologous immune cell is TIL therapy. In some embodiments, the non-autologous immune cell is adoptive cell therapy. In some embodiments, the autologous immune cell is adoptive cell therapy. In some embodiments, the adoptive cell is a CAR cell. In some embodiments, the adoptive cell is a TIL.

[0120] In some embodiments, the composition of the present invention and another therapeutic agent are administered simultaneously. In some embodiments, the composition of the present invention is administered before, after, or simultaneously with another therapeutic agent.

[0121] According to another aspect, a method for determining the suitability of a subject by the method of the present invention, the method comprising obtaining a sample from the subject and determining the level of HVEM in the sample, wherein positive expression of HVEM indicates that the subject is suitable for the method of treatment of the present invention.

[0122] In another aspect, a method for detecting HVEM in a sample is provided, the method comprising contacting the sample with the agent of the present invention, or an antibody of the present invention or an antigen-binding fragment thereof, thereby detecting HVEM.

[0123] In some embodiments, the contacting is under conditions suitable for the binding of an agent or antibody or antigen-binding fragment thereof that binds to HVEM. In some embodiments, the conditions are suitable for specific binding to HVEM. In some embodiments, the binding is to hybridize. The antibody / agent binding conditions can vary depending on the sample. One of ordinary skill in the art will understand that the conditions required for binding to a tissue sample (depending on the method / type of fixation) are different from the conditions for binding in a liquid solution such as a whole cell lysate. Such binding conditions are well known in the art, and one of ordinary skill in the art can select the appropriate conditions for a given sample.

[0124] In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a paraffin-embedded sample. In some embodiments, the sample is a perfusion sample. In some embodiments, the sample is from a subject. In some embodiments, the sample is a healthy sample. In some embodiments, the sample is a diseased sample. In some embodiments, the sample is a diagnostic sample.

[0125] In some embodiments, the method further comprises detecting an agent of the invention, or an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the detection is by immunohistochemical detection. In some embodiments, the detection is by immunofluorescent detection. In some embodiments, the detection is by FACS detection. In some embodiments, the detection further comprises contacting the sample with a secondary antibody that recognizes an agent or antibody of the invention. In some embodiments, the detection comprises detecting the secondary antibody. In some embodiments, the detection comprises microscopy.

[0126] In some embodiments, the subject has a disease or condition. In some embodiments, the subject is suspected of having a disease or condition. In some embodiments, the subject is at risk of developing a disease or condition. In some embodiments, the subject has a disease or condition that may include an increase in HVEM expression. In some embodiments, the subject has a disease or condition that may be characterized by an increase in HVEM expression. In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer that did not respond to first-line therapy. In some embodiments, the cancer is a cancer recurrence. In some embodiments, the cancer is a cancer that did not respond to PD-1 / PD-L1 therapy.

[0127] In some embodiments, the sample is a disease sample. In some embodiments, the sample is a biological fluid. In some embodiments, the biological fluid is selected from blood, serum, plasma, urine, feces, bile, semen, tumor fluid, and cerebrospinal fluid. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a cell sample. In some embodiments, the sample contains cells. In some embodiments, the sample contains diseased cells. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy.

[0128] In some embodiments, positive expression of HVEM includes HVEM expression. In some embodiments, positive expression of HVEM includes elevated HVEM levels. In some embodiments, positive expression of HVEM includes increased HVEM levels. In some embodiments, positive expression of HVEM includes overexpression of HVEM. In some embodiments, positive expression of HVEM is compared to a healthy sample. In some embodiments, a healthy sample is from healthy tissue and / or diseased tissue and / or cells adjacent to cells. In some embodiments, a healthy sample includes uninfected cells. In some embodiments, a healthy sample is for a healthy donor. In some embodiments, positive expression of HVEM is compared to a predetermined threshold.

[0129] According to another aspect, there is provided a pharmaceutical composition of the present invention or a kit comprising the agent of the present invention.

[0130] In some embodiments, the kit further comprises a PD-1-based therapy. In some embodiments, the kit further comprises a PD-L1-based therapy. In some embodiments, the therapy is immunotherapy. In some embodiments, the therapy is an anti-PD-1 therapy. In some embodiments, the therapy is an anti-PD-L1 therapy. In some embodiments, the therapy is a PD-1 / PD-L1 blockade therapy. In some embodiments, the therapy is a blocking antibody. In some embodiments, the therapy is an anti-PD-1 antibody. In some embodiments, the therapy is an anti-PD-L1 antibody. In some embodiments, the antibody is a blocking antibody.

[0131] In some embodiments, the kit further comprises a label indicating that the pharmaceutical composition of the present invention is for use with a PD-1 and / or PD-L1-based therapy. In some embodiments, the kit further comprises a label indicating that the agent of the present invention is for use in a PD-1 and / or PD-L1-based therapy.

[0132] In some embodiments, the kit further comprises a secondary detection molecule. In some embodiments, the detection molecule is for detecting the agent of the present invention. In some embodiments, the secondary detection molecule is an antibody that binds to the agent of the present invention. In some embodiments, the detection molecule comprises a detectable moiety. Examples of detectable moieties include, but are not limited to, fluorescent moieties, tags, radiolabels, dyes, and chemiluminescent moieties. In some embodiments, the detectable moiety is a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, GFP, RFP, YFP, APC, CY5, CY7, and Pacific Blue. Secondary detection molecules are well known in the art, and any such molecule that detects the agent of the present invention can be used.

[0133] As used herein, the term "about," when combined with a value, refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0134] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. Further, it should be noted that the claims may be drafted to exclude any element. Thus, this description is intended to serve as a precedent for the use of exclusive terms such as "solely," "only," etc. in connection with the recitation of claim elements or the use of "negative" limitations.

[0135] When conventions similar to "at least one of A, B, and C, etc." are used, generally, such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (for example, "a system having at least one of A, B, and C" includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by one of ordinary skill in the art that substantially any disjunctive and / or phrase presenting two or more alternative terms, regardless of whether in the description, claims, or drawings, is intended to contemplate the possibility of including one of the terms, any of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0136] For clarity, it is understood that certain features of the invention that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable partial combination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and are disclosed herein as if every possible combination were individually and explicitly disclosed. Additionally, all partial combinations of the various embodiments and their elements are also specifically encompassed by the present invention and are disclosed herein as if every such partial combination were individually and explicitly disclosed herein.

[0137] Additional objects, advantages, and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and claimed in the following claims section finds experimental support in the following examples.

[0138] Various embodiments and aspects of the invention as described above and claimed in the sections of the following claims find experimental evidence in the following examples.

Example

[0139] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well explained in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994), Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998), the methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994), "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition, "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994), Stites et al.(Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (Eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), are hereby incorporated by reference in their entirety. Other general references are provided throughout this book.

[0140] Materials and Methods Generation of scFv against HVEM by phage display: A naive human scFv library was incubated with a synthetic / recombinant tag to subtract tag-specific clones and with control CHO-K1 empty cells to subtract CHO-K1 cell-binding clones. The phages were then subjected to three rounds of biopanning via incubation with CHO-K1 cells overexpressing hrHVEM. Clones were selected, rescued with the help of helper phage, and screened by phage ELISA against hrHVEM, mrHVEM, and crHVEM. The sequences of the clones were determined, and positive unique clones were subcloned into the pET25b protein expression vector. Lysates were prepared from 200 ml of induced cultures. Bacterial cells were pelleted, washed with PBS, and lysed with a French press. Insoluble fragments were removed by high-speed centrifugation, and lysates containing soluble proteins including scFv were collected. The scFv was cloned with a HIS tag.

[0141] Generation of transfected CHO-K1 cells: CHO-K1 cells were seeded in 6-well plates at a density of 200K cells / well in Nutrient Mixture F-12 (Ham's) with L-glutamine medium (Biological Industries, Israel) supplemented with 10% FBS (Biological Industries, Israel) and incubated overnight at 37°C. After incubation, 2 μg of plasmid DNA was transfected using 4 μl of jetPRIME transfection reagent (Polyplus-transfection, France) according to the manufacturer's instructions. Selection with 1 mg / ml G418 sulfate antibiotic (Millipore, Germany) was performed on the second day after transfection, and the cells were cultured as normal with the antibiotic added.

[0142] Flow cytometry: Surface cell staining was performed on ≤1x10 5 cells diluted in FACS buffer (PBS, 0.02% sodium azide, 0.5% BSA) with the appropriate pure specific antibody on ice for 30 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes at 4°C and the supernatant was removed. The cells were incubated on ice for 30 minutes covered with the appropriate fluorophore-conjugated secondary antibody. In the case of fluorophore-conjugated primary antibody, direct staining was performed without secondary antibody. After incubation, the cells were washed and resuspended in 200 μl of FACS buffer for flow cytometry analysis.

[0143] mAb production: a. Luciferase reporter assay: Luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega, USA) according to the manufacturer's instructions. Briefly, the luciferase assay reagent was prepared by combining and mixing 1 bottle of buffer and 1 bottle of substrate. Equal volumes of reagent and sample were mixed, and luminescence was measured 3 or 10 minutes after adding the reagent using appropriate instrument settings. b. Plasmid Preparation: scFv2 was converted to a complete IgG4 antibody containing the S228P mutation. The target DNA sequence was designed, optimized, and synthesized. The complete sequence was subcloned into the pcDNA3.4 vector. Transfection-grade plasmids were maxi-prepared for Expi293F cell expression. c. Cell Culture and Transient Transfection: Expi293F cells were cultured in serum-free Expi293FTM expression medium (Thermo Fisher Scientific, USA). The cells were maintained in Erlenmeyer flasks (Corning Inc., USA) on an orbital shaker (VWR Scientific, USA) at 37 °C with 8% CO2. One day prior to transfection, the cells were seeded into Corning Erlenmeyer flasks at an appropriate density. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and added to the flask with cells ready for transfection. The recombinant plasmid encoding the target protein was transiently transfected into the suspension Expi293F cell culture. The cell culture supernatant collected on the 6th day after transfection was used for purification.

[0144] Purification and Analysis: After centrifuging the cell culture, it was filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and eluting with an appropriate buffer, the eluted fractions were pooled and the buffer was exchanged to the final formulation buffer. The purified protein was analyzed for molecular weight and purity measurement by SDS-PAGE and Western blotting analysis. The concentration was measured by the A280 method (for antibodies).

[0145] Cytotoxicity assay: Melanoma cells stained with nuclear RFP were seeded and allowed to adhere overnight to incubate. Next, the melanoma cells were pre-incubated with 20 μg / ml of the mAb of the present invention or an hIgG4 isotype control antibody (Invivogen, France), and the TILs were pre-incubated with 20 μg / ml of an anti-PD1 mAb (nivolumab, BMS) or an hIgG4 isotype control antibody (Invivogen, France). Media containing only the pre-incubated TILs or anti-PD1 or hIgG4 isotype control mAb were added to the melanoma cells together with the CellEvent Caspase-3 / 7 Green Detection Reagent (Invitrogen, USA). The plates were placed in an Incucyte ZOOM system (ESSEN Bioscience, USA) and scanned every hour. Caspase 3 / 7 positive events reflecting specific killing of melanoma were counted. The same protocol was used for the cytotoxicity assay experiment using RCC cells and the cytotoxicity assay experiment using MAB356.

[0146] T cell activation assay by flow cytometry: Melanoma cells or media alone were incubated at 37°C for 1 hour with 20 μg / ml of the mAb of the present invention or an hIgG4 isotype control antibody (Invivogen, France). After 1 hour, TILs were added to the melanoma cells or media and co-cultured at 37°C for 4 hours. After 4 hours, the cells were surface stained with anti-CD3 (Biolegend, USA) and anti-41BB (Biolegend, USA), and the expression was evaluated by flow cytometry using a CytoFLEX instrument (Beckman Coulter, USA), and data analysis was performed using FCS Express 6 software (DeNovo Software, US).

[0147] T cell activation assay by Incucyte ZOOM system: Nucleus RFP-stained melanoma cells were seeded and allowed to adhere for overnight incubation. After overnight incubation, the melanoma cells were incubated with 20 μg / ml of the mAb of the present invention or hIgG4 isotype control at 37 °C for 1 hour. MART-1 electroporated PBMC or medium was added to the melanoma cells together with anti-41BB (Biolegend, USA) labeled with FabFlour-488 reagent (ESSEN Bioscience, USA). The plates were placed in the Incucyte ZOOM system and scanned every hour.

[0148] T cell cluster assay: Images were acquired by the Incucyte ZOOM system. The number of clusters was calculated by image analysis using ImageJ version 1.52a. After the images were smoothed and the contrast was enhanced, they were converted to 8-bit images. After binarization of the images, the images were processed by applying the "Close", "Fill holes", and "Erode" commands. Next, the "Analyze particles" command was applied with the following parameters: size 500 mm^2 - infinity and circularity 0.1 - 1.

[0149] Example 1: Identification of HVEM as an immune checkpoint target A high-throughput genome-wide primary siRNA screening consisting of approximately 8,300 genes was performed in two patient-derived melanoma cell lines. Using an LDH-based cytotoxicity assay, the effect on cell death was evaluated 96 hours after siRNA transfection by co-culturing with TIL overnight. The success of the knockdown of the positive control was evaluated by FACS. A confirmation screen consisting of 360 genes was verified in 8 melanoma cell lines using the same design as the primary screen. Positive hits were defined based on the Z-score and the ratio of enhanced cell death, with the effect of the siRNA on proliferation being corrected. Finally, a validation screen consisting of 54 genes was performed in 5 melanoma cell lines. HVEM was discovered as a strong positive hit as a potential immunotherapy target.

[0150] Example 2: Selection of scFv against HVEM After establishing HVEM as a potential target for therapeutic immune blockade in cancer treatment, single-chain antibody fragments (scFvs) that can bind to HVEM were isolated and assayed. The scFvs were isolated as described above (Materials and Methods). Six selected scFvs were screened for binding to recombinant human, mouse, and cynomolgus monkey HVEM (see Table 1). Five out of six showed strong binding to human recombinant HVEM (hrHVEM) expressed in CHO-K1 cells as measured by cell ELISA. CHO-K1 empty cells were used as a negative control. Two of these five showed very strong binding to mouse recombinant HVEM (mrHVEM), while the other three showed much weaker binding. One of the five also showed strong binding to monkey (cynomolgus) HVEM, while the other three showed much weaker binding. These results were confirmed using FACS analysis. The scFvs contained HIS tags, and an anti-HIS secondary APC-conjugated antibody was used for detection of surface binding. As predicted by ELISA, it was found that five scFvs bound to surface hrHVEM of CHO-K1 HVEM-overexpressing cells, while two scFvs had insufficient binding (Figure 1).

[0151] Table 1. Cell ELISA using lysates containing scFv against CHO-K1 cells overexpressing HVEM. Numbers represent OD values. [Table 1]

[0152] Next, the ability of the scFv to inhibit the interaction between HVEM and its two ligands, BTLA and LIGHT (TNFSF14), was confirmed. ELISA plates were coated with 2.5 μg / ml of hrHVEM and incubated overnight at 4°C. The plates were then washed and blocked with 2% BSA for 1 hour at room temperature. The scFv1-6 lysates and control lysates were added to the ELISA plates and incubated for 1 hour at room temperature. The plates were washed, blocked for 10 minutes, and then washed again. hrBTLA-Fc or hrLIGHT-HIS was added to the ELISA plates and incubated for 2 hours at room temperature. The plates were washed, blocked, and incubated with mouse anti-BTLA biotin mAb or goat anti-LIGHT pAb for 1 hour at room temperature. The plates were washed and incubated with streptavidin peroxidase or goat anti-goat HRP antibody for 30 minutes at room temperature. Finally, the plates were washed and TMB followed by a sulfuric acid stop solution was added. Absorbance was read at 450 nM and 570 nM using a Spark 10M plate reader. Unexpectedly, scFvs 1, 3, 4, and 6 bound to hrHVEM, but these antibodies did not inhibit HVEM-BTLA binding. In contrast, scFv2 almost completely inhibited BTLA binding (Figure 2A). Notably, none of the antibodies significantly inhibited LIGHT binding to HVEM (Figure 2B). scFv2 was selected for all future work because it binds to human, mouse, and monkey rHVEM and has the ability to block the HVEM-BTLA interaction.

[0153] Example 3: Anti-HVEM IgG Antibody The single-chain antibody scFv2 was effective in blocking the HVEM-BTLA interaction, but a full IgG monoclonal antibody (mAb) was designed from the light and heavy chains of scFv2, particularly the CDRs. scFv2 was sequenced and the light and heavy chains were cloned into an IgG4 backbone, excluding the S228P mutation. The complete sequence of the heavy chain of this mAb is shown in SEQ ID NO: 9, and the light chain is shown in SEQ ID NO: 10. The presence of the full IgG antibody was confirmed by SDS-PAGE and Western blot (Figure 3).

[0154] Next, the binding of the new mAbs to recombinant HVEM from human, mouse, and monkey was evaluated as before. Binding to hrHVEM was evaluated by ELISA as before. The new mAbs bound strongly to hrHVEM (Figure 4A). Binding was also evaluated by FACS as before. CHO-K1 empty, CHO-K1 hrHVEM, CHO-K1 mrHVEM, and CHO-K1 crHVEM cells were incubated on ice for 1 hour with 20 μg / ml of the antibody or hIgG4 isotype control antibody, and then stained with anti-human IgG FC biotin and anti-biotin FITC. Expression was evaluated by flow cytometry using a CytoFLEX instrument and data analysis using FCS Express 6 software. The new mAbs bound strongly to rHVEM in all three species (Figure 4B).

[0155] Since the new mAb can bind to recombinant human HVEM, its ability to bind to endogenous HVEM on the surface of healthy and cancer cells was tested. Initial immunohistochemistry was performed on healthy human colon and spleen tissues using the mAb of the invention, where strong staining was observed (Figure 5A). Next, melanoma cells from three primary patient samples known to express HVEM (Figure 5B) were incubated on ice for 1 hour with 20 μg / ml of the mAb of the invention, followed by staining with anti-human IgG FC biotin and anti-biotin FITC. Expression was evaluated by FACS as before. The new mAb bound to all three melanoma samples tested (Figure 5C). Binding to renal cell carcinoma (RCC) patient samples was also tested. In this case, a commercially available anti-HVEM antibody (R&D Systems AF356) was used to confirm that the cancer cells expressed HVEM (Figure 5D). Binding of the new mAb to these surfaces was equivalent to that of the commercially available antibody (Figure 5E). Finally, tumor microarrays were probed with the mAb of the invention (Figure 5F). Twelve different indications, including one healthy tissue sample and five different tumor samples for each indication, were tested. Healthy pancreas, lung, breast, bladder, and ovary were HVEM negative. On the other hand, lymph nodes, larynx / oral cavity / tongue, brain, kidney, liver, and stomach were all positive to varying degrees. Not all tumors expressed HVEM, but at least one tumor sample from each organ type was positive.

[0156] The ability of the full mAb to inhibit BTLA binding to HVEM was also evaluated as before. In the presence of the mAb of the present invention, as measured by ELISA, BTLA did not bind to HVEM (Figure 6A). The addition of the isotype control IgG functioned as a positive control for strong binding of BTLA to HVEM. Also, as seen with the scFv, the mAb did not inhibit the binding of LIGHT to HVEM (Figure 6B). In contrast, the commercially available anti-HVEM mAB, MAB356, was able to bind strongly to hrHVEM as expected (Figure 6C), but the presence of MAB356 only slightly interfered with the interaction between HVEM and BTLA as compared to the addition of the mIgG1 isotype control antibody (Figure 6D). Thus, the mAb of the present invention is far superior to MAB356 with respect to inhibition of the BTLA-HVEM interaction.

[0157] Next, downstream signaling via HVEM was examined. Activation of HVEM by ligand binding induces NF-kB signaling. To evaluate the effect of the mAb of the present invention on HVEM signaling, Jurkat T cells expressing hrHVEM and NF-kB luciferase reporter were pre-incubated on ice for 1 hour with 20 μg / ml of the mAb or hIgG4 isotype control. After washing the cells, they were co-cultured at 37 °C for 6 hours with CHO-K1 empty, CHO-K1-hrBTLA, or no cells. Luciferase activity was tested using the Bright-Glo luciferase assay system, and luminescence was measured using a Spark 10M plate reader 10 minutes after reagent addition. Luciferase was induced in Jurkat cells pre-incubated with the control antibody only when CHO cells expressed BTLA (Figure 7, right). This is expected because NF-kB signaling should be induced only by the ligand of HVEM. Interestingly, when pre-incubation was performed with the mAb of the present invention and co-incubation was performed with CHO-K1-hrBTLA cells, no very strong induction of luciferase was observed (Figure 7, left middle bar). This is because the mAb blocks the interaction of HVEM-BTLA. However, the level of luciferase after pre-incubation with the mAb is also not the baseline. Rather, the mAb itself induces mild activation of HVEM and NF-kB signaling (Figure 7, left). The same level of activation is seen regardless of whether CHO cells are co-incubated. This confirms that this activation is caused by the mAb itself rather than the cells.

[0158] Example 4: The mAb of the present invention enhances the cytotoxicity of T cells against cancer cells. The mAb binds to HVEM and blocks binding to BTLA, and was theorized to reduce the inhibitory effect of BTLA signaling on T cells. To test this, adherent melanoma cells were incubated with 20 μg / ml of the mAb of the invention or an hIgG4 isotype control antibody at 37° C. for 1 hour. After 1 hour, tumor infiltrating lymphocytes (TILs) were added to the melanoma cells and co-incubated at 37° C. for 24 hours. After incubation, supernatants were collected from co-culture samples or samples of TILs only as controls for background IFNγ secretion. IFNγ secretion was measured by ELISA using a Human IFNγ ELISA Max Deluxe kit. Pre-incubation with the mAb of the invention resulted in almost a 2-fold increase in the amount of IFNγ secreted in one melanoma cell line and, still importantly, an increase, albeit lower, in a second line (FIG. 8), indicating that T cell inhibition is indeed reduced by the antibody.

[0159] Next, specific killing of primary patient melanoma cells in the presence of autologous TILs was measured. Adherent melanoma cells from two patients were pre-incubated with the mAb of the invention or an isotype control. At the same time, TILs were pre-incubated with an anti-PD-1 antibody or an isotype control. Next, melanoma cells and TILs were co-incubated in all possible combinations and specific killing of the melanoma cells was measured every hour for 11 hours (FIGS. 9A-B). In the first cell line, addition of the mAb of the invention resulted in an almost 30% increase in total cell killing after 11 hours compared to cells treated with the isotype control. Similar levels of killing were observed with anti-PD1 treatment (33% increase). Combination treatment with both antibodies resulted in an additive increase in total killing of 60%. The combined effect was strengthened by the results obtained with cells from the second patient. Again, the mAb of the invention and anti-PD1 showed similar effects, increasing killing by 67% and 64% respectively. Unexpectedly, combination treatment with both antibodies resulted in a synergistic effect, with total killing increasing 180% compared to control co-cultures. This indicates that the two antibodies can work together to provide a synergistic effect on immune checkpoint inhibition.

[0160] Next, MAB356 was tested in the same system. The killing of melanoma cells by TILs was tested as described above using the melanoma cells of the first patient. Melanoma cells were pre-incubated with MAB356 or the mAb of the present invention or an appropriate isotype control and then mixed with autologous TILs. Specific killing of melanoma cells was measured every hour for 15 hours. The addition of the mAb of the present invention resulted in a 22% increase in total cell killing after 15 hours compared to cells treated with the hIgG4 isotype control (Figure 9D), while the addition of commercially available MAB356 resulted in a 9% decrease in total cell killing compared to cells treated with the mIgG1 isotype control. (Figure 9E). This clearly shows that the mAb of the present invention is superior to MAB356.

[0161] A similar experiment was conducted with cells from RCC patients. Before co-culturing with TILs from the same patient, the cancer cells were allowed to adhere overnight and pre-incubated with the mAb of the present invention or an isotype control for 1 hour. Cell death of the cancer cells was measured by caspase 3 / 7 positive events, and after 26 hours, the mAb of the present invention was found to increase total cell killing by 47% compared to the isotype control (Figure 9C).

[0162] Example 5: The mAb of the present invention enhances the cytotoxicity of non-autologous immune cells against cancer cells. Since it was shown that the cytotoxicity of TILs from cancer patients can be enhanced using the mAb of the present invention, the ability to enhance non-autologous immune cells was tested. PBMCs were collected from healthy donors and made to express the alpha and beta chains of the human MART1 TCR. Nucleus RFP-stained melanoma cells expressing MART1 were allowed to adhere overnight and pre-incubated with the mAb of the present invention or an isotype control for 1 hour before co-culturing with PBMCs. Cell death of the cancer cells was measured by caspase 3 / 7 positive events, and after 26 hours, the mAb of the present invention was found to increase total cell killing by 28% compared to the isotype control (Figure 10A).

[0163] Inflammatory cytokine secretion from PBMCs was also measured. Specifically, interferon gamma (IFNγ) secretion was measured in the medium from PBMCs cultured in a medium containing the mAb or isotype control of the present invention, or cultured with melanoma cells pre-incubated with the mAb or isotype control of the present invention. IFNγ levels were measured 24 hours after the start of culture. As expected, incubation with melanoma cells significantly increased IFNγ secretion, but incubation with melanoma cells pre-incubated with the mAb of the present invention further increased secretion by 32% compared to melanoma cells pre-incubated with the isotype control (Figure 10B).

[0164] To rule out the possibility that the increased cytotoxicity was due to an increase in T cell death and was directly caused by the antibody, the direct cytotoxic effect of the mAb was measured. Adherent melanoma cells stained with nuclear RFP were incubated with 20 μg / ml of the mAb of the present invention or the hIgG4 isotype control antibody at 37°C for 1 hour. After 1 hour, the CellEvent Caspase-3 / 7 Green Detection Reagent was added to the melanoma cells. The plates were placed in an Incucyte ZOOM system (ESSEN Bioscience, USA) and scanned every hour for 15 hours. No increase in cell death was observed in two different melanoma cells incubated with the mAb (Figure 11). This indicates that the antibody itself is not cytotoxic, and the increased cancer cell death observed in the last experiment is rather due to an increase in the cytotoxicity of T cells.

[0165] Example 6: The mAb of the present invention induces basic gentle activation of TILs. While reducing BTLA-induced suppression in T cells, the mAbs of the invention also bind to HVEM on the T cells themselves. Since the mAbs increase NF-kB-induced transcription via HVEM signaling, the direct binding of the mAbs can directly increase the activation of T cells. To test this, TILs were stimulated with the mAbs or isotype controls alone and in the presence of autologous melanoma cells, and the activation of T cells was monitored (Figure 12A). The percentage of activated T cells increased in the presence of the mAbs, regardless of the presence or absence of melanoma cells. This indicates that the binding of the antibody to HVEM on T cells induces activation by itself. Thus, the antibody has at least a dual effect, both of which block the cancer cell's immune surveillance evasion mechanism while directly activating T cells.

[0166] Signaling via HVEM was also tested by measuring T cell clustering. Clustering of cells is characteristic of activated T cells. TILs from two different melanoma patients were incubated with the mAbs of the invention or hIgG4 isotype control for 20 hours and then the number of clusters was measured. For both samples tested, the mAbs of the invention significantly increased T cell clustering (Figure 12B). When MAB356 was tested, no change in T cell clustering was observed compared to the mIgG1 control (Figure 12C). This indicates that the binding of the mAbs of the invention to HVEM on T cells induces signaling and low levels of activation, while the binding of commercially available MAB356 does not.

[0167] Activation of PBMCs in the presence of melanoma cells was also measured. As before, PBMCs expressed a MART-1 specific TCR to target melanoma cells. For these non-self cells, the mAbs significantly increased activation, as measured by 41BB expression on the cells (Figure 12D).

[0168] When administering immune checkpoint inhibitors, cytokine release syndrome is always a concern. As shown above, the mAb of the present invention can induce mild activation of TILs even in the absence of cancer cells. To further elucidate the degree of this activation, a cytokine release assay was performed. Whole blood was collected from 10 healthy donors and tested with the mAb of the present invention at concentrations of 0.1, 1, 10, and 100 μg / ml. After 24 hours, the expression of five cytokines (IL-6, IL-8, IL-10, IFNγ, TNFα) in the samples was measured. Two commercially available anti-cancer antibodies, Erbitux (EGFR inhibitor) and Remtrada (anti-CD52), were used as controls. The results of the analysis are summarized in Figure 13. Unlike Remtrada, which induced a strong cytokine response, the mAb of the present invention produced only a mild response comparable to Erbitux.

[0169] Example 7: In Vivo Cancer Treatment with the mAb of the Present Invention. Human melanoma cells were subcutaneously transplanted into the flanks of SCID-NOD mice (day 0). When the tumors reached approximately 45 mm^3 (day 17), the mice were assigned to four treatment groups (3 mice / group) and human autologous TILs were administered intravenously. TILs were administered again on day 34. On day 17, simultaneously with TIL administration, hIL-2 was administered subcutaneously for 5 consecutive days and then twice a week. On day 18, and then twice a week, the mice were administered intravenously with hIgG4 isotype control (black circles), the mAb of the present invention (white squares), anti-PD1 (black triangles), or a combination of both antibodies (black diamonds). Treatment with the mAb of the present invention resulted in 31% tumor growth inhibition (TGI) compared to treatment with the isotype control, while treatment with anti-PD1 resulted in only 11% TGI. The combination treatment had a synergistic effect, and the TGI was 48% (Figure 14). No harmful side effects were observed in any of the mice. These results demonstrate that the antibody of the present invention is effective in the in vivo treatment of cancer and that combination treatment with an anti-PD1 antibody has a synergistic effect that enhances the treatment.

[0170] Although the present invention has been described in conjunction with its specific embodiments, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. 1. An antibody or antigen-binding fragment thereof that binds to herpes virus entry mediator (HVEM) and inhibits the interaction between said HVEM and B and T lymphocyte attenuator (BTLA), comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6 (QQYGSPPYT); Optionally, the heavy chain may comprise the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7) and the light chain may comprise the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8); An antibody or an antigen-binding fragment thereof.

2. Sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCCSRSTSESTAALGCLVKDYFPEVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM , and a heavy chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSG 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain comprising SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

3. An antibody or antigen-binding fragment thereof that inhibits the interaction between HVEM and BTLA, and competes with the antibody of claim 2 for binding to HVEM.

4. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharma- ceutically acceptable carrier, excipient or adjuvant.

5. 5. The pharmaceutical composition of claim 4 for use in the treatment of a disease or condition characterized by HVEM-positive disease cells, comprising: The disease is a. HVEM-positive cancer or precancerous lesion; b. HVEM-positive cancer selected from melanoma, renal cancer, cervical cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, and head and neck cancer; and c. Infection in which the infected cells contain HVEM expression; Selected from: Pharmaceutical compositions.

6. The pharmaceutical composition of claim 5 for use in combination with anti-PD-1 / PD-L1 based immunotherapy.

7. 6. The pharmaceutical composition of claim 5 for use in combination with adoptive cell therapy.

8. The adoptive cell therapy is selected from adoptive tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-expressing immune cell therapy, wherein the CAR targets a non-HVEM protein on the surface of the HVEM-expressing cell.

8. The pharmaceutical composition of claim 7.

9. The pharmaceutical composition according to any one of claims 5 to 8, wherein the disease is an HVEM-positive cancer selected from melanoma and renal cell carcinoma.

10. A pharmaceutical composition according to claim 4 ; a. Anti-PD-1 / PD-L1 based immunotherapy; b. a label indicating that the pharmaceutical composition of the invention is for use with anti-PD-1 / PD-L1 based immunotherapy; and c. A secondary detection molecule for detecting at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and at least one of:

11. A pharmaceutical composition for use in combination with anti-PD-1 / PD-L1 based immunotherapy to treat HVEM-positive cancer, comprising an antibody or antigen-binding fragment thereof that binds to HVEM and inhibits the interaction between said HVEM and BTLA, and a pharma- ceutical acceptable carrier, excipient or adjuvant.

12. 12. The pharmaceutical composition of claim 11, wherein the anti-PD-1 / PD-L1 based immunotherapy is an antibody that binds to PD-1 or PD-L1 and inhibits the interaction between PD-1 and PD-L1.

13. 13. The pharmaceutical composition of claim 11 or 12, wherein the anti-PD-1 / PD-L1 based immunotherapy is selected from nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, pidilizumab, AMP-224, AMP-514, and PDR001.

14. 14. The pharmaceutical composition of claim 13, wherein the anti-PD-1 / PD-L1 based immunotherapy is nivolumab.

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