Anti-HVEM antibodies and uses thereof

Abstract

Provided are agents that bind to HVEM, inhibit HVEM-BTLA interactions, and activate downstream HVEM signaling. Also provided are methods of treating disease using those agents and kits containing those agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to 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 contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of immunotherapy. Background Art

[0004] Immunotherapies designed to enhance immune responses are considered activating immunotherapies and are at the forefront of cancer treatment. Currently, immune checkpoint blockade therapy is successfully used for the treatment of advanced non-small cell lung cancer (NSCLC), metastatic melanoma, and advanced renal cell carcinoma (RCC). Some such drugs developed and produced by different companies, including antibodies against immune checkpoint programmed cell death protein 1 receptor (PD1), programmed cell death protein 1 ligand (PDL1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), have shown great promise in different cancer types and have been approved by the FDA. However, the response rate of patients is still not optimal, because usually only 10% to 40% of the treated patients benefit, and at the same time patients may suffer from side effects after immunotherapy treatment (Post Immunotherapy Treatments Side-Effects). In addition, treatment with these agents may induce resistance through the upregulation of additional immune checkpoints. Compared with single agents, the combination of anti-PD1 and anti-CTLA-4 therapy in melanoma patients showed a higher response rate (60%), however, this combination therapy also involves serious treatment-related adverse reactions. Therefore, the need for new activators of antitumor immunity is clear.

[0005] Herpesvirus entry mediator (HVEM) is a protein found on the surface of various cell types, including hematopoietic and non-hematopoietic cells. HVEM acts as a receptor for classical TNF-related ligands (such as LIGHT and LTα), and therefore acts as a signal transduction receptor. However, it also acts as a ligand for immunoglobulin (Ig) superfamily molecules (such as inhibitory receptors BTLA and CD160). Therefore, bidirectional signal transduction is possible for the signal transduction network mediated by HVEM, and the signal transduction network mediated by HVEM can participate in positive or negative immune responses in different situations. The dysregulation of this network is involved in the pathogenesis of autoimmune diseases, inflammatory diseases, and cancer, making HVEM a target for immunotherapy. Summary of the invention

[0006] The present invention provides agents that bind to HVEM, inhibit HVEM-BTLA interaction, inhibit downstream BTLA signaling, and activate downstream HVEM signaling. Also provided are methods of treating disease using those agents and kits containing those agents.

[0007] According to a first aspect, there is provided an agent that specifically binds to a herpes virus invasion mediator (HVEM) on a cell,

[0008] a. inhibiting the interaction between HVEM and B- and T- lymphocyte attenuator (BTLA); and

[0009] b. Activate downstream signaling through HVEM in cells.

[0010] According to another aspect, an antibody or an antigen-binding fragment thereof is provided comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence listed in SEQ ID NO:1 (SYAMS), CDR-H2 comprises the amino acid sequence listed in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence listed in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence listed in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence listed in SEQ ID NO:5 (GASSRAT), and CDR-L3 comprises the amino acid sequence listed in SEQ ID NO:6 (QQYGSSPPYT).

[0011] According to another aspect, a pharmaceutical composition is provided, which comprises an agent of the invention, or an antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0012] According to another aspect, a method of treating a disease or condition characterized by HVEM-positive cells is provided, the method comprising administering a pharmaceutical composition of the present invention, thereby treating the disease or condition.

[0013] According to another aspect, there is provided a method of treating a HVEM-positive disease or condition, the method comprising:

[0014] a. inhibiting the interaction between HVEM and BTLA; and

[0015] b. Activation of HVEM signaling in immune cells, disease cells, or both;

[0016] Thereby treating a HVEM-positive disease or condition.

[0017] According to another aspect, a method of determining suitability of a subject to be treated by the methods of the invention is provided, 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 treatment methods of the invention.

[0018] According to another aspect, a method for detecting HVEM in a sample is provided, the method comprising contacting the sample with the reagent of the present invention or the antibody or antigen-binding fragment thereof of the present invention, thereby detecting HVEM.

[0019] According to another aspect, a kit is provided, comprising a pharmaceutical composition of the present invention and at least one of:

[0020] a. Immunotherapy based on anti-PD-1 / PD-L1;

[0021] b. a label indicating that the pharmaceutical composition of the present invention is used in conjunction with an anti-PD-1 / PD-L1 based immunotherapy; and

[0022] c. A secondary detection molecule for detecting the reagent of the present invention or the antibody or antigen-binding fragment thereof of the present invention.

[0023] According to some embodiments, the agent does not substantially inhibit the interaction between HVEM and tumor necrosis factor superfamily member 14 (TNFSF14).

[0024] According to some embodiments, the agent inhibits the interaction between HVEM and CD160, lymphotoxin alpha (LTα), or both.

[0025] According to some embodiments, the agent does not directly induce apoptosis in a HVEM-expressing cell upon binding to HVEM expressed by the cell.

[0026] According to some embodiments, the agent is an antibody or an antigen-binding fragment thereof.

[0027] According to some embodiments, the reagent of the present invention comprises IgG2, IgG4, or a modified IgG1 or IgG3 that is not cytotoxic to cells.

[0028] According to some embodiments, the agent is a single chain antibody (scFv).

[0029] According to some embodiments, downstream signaling by HVEM includes nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB)-mediated transcription.

[0030] According to some embodiments, downstream signaling by HVEM includes increased cytotoxicity of immune cells expressing HVEM.

[0031] According to some embodiments, the increased cytotoxicity comprises increased secretion of pro-inflammatory cytokines.

[0032] According to some embodiments, the cell is an immune cell and the signaling induces immune activation.

[0033] According to some embodiments, the immune cells are tumor infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC).

[0034] According to some embodiments, the cell is a cancer cell and the signaling comprises an anti-tumor effect.

[0035] According to some embodiments, the agents of the invention are used to treat a disease characterized by disease cells expressing HVEM.

[0036] According to some embodiments, the disease is a HVEM-positive cancer or precancerous lesion.

[0037] 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.

[0038] According to some embodiments, the disease is an infectious disease and wherein the infected cells comprise HVEM expression.

[0039] 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 listed in SEQ ID NO:1 (SYAMS), CDR-H2 comprises the amino acid sequence listed in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence listed in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence listed in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence listed in SEQ ID NO:5 (GASSRAT), and CDR-L3 comprises the amino acid sequence listed in SEQ ID NO:6 (QQYGSSPPYT).

[0040] According to some embodiments, an agent of the invention comprises a heavy chain comprising the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7).

[0041] According to some embodiments, an agent of the invention comprises a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8).

[0042] According to some embodiments, the agent of the invention comprises: a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO: 9); and a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10).

[0043] According to some embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain comprising the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7).

[0044] According to some embodiments, the antibodies or antigen-binding fragments of the invention comprise a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8).

[0045] According to some embodiments, the antibody or antigen-binding fragment of the invention comprises: a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 9); and a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10).

[0046] According to some embodiments, the method of the present invention further comprises administering an anti-PD-1 / PD-L1 based immunotherapy.

[0047] According to some embodiments, the method of the present invention further comprises administering adoptive cell therapy.

[0048] According to some embodiments, adoptive cell therapy comprises adoptive TIL therapy.

[0049] According to some embodiments, adoptive cell therapy comprises administering immune cells expressing a chimeric antigen receptor (CAR), wherein the CAR targets a non-HVEM protein on the surface of a cell expressing HVEM.

[0050] According to some embodiments, the method of the present invention further comprises inhibiting the interaction between PD-1 and PD-L1.

[0051] According to some embodiments, the methods of the present invention further comprise not substantially inhibiting the interaction between HVEM and TNFSF14.

[0052] According to some embodiments, the disease or condition is a HVEM-positive cancer or precancerous lesion.

[0053] According to some embodiments, the HVEM-positive cancer is selected from melanoma and renal cell carcinoma.

[0054] According to some embodiments, the disease or condition is an infectious disease and wherein the infected cells comprise HVEM expression.

[0055] According to some embodiments, the immune cells are TILs or PBMCs.

[0056] According to some embodiments, the immune cell is a CAR-T cell.

[0057] According to some embodiments, the methods of the present invention comprise administering a pharmaceutical composition of the present invention.

[0058] According to some embodiments, positive expression of HVEM comprises elevated levels of HVEM compared to a healthy sample or a predetermined threshold.

[0059] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given only as examples, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art through this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1. Binding of lysates containing scFv to CHO-K1 cells overexpressing HVEM: CHO-K1 cells overexpressing human HVEM (CHO-K1 hrHVEM), CHO-K1 cells overexpressing mouse HVEM (CHO-K1mrHVEM), CHO-K1 cells overexpressing cynomolgus monkey HVEM (CHO-K1 crHVEM), or CHO-K1 cells overexpressing empty vector as a control (CHO-K1 empty) were incubated with 5 μg / ml of lysates containing HIS-tagged scFv1-6 or control lysates on ice for 1 hour, followed by staining with anti-HIS APC antibody (R&D Systems, USA) or isotype control antibody (R&D Systems, USA). Expression was assessed by flow cytometry using a CytoFLEX instrument (Beckman Coulter, USA) and data analysis using FCS Express 6 software (DeNovo Software, USA). The solid black line represents background staining of CHO-K1 null cells (i.e., isotype control antibody), the dash-dot gray line represents specific antibody staining of CHO-K1 null cells, the dash-dot black line represents specific antibody staining of CHO-K1 hrHVEM cells, the solid gray line represents specific antibody staining of CHO-K1 mrHVEM cells, and the dashed black line represents specific antibody staining of CHO-K1 crHVEM cells.

[0061] Figure 2A-2B . Bar graph of ELISA binding of hrHVEM to (2A) hrBTLA and (2B) hrLIGHT in the presence of six scFvs.

[0062] Figure 3 SDS-PAGE and Western Blot Analysis of .scFv2-IgG4(S228P): The purified protein was analyzed by SDS-PAGE (left panel) and Western Blot (right panel) to measure the molecular weight and purity. Lanes M1 and M2 represent protein markers. Lanes 1 and 2 represent reducing and non-reducing conditions, respectively. Lane P represents human IgG1-k as a positive control.

[0063] Figure 4A-4B.(4A) Histogram of mAb of the present invention binding to hrHVEM measured by ELISA. (4B) Histogram of the binding of mAb of the present invention or hIgG4 isotype control antibody to hrHVEM, mrHVEM and crHVEM expressed on CHO-K1 cells. The black and gray solid lines represent the background staining of the hIgG4 isotype control antibody and mAb of the present invention, respectively (i.e., the second anti-biotin FITC antibody). The gray dotted line and the black dashed line represent the specific antibody staining of the hIgG4 isotype control antibody and mAb of the present invention, respectively (i.e., anti-human IgG Fc biotin antibody + anti-biotin FITC antibody).

[0064] Figure 5A-5F .(5A) Photomicrographs of HVEM staining of healthy human colon and spleen tissue samples using mAbs of the present invention. (5B) Histograms of binding of a commercially available anti-HVEM antibody (R&D Systems AF356) to two different melanoma patient primary cell samples. The black and gray solid lines represent background staining (i.e., second anti-mouse APC antibody) and specific antibody staining, respectively. (5C) Histograms of binding of the mAbs of the present invention to three different melanoma patient primary cell samples. The solid and dotted lines represent background staining (i.e., second anti-biotin FITC antibody) and specific antibody staining (i.e., anti-human IgG Fc biotin antibody + anti-biotin FITC antibody), respectively. (5D) Histograms of the same as in 5B, but staining of cells from patients with renal cell carcinoma (RCC). (5E) Histograms of the same as in 5C, but staining of cells from patients with RCC. (5F) Table of HVEM expression in various healthy and tumor samples measured using mAbs of the present invention. “NA”: “Not Applicable” due to absence / destruction of the organizational core.

[0065] Figure 6A-6D .Bar graphs of binding ELISA of hrHVEM to (6A) hrBTLA and (6B) hrLIGHT in the presence of mAbs of the invention or hIgG4 isotype control antibody, and binding ELISA of hrHVEM (6C) to a commercially available anti-HVEM mAb (R&D Systems, MAB356) and (6D) hrBTLA in the presence of MAB356 or mIgG1 isotype control antibody.

[0066] Figure 7 . Histogram of luciferase output of Jurkat cells expressing hrHVEM and NF-kB luciferase reporter genes after preincubation with mAbs of the invention (left) and control hIgG4 (right) followed by co-incubation with empty CHO-K1 cells, CHO-K1 cells expressing hrBTLA, and no cells.

[0067] Figure 8 . Bar graph showing IFNγ secretion by TILs after co-culture with melanoma cells from two separate patients pre-incubated with mAbs of the invention or hIgG4 isotype control.

[0068] Figure 9A-9E .(9A-9B). Line graphs of specific killing of melanoma cells from (9A) the first primary patient sample, (9B) the second primary patient sample, and (9C) RCC cells from the primary patient sample at various time points during co-incubation with autologous TILs. Black circles, white squares, black triangles, and black diamond lines represent incubations with hIgG4 isotype control antibodies, mAbs of the invention, anti-PD1 mAbs, or mAbs of the invention + anti-PD1 mAbs, respectively. (9D-9E) Line graphs of specific killing of melanoma cells from primary patient samples pre-incubated with (9D) mAbs of the invention or (9E) MAB356 at various time points during co-incubation with autologous TILs. Black circles and white squares represent incubations with isotype control antibodies and mAbs, respectively.

[0069] Figure 10A-10B (10A) Line graph of specific killing of melanoma cells from primary patient samples at various time points during co-incubation with non-autologous PBMCs from healthy donors. Black circles represent hIgG4 isotype control antibodies and white squares represent mAbs of the invention. (10B) Bar graph of IFNγ secretion from PBMCs cultured alone or with melanoma cells incubated with hIgG4 isotype control antibodies or mAbs of the invention.

[0070] Fig.11 . Bar graph showing the percentage of caspase 3 / 7 positive events among the total number of melanoma cells after 15 hours of incubation, reflecting apoptosis of melanoma cells incubated with mAbs of the invention and hIgG4 isotype control from two primary samples.

[0071] Figures 12A-12D.(12A) Histogram of 41BB FACS staining in T cells as a measure of activation. The thick black solid line represents background staining (i.e., APC-coupled isotype control antibody), the black dashed line represents 41BB staining of TIL incubated with medium containing hIgG4 isotype control, the thin black solid line represents 41BB staining of TIL incubated with medium containing mAb of the present invention, the gray dashed line represents 41BB staining of TIL incubated with melanoma cells pre-incubated with hIgG4 isotype control, and the thin gray solid line represents 41BB staining of TIL incubated with melanoma cells pre-incubated with mAb of the present invention. (12B-12C) Histograms of the average number of T cell clusters observed after TIL from melanoma patients were incubated for 20 hours with (12B) mAb of the present invention or hIgG4 isotype control antibody and (12C) MAB356 or mIgG1 isotype control. (12D) Expression of 41BB on PBMCs cultured with melanoma cells pre-incubated with hIgG4 isotype control or with mAbs of the invention measured by microscopy at various time points.

[0072] Fig.13 Table of cytokine secretion following cytokine release assays using mAbs of the invention and two commercially available antibodies.

[0073] Fig.14 . Line graph of mean tumor volume at various time points in mice implanted with human melanoma cells and treated with human autologous TILs. Black circles, white squares, black triangles, and black diamond lines represent incubations with hIgG4 isotype control antibody, mAbs of the invention, anti-PD1 mAbs, or mAbs of the invention + anti-PD1 mAbs, respectively. DETAILED DESCRIPTION

[0074] The present invention, in some embodiments, provides agents that bind to HVEM, inhibit HVEM-BTLA interaction, and are also HVEM agonists. The present invention further relates to methods of treating HVEM-positive diseases and kits comprising these agents.

[0075] It is well known that HVEM-positive cancers can escape immune surveillance by binding to BTLA on the surface of immune cells. Engagement of BTLA generates inhibitory signals within immune cells that reduce T cell proliferation and increase cancer survival. It is known to inhibit this signaling with antibodies that bind to HVEM or antibodies that bind to BTLA. The present invention is based on the surprising discovery that antibodies that specifically bind to HVEM and activate HVEM signaling provide another benefit for combating cancer: increased immune cell activation. HVEM is also expressed on the surface of immune cells, however, its role is poorly understood. The antibodies of the present invention not only block the HVEM-BTLA interaction by binding to HVEM on pathogenic cells, thereby leaving immune cells uninhibited, but it also binds to HVEM on immune cells and induces NF-kB signaling in those immune cells that enhances activation properties.

[0076] In a first aspect, an agent is provided that binds to herpes virus entry mediator (HVEM) and inhibits the interaction between HVEM and B- and T-lymphocyte attenuator (BTLA).

[0077] In some embodiments, the agent activates downstream signaling through HVEM. In some embodiments, the agent inhibits downstream signaling through BTLA. In some embodiments, the agent activates downstream signaling through HVEM and inhibits downstream signaling through BTLA. In some embodiments, inhibiting the interaction between HVEM and BTLA inhibits downstream signaling through BTLA.

[0078] In some embodiments, HVEM is mammalian HVEM. In some embodiments, HVEM is rodent HVEM. In some embodiments, HVEM is monkey HVEM. In some embodiments, HVEM is human HVEM. In some embodiments, HVEM is any one of mouse, monkey and human HVEM. In some embodiments, HVEM is membrane-bound HVEM. In some embodiments, HVEM is HVEM on cells. In some embodiments, HVEM is HVEM on the surface of cells. In some embodiments, HVEM is soluble HVEM.

[0079] In some embodiments, the cell is a pathogenic cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a cell of a pathogen. 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 by a pathogen. In some embodiments, the cell is a cell infected by a bacteria. In some embodiments, the cell is a cell infected by a virus. In some embodiments, the pathogen is selected from bacteria, viruses and fungi.

[0080] 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.

[0081] In some embodiments, the immune cell is an immune cell expressing a chimeric antigen receptor (CAR). In some embodiments, CAR is a CAR-T cell. In some embodiments, CAR is a CAR-NK cell. As used herein, the term "CAR" refers to an engineered receptor that is specific to at least one protein of interest (e.g., a protein expressed by a cell expressing HVEM) and is transplanted to an immune effector cell (such as a T cell or NK cell). In some embodiments, CAR-T cells have the specificity of monoclonal antibodies transplanted to T cells. In some embodiments, CAR-NK cells have the specificity of monoclonal antibodies transplanted to NK cells. In some embodiments, T cells are selected from cytotoxic T lymphocytes and regulatory T cells. MART1 is an example of a target protein co-expressed with HVEM on a target cell. In some embodiments, CAR targets a protein expressed by a cell expressing HVEM. In some embodiments, the protein is not HVEM. In some embodiments, CAR targets a protein on the surface of a cell expressing HVEM. In some embodiments, the protein is an antibody. In some embodiments, CAR targets antibodies. In some embodiments, CAR targets antibodies of the present invention. In some embodiments, CAR targets cytotoxic antibodies. In some embodiments, CAR targets antibody constant domains. In some embodiments, CAR targets Fc domains. In some embodiments, CAR therapy further includes administering antibodies targeting cells expressing HVEM. In some embodiments, the antibody targeted by CAR is not an antibody of the present invention.

[0082] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target proteins bound by receptors and are cytotoxic to them. In some embodiments, CAR-T or CAR-NK cells target at least one cancer protein. In some embodiments, CAR-T or CAR-NK cells target multiple cancer proteins.

[0083] The construction of CAR-T cells is well known in the art. In a non-limiting example, a monoclonal antibody against a cancer protein can be prepared, and then a vector encoding the antibody will be constructed. The vector will also include a costimulatory signal region. In some embodiments, the costimulatory signal region comprises an intracellular domain of a known T cell or NK cell stimulating molecule. In some embodiments, the intracellular domain is selected from at least one of the following: 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 a ligand specifically bound to 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.

[0084] In some embodiments, HVEM is a tumor necrosis factor receptor superfamily member 14 (TNFRSF14). In some embodiments, HVEM is CD270. In some embodiments, HVEM is a receptor for BTLA. In some embodiments, HVEM is a ligand for BTLA. In some embodiments, BTLA is CD272. In some embodiments, HVEM is a receptor for tumor necrosis factor superfamily member 14 (TNFSF14). In some embodiments, HVEM is a ligand for TNFSF14. In some embodiments, TNFSF14 is LIGHT. In some embodiments, TNFSF14 is CD258. In some embodiments, HVEM is a receptor for CD160. In some embodiments, HVEM is a ligand for CD160. In some embodiments, HVEM is a receptor for lymphotoxin alpha (LTα). In some embodiments, HVEM is a ligand for LTα. In some embodiments, LTα is TNF-β.

[0085] In some embodiments, the agent specifically binds to HVEM. In some embodiments, the agent does not bind to other proteins besides 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 blocks 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 prevents the bound HVEM from further binding to BTLA. In some embodiments, the agent inhibits downstream signaling through BTLA. In some embodiments, the agent reduces downstream signaling through BTLA.

[0086] 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 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 does inhibit 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, inhibition is substantial inhibition. In some embodiments, 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 present 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.

[0087] In some embodiments, the agent activates signal transduction through HVEM. In some embodiments, the agent is a HVEM agonist. In some embodiments, the agent induces signal transduction through HVEM. In some embodiments, signal transduction through HVEM is HVEM downstream signal transduction. In some embodiments, the agent binds to HVEM on the cell and activates / induces HVEM signal transduction in the cell. In some embodiments, HVEM signal transduction includes activation of nuclear factor activated B cell kappa-light chain enhancer (NF-kB). In some embodiments, NF-kB signal transduction includes control of a gene, having an NF-kB response element in the promoter of the gene. In some embodiments, signal transduction includes increased cytotoxicity of the cell. In some embodiments, signal transduction includes increased cytotoxicity of cells expressing HVEM contacted by the agent. In some embodiments, increased cytotoxicity includes increased secretion of proinflammatory cytokines. In some embodiments, the proinflammatory 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 proinflammatory cytokine is IFNγ. In some embodiments, the increase is compared to cells that are not contacted by the reagent of the present invention. In some embodiments, the increase is 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% increase. Each possibility represents a separate embodiment of the present invention.

[0088] In some embodiments, the cell is an immune cell and 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 γ / δT cell. In some embodiments, the immune cell is a γ / δT cell. In some embodiments, the immune cell is a NK cell. In some embodiments, activating HVEM signaling includes activating an immune cell. In some embodiments, immune activation includes increased proliferation. In some embodiments, immune activation includes increased cytotoxicity. In some embodiments, immune activation includes increased 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 T regulatory cells. In some embodiments, immune activation includes expression of markers selected from: 41BB, CD69, CD25, CD107a, HLA-DR and increased secretion of cytokines. In some embodiments, cytokines are proinflammatory cytokines. In some embodiments, T cell activation includes increased T cell clustering.

[0089] In some embodiments, the cell is a cancer cell and the signaling comprises an anti-tumor effect. In some embodiments, the anti-tumor effect comprises increased apoptosis. In some embodiments, the anti-tumor effect comprises reduced proliferation. In some embodiments, the anti-tumor effect comprises increased chemosensitivity. In some embodiments, the anti-tumor effect comprises reduced mobility. In some embodiments, the anti-tumor effect comprises reduced invasion. In some embodiments, the anti-tumor effect comprises reduced metastasis. In some embodiments, the anti-tumor effect comprises reduced self-renewal. In some embodiments, the effect is compared to cancer cells that have not been contacted by an agent of the invention. In some embodiments, the reduction is a reduction 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.

[0090] 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, "direct induction" refers to the result that occurs as a direct result of the combination of the agent, and is not characterized by downstream signaling in the combined cell. In some embodiments, the agent has no cytotoxicity. In some embodiments, the agent itself has no cytotoxicity. In some embodiments, the agent does not induce antibody-directed cellcytotoxicity (ADCC). In some embodiments, the agent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the agent does not include a cytotoxic part. In some embodiments, the agent does not induce apoptosis of the cell after binding to HVEM expressed by the cell expressing HVEM. In some embodiments, the agent does not induce apoptosis via interaction with another cell. In some embodiments, the agent does not directly induce the killing of cells expressing HVEM. In some embodiments, the agent does not target cells for killing. In some embodiments, the agent does induce indirect apoptosis. In some embodiments, indirect apoptosis is apoptosis induced by downstream signaling through the HVEM receptor that leads to apoptosis. In some embodiments, indirect apoptosis is apoptosis that requires signaling. In some embodiments, indirect apoptosis is apoptosis that does not require the participation 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.

[0091] 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.

[0092] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its fragment is a fab fragment. In some embodiments, the antibody or its fragment is a single-chain antibody (scFv). In some embodiments, the antibody or its fragment 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.

[0093] As used herein, the term "antibody" refers to a polypeptide or polypeptide group comprising at least one binding domain, which is formed by folding a polypeptide chain with a three-dimensional binding space, the internal surface shape and charge distribution of which are complementary to the characteristics of the antigenic determinant of the antigen. Antibodies usually have a tetrameric form, including two pairs of identical polypeptide chains, each pair having a "light" chain and a "heavy" chain. The variable region of each light / heavy chain pair forms an antibody binding site. Antibodies can be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, camelid antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, fully human antibodies, anti-idiotypic antibodies, and antibodies and fragments that can be labeled in soluble form or in a bound form, including epitope binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can 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), dimer variable regions (diabodies) and disulfide-linked variable regions (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to the Fc region or a fragment thereof. The skilled person will further appreciate that other fusion products may be produced, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH) to Fc fusions, and scFv-scFv-Fc fusions.

[0094] The immunoglobulin molecule can 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 embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG4. In some embodiments, the antibody comprises IgG1. In some embodiments, the antibody comprises IgG3. In some embodiments, the antibody comprises a modified IgG1 or IgG3 with reduced toxicity.

[0095] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains, which are linked together by non-covalent association and by disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are five human antibody classes (IgG, IgA, IgM, IgD and IgE), and within these classes, various subclasses are recognized based on structural differences (such as the number of immunoglobulin units in a single antibody molecule, the disulfide bond structure of the independent units, and the differences in chain length and sequence). The class and subclass of an antibody are its isotype.

[0096] The amino terminal regions of heavy and light chains are more diverse in sequence than the carboxyl terminal regions, and are therefore referred to as variable domains. This part of the antibody structure confers the antigen binding specificity of the antibody. The heavy chain variable (VH) domain and the light chain variable (VL) domain together form a single antigen binding site, and therefore, the basic immunoglobulin unit has two antigen binding sites. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 824592-4596).

[0097] The carboxyl-terminal portions of the heavy and light chains form the constant domains, CH1, CH2, CH3, CL. Although these domains are much less diverse, they vary from one animal species to another, and within the same individual, there are several different antibody isotypes, each with different functions.

[0098] The term "framework region" or "FR" refers to the amino acid residues in the variable domain of an antibody that are different from the amino acid residues in the hypervariable region defined herein. As used herein, the term "hypervariable region" refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of the antigen. The scope of FR and CDR has been precisely defined (see Kabat et al.).

[0099] Immunoglobulin variable domains can also be identified using the IMGT information system (www: / / imgt.cines.fr / ) ( / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al., Nucl. Acids Res. J6: W503-508 (2008).

[0100] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to improve similarity to a human antibody. Humanized antibodies can be produced by recombinant DNA that produces CDRs of non-human antibodies that are surrounded by sequences similar to human antibodies. In some embodiments, humanized antibodies are chimeric antibodies. In some embodiments, humanization includes inserting CDRs of the present invention into a human antibody support or skeleton. Humanized antibodies are well known in the art, and any method for producing these antibodies that retains CDRs of the present invention can be used.

[0101] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants that may occur during the production of the monoclonal antibody, which variants are usually present in small amounts, the individual antibodies constituting the population are all identical and / or bind the same epitope. Unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as being produced by any particular preparation method. The monoclonal antibodies used according to the methods provided herein can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can 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).

[0102] mAb of the present invention can be any immunoglobulin class including IgG, IgM, IgD, IgE or IgA. The hybridoma producing mAb can be cultured in vitro or in vivo. High titer mAb can be obtained in vivo production, and cells from a separate hybridoma are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites containing high concentrations of required mAb. Column chromatography well known to those skilled in the art can be used to purify mAb of isotype IgM or IgG from such ascites or from culture supernatant.

[0103] "Antibody fragments" include 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. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); and one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed by antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (bi, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0104] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0105] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region is composed of a dimer (non-covalent association) of a heavy chain variable domain and a light chain variable domain in a tight manner. It is in this configuration that the three surfaces of the VH-VL dimer. The six hypervariable regions together give the antibody antigen binding specificity. However, even if a single variable domain (or only half of an Fv containing three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, its affinity is lower than that of the entire binding site.

[0106] 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 some residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein, wherein the cysteine ​​residue(s) of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0107] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0108] According to the amino acid sequence of the constant domain of the heavy chain of the antibody, the antibody can be designated as different classes. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the antibodies of different classes are respectively referred to as α, δ, e, γ and μ. The subunit structure and three-dimensional configuration of the immunoglobulins of different classes are well known.

[0109] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired antigen-binding structure. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0110] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Diabody production is known in the art and is described in Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0111] The monoclonal antibodies of the present invention can be prepared using methods known in the art. Examples include various techniques such as 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).

[0112] In addition to the conventional methods of producing antibodies in vivo, phage display technology can also be used to produce antibodies in vitro. Compared with conventional antibody production, the production of this recombinant antibody is much faster, and this recombinant antibody can be produced for a large number of antigens. In addition, when using conventional methods, many antigens are shown to be non-immunogenic or highly toxic, and therefore cannot be used to produce antibodies in animals. In addition, the affinity maturation (i.e., improving affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies for specific antigens can be produced in a selection procedure. In order to produce recombinant monoclonal antibodies, people can use various methods based on display libraries to produce a large number of antibodies with different antigen recognition sites. Such libraries can be prepared in several ways: people can produce synthetic libraries by cloning synthetic CDR3 regions in the heavy chain germline gene library, thereby producing a large number of antibody libraries, from which recombinant antibody fragments with various specificities can be selected. People can use human lymphocyte libraries as the starting material for constructing antibody libraries. Naive repertoires of human IgM antibodies can be constructed to create a human library with great diversity. This method has been widely and successfully used to select large numbers of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided 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.

[0113] Non-human antibodies can be humanized by any method known in the art. In one method, non-human complementary determining regions (CDRs) are inserted into human antibodies or consensus antibody framework sequences. Further changes can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0114] In some embodiments, antibodies and parts 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 Fv, Fab, F(ab')2, scFV or scFV2 ​​or scFv4 fragments.

[0115] In some embodiments, the invention provides nucleic acid sequences encoding the antibodies or antigen-binding portions of the invention.

[0116] For example, a polynucleotide can encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH), but also a heavy chain constant region (CH), which will generally include three constant domains: CH1, CH2, and CH3; and a "hinge" region. In some cases, it is desirable to have a constant region.

[0117] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAb"), Fv, scFv, Fab' and CHI and CK or CL domains have been removed. Since mini antibodies are smaller than conventional antibodies, they should achieve better tissue permeability in clinical / diagnostic applications, but since they are bivalent, they should retain higher binding affinity compared to monovalent antibody fragments (such as dAb). Therefore, unless the context indicates otherwise, the term "antibody" used herein not only covers complete antibody molecules, but also covers antigen-binding antibody fragments of the types discussed above. Relative to the corresponding human receptor framework, each framework region present in the encoded polypeptide may include at least one amino acid substitution. Thus, for example, relative to the receptor framework region, the framework region may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Taking into account the characteristics of the individual amino acids constituting the disclosed protein product, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved, ie, "conservative substitutions."

[0118] Suitably, the polynucleotides described herein may be isolated and / or purified.In some embodiments, the polynucleotide is an isolated polynucleotide.

[0119] As used herein, the term "non-naturally occurring" substances, ingredients, entities and / or any combination of substances, ingredients, or entities, or any grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those substances, ingredients, entities and / or any combination of substances, ingredients or entities that are known to one of ordinary skill in the art as "naturally occurring" or that are determined or interpreted by a judge or administrative or judicial body as possibly "occurring in nature" at any time.

[0120] 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 a mutated IgG1 and / or IgG3, wherein the mutation inhibits the induction of ADCC, CDC or both. In some embodiments, the mutation is in the FcRγ binding motif.

[0121] 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 are according to the KABAT numbering system.

[0122] In another aspect, an antibody or antigen-binding fragment thereof is provided, 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 (QQYGSSPPYT). In some embodiments, the CDRs are according to the KABAT numbering system.

[0123] 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: 11 (GFTFSSYA), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 12 (ISGSGGST), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 13 (AKAPGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (QSVSSY), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (GAS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 16 (QQYGSSPPYT). In some embodiments, the CDRs are according to the IMGT numbering system.

[0124] In another aspect, an antibody or antigen-binding fragment thereof is provided, 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: 11 (GFTFSSYA), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 12 (ISGSGGST), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 13 (AKAPGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (QSVSSY), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (GAS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 16 (QQYGSSPPYT). In some embodiments, the CDRs are according to the IMGT numbering system.

[0125] 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 embodiments, the antibody comprises a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO: 9.

[0126] 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 the sequence of SEQ ID NO: 8. In some embodiments, 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.

[0127] In some embodiments, the heavy chain and light chain are combined by a connector. In some embodiments, the connector is an amino acid connector. In some embodiments, the connector 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 long. Every possibility represents a separate embodiment of the present invention. In some embodiments, the connector 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 long. Every possibility represents a separate embodiment of the present invention. In some embodiments, the linker is between 1-25, 5-25, 10-25, 15-25, 20-25, 1-20, 5-20, 10-20, 15-20, 1-15, 5-15, 10-15, 1-10, 5-10, or 1-5 amino acids long. Each possibility represents a separate embodiment of the invention.

[0128] In another aspect, a pharmaceutical composition comprising an agent of the invention is provided.

[0129] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an agent of the present invention.

[0130] As used herein, the term "carrier", "excipient" or "adjuvant" refers 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 saline. Some examples of materials that can be used 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 carboxymethylcellulose, ethylcellulose and cellulose acetate, powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffered saline solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used 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 saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as colorants, 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, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (which are incorporated herein by reference in their entirety).Examples of pharmaceutically acceptable excipients, carriers and diluents for use in the present compositions include distilled water, physiological saline, Ringer's solution, glucose solution, Hank's solution and DMSO. These additional inactive components and 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 edition, Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described herein may also be contained in artificially generated structures such as liposomes, ISCOMS, slow-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, etc. The liposomes used with the peptides described herein are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids generally depends on considerations such as liposome size and stability in blood. For example, Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, review various methods that can be used to prepare liposomes, and also see U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0131] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions set forth herein.

[0132] The term "therapeutically effective amount" refers to an amount of a drug that is effective in treating a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount that is effective in achieving the desired therapeutic or preventive result at the necessary dosage and for the necessary period of time. The exact dosage form and regimen will be determined by the physician based on the patient's condition.

[0133] In some embodiments, the agent is for treating a disease or condition. In some embodiments, the pharmaceutical composition is for treating a disease or condition. In some embodiments, the disease or condition is characterized by cells expressing HVEM. In some embodiments, the cell is a disease cell characterized by expressing HVEM. In some embodiments, the cell is characterized by overexpressing HVEM. In some embodiments, overexpression is compared to healthy cells. In some embodiments, healthy cells are non-diseased cells. In some embodiments, overexpression is increased expression. In some embodiments, the disease or condition is a HVEM-positive disease or condition. In some embodiments, the disease or condition is a BTLA-positive disease or condition.

[0134] In some embodiments, the disease or condition is a HVEM-positive cancer. In some embodiments, the disease or condition is a BTLA-positive cancer. In some embodiments, the disease or condition is a HVEM-positive precancerous lesion. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a 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 a renal cell carcinoma. The skilled person will understand that any cancer expressing HVEM can be a therapeutic target. In some embodiments, the disease is an infectious disease. In some embodiments, the infected cell comprises HVEM expression. In some embodiments, the infected cell comprises HVEM expression. In some embodiments, the infectious disease is a bacterium and the bacterial cell comprises HVEM expression. In some embodiments, the infectious disease is a fungal infection and the fungal cell comprises HVEM expression. In some embodiments, the infectious disease is a virus and the virus-infected subject cell comprises HVEM expression. In some embodiments, the virus is a herpes virus. In some embodiments, HVEM expression is HVEM overexpression. In some embodiments, the HVEM expression is increased HVEM expression.

[0135] In another aspect, a method of treating a HVEM-positive disease or condition is provided, the method comprising inhibiting the interaction of HVEM and BTLA and activating HVEM signaling in immune cells, disease cells, or both.

[0136] In some embodiments, the methods comprise administering an agent of the invention. In some embodiments, the methods comprise administering a pharmaceutical composition of the invention.

[0137] In another aspect, a method of treating a HVEM-positive disease or condition is provided, the method comprising administering an agent of the invention.

[0138] In another aspect, provided is a method of treating a HVEM-positive disease or condition comprising administering a pharmaceutical composition of the invention.

[0139] In some embodiments, the method further comprises not inhibiting HVEM-TNFSF14 interaction. In some embodiments, the method further comprises not substantially inhibiting HVEM-TNFSF14 interaction.

[0140] 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 of PD-1 with PD-L1. In some embodiments, the method further comprises inhibiting the interaction of PD-1 with PD-L2. In some embodiments, the method further comprises inhibiting the interaction of PD-1 with PD-L1 or PD-L2. In some embodiments, inhibiting the interaction comprises administering an agent that inhibits the interaction of PD-1 with PD-L1 or PD-1 with 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, at least one ligand is two ligands. In some embodiments, inhibiting the interaction comprises PD-1 / PD-L1 blockade. In some embodiments, inhibiting the interaction comprises PD-1 / PD-L1 or PD-L2 blockade. In some embodiments, inhibiting the interaction comprises anti-PD-1 / PD-L1 therapy. In some embodiments, inhibiting 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.

[0141] In some embodiments, the method of treatment further includes administering another therapeutic agent (therapeutic) to cells expressing HVEM. In some embodiments, other therapeutic agents are anticancer therapeutic agents. In some embodiments, the therapeutic agent is a non-autologous immune cell. In some embodiments, other therapeutic agents are autologous immune cells. In some embodiments, immune cells are CAR-expressing immune cells. In some embodiments, CAR is CAR-T. In some embodiments, CAR is CAR-NK. In some embodiments, autologous immune cells are TIL therapy. In some embodiments, non-autologous immune cells are adoptive cell therapy. In some embodiments, autologous immune cells are adoptive cell therapy. In some embodiments, adoptive cells are CAR cells. In some embodiments, adoptive cells are TIL.

[0142] In some embodiments, the composition of the invention and another therapeutic agent are administered simultaneously. In some embodiments, the composition of the invention is administered before, after, or simultaneously with another therapeutic agent.

[0143] In another aspect, a method of determining suitability of a subject to be treated by the methods of the invention is provided, 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 treatment methods of the invention.

[0144] In another aspect, a method of detecting HVEM in a sample is provided, the method comprising contacting the sample with an agent of the present invention, or an antibody or antigen-binding fragment thereof of the present invention, thereby detecting HVEM.

[0145] In some embodiments, contacting is performed under conditions suitable for binding, the binding being the binding of an agent or an antibody or antigen binding fragment thereof to HVEM. In some embodiments, the conditions are suitable for specific binding to HVEM. In some embodiments, the binding is hybridization. The conditions for antibody / agent binding will depend on the sample. The skilled person will appreciate that the conditions required for binding to a tissue sample (depending on the method / type of fixation) are different from those for binding in a liquid solution (e.g., whole cell lysate). Such binding conditions are well known in the art, and the skilled person can select appropriate conditions for a given sample.

[0146] 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.

[0147] In some embodiments, the method further comprises detecting an agent of the invention, or an antibody of the invention, or an antigen-binding fragment thereof. In some embodiments, the detection is an immunohistochemical detection. In some embodiments, the detection is an immunofluorescence detection. In some embodiments, the detection is a 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 a secondary antibody. In some embodiments, the detection comprises microscopy.

[0148] 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 increased HVEM expression. In some embodiments, the subject has a disease or condition that may be characterized by increased HVEM expression. In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer that is unresponsive to first-line therapy. In some embodiments, the cancer is a cancer recurrence. In some embodiments, the cancer is a cancer that is unresponsive to PD-1 / PD-L1 therapy.

[0149] 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 comprises cells. In some embodiments, the sample comprises disease cells. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy.

[0150] In some embodiments, the positive expression of HVEM includes HVEM expression. In some embodiments, the positive expression of HVEM includes elevated HVEM levels. In some embodiments, the positive expression of HVEM includes increased HVEM levels. In some embodiments, the positive expression of HVEM includes overexpression of HVEM. In some embodiments, the positive expression of HVEM is compared to a healthy sample. In some embodiments, the healthy sample is from healthy tissue and / or cells adjacent to the diseased tissue and / or cells. In some embodiments, the healthy sample comprises uninfected cells. In some embodiments, the healthy sample is for a healthy donor. In some embodiments, the positive expression of HVEM is compared to a predetermined threshold.

[0151] In another aspect, a kit comprising a pharmaceutical composition of the invention or an agent of the invention is provided.

[0152] 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 an 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 blocking 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.

[0153] In some embodiments, the kit further comprises a label indicating that the pharmaceutical composition of the present invention is used 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 used with a PD-1 and / or PD-L1-based therapy.

[0154] In some embodiments, the kit further comprises a second detection molecule. In some embodiments, the detection molecule is used to detect the reagent of the present invention. In some embodiments, the second detection molecule is an antibody that binds to the reagent of the present invention. In some embodiments, the detection molecule comprises a detectable portion. Examples of detectable portions include, but are not limited to, fluorescent moieties, labels, radioactive labels, dyes, and chemiluminescent moieties. In some embodiments, the detectable portion is a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, GFP, RFP, YFP, APC, CY5, CY7, and Pacific Blue. The second detection molecule is well known in the art, and any such molecule that will detect the reagent of the present invention can be used.

[0155] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0156] Note that as used herein and in 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, and reference to a "polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0157] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). One skilled in the art would further understand that, in practice, any antonymous conjunctions and / or phrases presenting two or more optional terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, any of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0158] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0159] After examining the following examples, other objects, advantages and novel features of the present invention will become apparent to those skilled in the art, and these examples are not intended to be limiting. In addition, as described above and as claimed in the following claims, each of the various embodiments and aspects of the present invention is experimentally supported in the following examples.

[0160] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0161] Example

[0162] 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 explained fully in the literature.See, e.g., “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, RM, 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”, Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology" Vols. I-III Coligan JE, 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); all of which are incorporated by reference. Other general references are provided throughout this document.

[0163] Materials and methods

[0164] scFv for HVEM was produced by phage display: the naive human scFv library was incubated with synthetic / recombinant tags to subtract tag-specific clones, and incubated with control CHO-K1 empty cells to subtract CHO-K1 cell binding clones. Then, by incubating with CHO-K1 cells overexpressing hrHVEM, phage was subjected to three rounds of biopanning. The clone was picked, rescued with the help of helper phage and screened by phage ELISA for hrHVEM, mrHVE and crHVEM. The clone was sequenced and the positive unique clone was subcloned into the pET25b protein expression vector. Lysate was prepared from 200ml of induced culture. The bacterial cells were precipitated, washed with PBS and lysed by a French press. Insoluble fragments were removed by high-speed centrifugation and lysates containing soluble proteins (including scFv) were collected. scFv was cloned and had a HIS tag.

[0165] Production of transfected CHO-K1 cells: CHO-K1 cells were seeded at a density of 200K cells / well in 6-well plates in Nutrient Mixture F-12 (Ham's) medium (Biological Industries, Israel) supplemented with 10% FBS (Biological Industries, Israel) containing L-glutamine and incubated overnight at 37°C. After incubation, 2 μg of plasmid DNA was transfected using 4 μl jetPRIME transfection reagent (Polyplus-transfection, France) according to the manufacturer's instructions. Two days after transfection, 1 mg / ml G418 sulfate antibiotic (Millipore, Germany) was used for selection and cells were cultured routinely with the addition of antibiotics.

[0166] Flow cytometry: ≤1x10 cells / well were analyzed on ice using appropriate purified specific antibodies diluted in FACS buffer (PBS, 0.02% sodium azide, 0.5% BSA). 5 Surface cell staining was performed on 10 cells 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 covered on ice and incubated with the appropriate secondary antibody conjugated with a fluorophore for 30 minutes. In the case of a fluorophore-conjugated primary antibody, direct staining was performed without a secondary antibody. After incubation, the cells were washed and resuspended in 200 μl FACS buffer for flow cytometry analysis.

[0167] mAb production:

[0168] A. Luciferase reporter gene assay: According to the manufacturer's instructions, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega, the U.S.). In short, a luciferase assay reagent was prepared by combining and mixing a bottle of buffer and a bottle of substrate. An equal volume of reagent and sample were mixed, and luminescence was measured using appropriate instrument settings 3 or 10 minutes after adding the reagent.

[0169] b. Plasmid preparation: scFv2 was converted into a full 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. A large number of transfection-grade plasmids were prepared (maxi-prepared) for expression in Expi293F cells.

[0170] c. Cell culture and transient transfection: Expi293F cells were grown in serum-free Expi293FTM expression medium (Thermo Fisher Scientific, USA). Cells were maintained in conical flasks (Corning Inc., USA) on an orbital oscillator (VWR Scientific, USA) at 37°C, 8% CO2. One day before transfection, cells were seeded in Corning conical flasks at an appropriate density. On the day of transfection, DNA and transfection reagent were mixed in an optimal ratio and then added to a flask containing cells prepared for transfection. Recombinant plasmids encoding target proteins were transiently transfected into suspension Expi293F cell cultures. Cell culture supernatants collected on the 6th day after transfection were used for purification.

[0171] Purification and analysis: The cell culture fluid was centrifuged and then filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with an appropriate buffer, the eluted fractions were combined and the buffer was exchanged for the final formulation buffer. The purified protein was analyzed for molecular weight and purity by SDS-PAGE and Western blot analysis. The concentration was determined by the A280 method (antibody).

[0172] Cytotoxicity assay: Melanoma cells inoculated with nuclear RFP staining were incubated overnight to allow them to adhere. Then, melanoma cells were pre-incubated with 20 μg / ml mAb of the present invention or hIgG4 isotype control antibody (Invivogen, France), and TILs were pre-incubated with 20 μg / ml anti-PD1 mAb (Nivolumab, BMS) or hIgG4 isotype control antibody (Invivogen, France). Pre-incubated TILs or culture medium containing only anti-PD1 or hIgG4 isotype control mAb were added to melanoma cells together with CellEvent Caspase-3 / 7 Green Detection Reagent (Invitrogen, USA). The plate was placed in the Incucyte ZOOM system (ESSEN Bioscience, USA) and scanned every hour. Caspase 3 / 7 positive events reflecting the specific killing of melanoma were counted. Similar protocols were used for cytotoxicity assays with RCC cells and for cytotoxicity assays with MAB356.

[0173] T cell activation assay by flow cytometry: Melanoma cells or culture medium alone were incubated with 20 μg / ml mAb of the present invention or hIgG4 isotype control antibody (Invivogen, France) for 1 hour at 37°C. After 1 hour, TILs were added to melanoma cells or culture medium and co-cultured for 4 hours at 37°C. After 4 hours, cells were surface stained with anti-CD3 (Biolegend, USA) and anti-41BB (Biolegend, USA), and expression was evaluated by flow cytometry using a CytoFLEX instrument (BeckmanCoulter, USA) and using FCS Express 6 software (DeNovo Software, USA).

[0174] T cell activation assay by Incucyte ZOOM system: Melanoma cells stained with nuclear RFP were inoculated and incubated overnight to allow them to adhere. After overnight incubation, melanoma cells were incubated with 20 μg / ml mAb or hIgG4 isotype controls of the present invention at 37°C for 1 hour. PBMCs or culture medium electroporated with MART-1 were added to melanoma cells together with anti-41BB (Biolegend, USA) labeled with FabFlour-488 labeling reagent (ESSEN Bioscience, USA). The plate was placed in the Incucyte ZOOM system and scanned every hour.

[0175] T cell cluster determination: Images were acquired by the Incucyte ZOOM system. The number of clusters was counted by image analysis with Image J version 1.52a. Images were smoothed, contrast enhanced, and then converted to 8-bit images. After image binarization, images were processed by applying the "close", "fill holes", and "erode" commands. The "grain analysis" command was then applied using the following parameters: size of 500 mm^2-infinity and circularity between 0.1-1.

[0176] Example 1: Identification of HVEM as an immune blocking target

[0177] A high-throughput genome-wide primary siRNA screening consisting of approximately 8,300 genes was run on 2 patient-derived melanoma cell systems. 96 hours after siRNA transfection by co-culture with TIL overnight, the killing effect was assessed using an LDH-based cytotoxicity assay. The successful knockdown of the positive control was assessed by FACS. Using the same design as the primary screening, where positive hits were defined based on their Z scores and percentage of killing enhancement, a confirmation screening consisting of 360 genes was validated on 8 melanoma cell systems, corrected for the effects of siRNA on proliferation. Finally, a validation screening consisting of 54 genes was performed on 5 melanoma cell systems. HVEM was found to have a strong positive hit as a potential immunotherapy target.

[0178] Example 2: Selection of scFv against HVEM

[0179] HVEM has been established as a potential target for therapeutic immune blocking in cancer therapy, and single-chain antibody fragments (scFv) that can be combined with HVEM are separated and measured. scFv is separated as described above (Materials and Methods). 6 selected scFvs were screened for combining with recombinant human, mouse and macaque HVEM (see Table 1). As measured by cell ELISA, five of the six showed strong combination with human recombinant HVEM (hrHVEM) expressed in CHO-K1 cells. CHO-K1-empty cells were used as negative controls. Two of the five showed very strong combination with mouse recombinant HVEM (mrHVEM), while the other three showed much weaker combination. One of the five also showed strong combination with monkey (macaque) HVEM, while the other three showed much weaker combination. FACS analysis was used to confirm these results. scFv contains HIS tag and anti-HIS second APC conjugated antibody is used to detect surface binding. As predicted by ELISA, five of the scFvs were found to bind to surface hrHVEM in CHO-K1 HVEM-overexpressing cells, although two of the scFvs performed poorly ( Figure 1 ).

[0180] Table 1. Cell ELISA using lysates containing scFv on CHO-K1 cells overexpressing HVEM. Numbers represent OD values.

[0181] Group Comparison people Mouse Macaque 1 0.044 1.194 0.140 0.049 2 0.045 1.507 1.366 1.112 3 0.042 1.052 0.403 0.209 4 0.048 1.110 0.161 0.140 5 0.055 0.395 0.057 0.046 6 0.044 1.207 0.951 0.486

[0182] Next, the scFvs were examined for their ability to inhibit the interaction between HVEM and two of its ligands: BTLA and LIGHT (TNFSF14). ELISA plates were coated with 2.5 μg / ml hrHVEM and incubated overnight at 4°C. The plates were then washed and blocked with 2% BSA for 1 hour at room temperature. ScFv1-6 lysates and control lysates were added to the ELISA plates and incubated for 1 hour at room temperature. The plates were washed and blocked for 10 minutes, followed by multiple washes. hrBTLA-Fc or hrLIGHT-HIS were 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-LIGHTpAb at room temperature for 1 hour. The plates were washed and incubated with streptavidin peroxidase or bovine anti-goat HRP antibodies for 30 minutes at room temperature. Finally, the plates were washed and sulfuric acid stop solution was added after TMB. The absorbance was read at 450nM and 570nM using a Spark 10M plate reader. Unexpectedly, although scFv 1, 3, 4, and 6 bound hrHVEM, 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 the binding of LIGHT to HVEM ( Figure 2B ). Due to its binding to human, mouse, and monkey rHVEM, and its ability to block the HVEM-BTLA interaction, scFv2 was chosen for all future work.

[0183] Example 3: Anti-HVEM IgG Antibodies

[0184] Although the single-chain antibody scFv2 was effective in blocking the HVEM-BTLA interaction, a complete IgG monoclonal antibody (mAb) was designed from the scFv2 light and heavy chains, specifically the CDRs. The scFv2 was sequenced and the light and heavy chains were cloned into an IgG4 backbone (excluding the S228P mutation). The complete sequence of this mAb heavy chain is presented in SEQ ID NO:9, and the light chain is presented in SEQ ID NO:10. The presence of the complete IgG antibody was confirmed by SDS-PAGE and Western blotting ( Figure 3 ).

[0185] Next, the new mAbs were evaluated for binding to recombinant HVEM from humans, mice, and monkeys as before. Binding to hrHVEM was evaluated by ELISA as before. The new mAbs strongly bound to hrHVEM ( Figure 4A Binding was also assessed by FACS as before. CHO-K1 empty, CHO-K1 hrHVEM, CHO-K1 mrHVEM, and CHO-K1 crHVEM cells were incubated with 20 μg / ml antibody or hIgG4 isotype control antibody on ice for 1 hour, followed by staining with anti-human IgG FC-biotin and anti-biotin FITC. Expression was assessed by flow cytometry using a CytoFLEX instrument and data analysis using FCS Express 6 software. The new mAb strongly bound to rHVEM from all three species ( Figure 4B ).

[0186] Since the new mAbs can bind to recombinant human HVEM, their ability to bind to endogenous HVEM on the surface of healthy and cancer cells was tested. The first immunohistochemistry was performed on healthy human colon and spleen tissues with the mAbs of the invention, where robust staining ( Figure 5A Next, melanoma cells known to express HVEM from three primary patient samples ( Figure 5B ) were incubated with 20 μg / ml mAb of the invention on ice for 1 hour, followed by staining with anti-human IgGFC-biotin and anti-biotin FITC. Expression was assessed 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. Again, expression of HVEM by cancer cells was confirmed using a commercially available anti-HVEM antibody (R&D Systems AF356). Figure 5D The binding of the new mAb to these surfaces was comparable to that of the commercial antibody ( Figure 5E Finally, the mAb of the present invention was used to probe the tumor microarray ( Fig. 5F ). Twelve different indications were tested, including 1 healthy tissue sample and 5 different tumor samples per indication. Healthy pancreas, lungs, chest, bladder, and ovaries were negative for HVEM; while lymph nodes, larynx / oral cavity / tongue, brain, kidney, liver, and stomach were all positive to varying degrees. Although not all tumors expressed HVEM, at least one tumor sample in each organ type was positive.

[0187] The ability of the intact mAb to inhibit BTLA binding to HVEM was also evaluated as before. In the presence of the mAb of the invention, BTLA did not bind to HVEM as measured by ELISA ( FIG. 6 ). The addition of an isotype control IgG served as a positive control, in which BTLA strongly bound to HVEM. It can also be seen with respect to the scFv that the mAb did not inhibit the binding of LIGHT to HVEM ( Figure 6B In contrast, the commercially available anti-HVEM mAB, MAB356, strongly bound hrHVEM as expected ( Figure 6C ), but the presence of MAB356 only slightly disrupted the HVEM and BTLA interaction compared to the addition of the mIgG1 isotype control antibody ( Fig.6D ). Therefore, the mAb of the present invention is much better than MAB356 in inhibiting BTLA-HVEM interaction.

[0188] Next, check the downstream signaling by HVEM.HVEM activation induced NF-kB signaling by ligand binding.To assess the impact of mAb of the present invention on HVEM signaling, Jurkat T cells expressing hrHVEM and NF-kB luciferase reporter genes were pre-incubated on ice for 1 hour with 20 μg / ml of mAb or hIgG4 isotype control.The washed cells were then incubated at 37°C with CHO-K1-empty, CHO-K1-hrBTLA or no cells for 6 hours.Luciferase activity was tested using the Bright-Glo luciferase assay system, and luminescence was measured using a Spark 10M plate reader after adding reagents for 10 minutes.Luciferase was induced in Jurkat cells pre-incubated with control antibodies only when CHO cells expressed BTLA ( Figure 7 , right). This would be expected since NF-kB signaling should only be induced by the ligands of HVEM. Interestingly, when pre-incubated with the mAbs of the invention, the very strong luciferase induction seen when co-incubated with CHO-K1-hrBTLA cells was not seen ( Figure 7 , left middle column). This is due to the mAb blocking the HVEM-BTLA interaction. However, the luciferase level after pre-incubation with the mAb was also not at baseline levels. Rather, the mAb itself induced a mild activation of HVEM and NF-kB signaling ( Figure 7 , left). The same level of activation was seen whether or not CHO cells were co-incubated, confirming that this activation was caused by the mAb itself and not by the cells.

[0189] Example 4: The mAbs of the present invention increase the cytotoxicity of T cells against cancer cells.

[0190] Since the mAb binds to HVEM and blocks its binding to BTLA, it should theoretically alleviate 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 present invention or hIgG4 isotype control antibody at 37°C for 1 hour. After 1 hour, tumor infiltrating lymphocytes (TILs) were added to the melanoma cells and incubated at 37°C for 24 hours. After incubation, supernatants were collected from the co-culture samples or from the TIL samples alone as a control for background IFNγ secretion. IFNγ secretion was measured by ELISA using the Human IFNγ ELISA Max Deluxe kit. Pre-incubation with the mAb of the present invention resulted in almost doubling of IFNγ secretion in one melanoma cell system and a significant increase (although lower) in the second system. Figure 8 ), indicating that T cell suppression was indeed relieved by the antibody.

[0191] 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 mAbs of the invention or isotype controls. At the same time, TILs were pre-incubated with anti-PD-1 antibodies or isotype controls. Melanoma cells and TILs were then co-incubated in all possible combinations, and specific killing of melanoma cells was measured every hour for 11 hours ( Fig.9A - Figure B). In the first cell system, the addition of the mAb of the present invention resulted in a nearly 30% increase in total cell killing after 11 hours compared to cells treated with isotype controls. Similar levels of killing were observed with anti-PD1 treatment (increased by 33%). The combined treatment of the two antibodies resulted in an additive increase of 60% in overall killing. The combined effect was enhanced by the results obtained using cells from the second patient. Once again, the mAb of the present invention and anti-PD1 had similar effects, increasing killing by 67% and 64%, respectively. Unexpectedly, the combined treatment of the two antibodies had a synergistic effect, with a total killing increase of 180% compared to the control co-culture. This proves that the two antibodies together can produce a synergistic effect on immune checkpoint inhibition.

[0192] Next, MAB356 was tested in the same system. Melanoma cell killing by TILs was tested as described above using melanoma cells from the first patient. Melanoma cells were pre-incubated with MAB356 or mAbs of the invention or appropriate isotype controls and then mixed with autologous TILs. Specific killing of melanoma cells was measured every hour for 15 hours. Addition of the mAbs of the invention resulted in a 22% increase in total cell killing after 15 hours compared to cells treated with the hIgG4 isotype control (Figure 2A). Fig.9D), whereas the addition of commercial MAB356 resulted in a 9% decrease in total cell killing compared to cells treated with the mIgG1 isotype control ( Fig.9E ). This clearly shows that the mAbs of the present invention are superior to MAB356.

[0193] Similar experiments were performed using cells from RCC patients. Cancer cells were allowed to adhere overnight, pre-incubated with mAbs of the invention or isotype controls for 1 hour, and then co-cultured with TILs from the same patient. Cell death in cancer cells was measured by caspase 3 / 7 positive events, and after 26 hours, mAbs of the invention were found to increase total cell killing by 47% (95% CI, 2.3-1.4) compared to isotype controls. Fig. 9C ).

[0194] Example 5: The mAbs of the present invention increase the cytotoxicity of non-autologous immune cells against cancer cells.

[0195] It has been shown that the use of mAbs of the present invention can enhance the cytotoxicity of TILs from cancer patients, and the ability to enhance non-autologous immune cells was tested. PBMCs from healthy donors were collected and expressed the α and β chains of the human MART1 TCR. Melanoma cells expressing nuclear RFP staining of MART1 were allowed to adhere overnight and pre-incubated with mAbs of the present invention or isotype controls for one hour before co-culture with PBMCs. Cell death in cancer cells was measured by caspase 3 / 7 positive events, and after 26 hours, mAbs of the present invention were found to increase total cell killing by 28% ( 100% CI 2.0-3.0) compared to isotype controls. Fig. 10A ).

[0196] Proinflammatory cytokine secretion from PBMCs was also measured. Specifically, interferon gamma (IFNγ) secretion was measured in culture medium from PBMCs cultured with culture medium containing mAb of the present invention or isotype control, or cultured with melanoma cells pre-incubated with mAb of the present invention or isotype control. IFNγ levels were measured 24 hours after the start of culture. As expected, incubation with melanoma cells greatly increased IFNγ secretion, but incubation with melanoma cells pre-incubated with mAb of the present invention further increased secretion by 32% (10.3% vs. 1.5% vs. 2.5% IFNγ) compared to melanoma cells pre-incubated with isotype control. Fig. 10B ).

[0197] To exclude the possibility that the increased cytotoxicity was directly caused by the antibody, rather than due to increased T cell killing, the direct cytotoxic effect of the mAb was measured. Nuclear RFP-stained adherent melanoma cells were incubated with 20 μg / ml of the mAb of the present invention or hIgG4 isotype control antibody for 1 hour at 37°C. After 1 hour, CellEvent caspase-3 / 7 green detection reagent was added to the melanoma cells. The plate was 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 ( Fig.11 ), indicating that the antibody itself is not cytotoxic and that the increased cancer killing observed in the previous experiment is due to increased T cell cytotoxicity.

[0198] Example 6: mAbs of the present invention induce basal mild activation of TILs.

[0199] While alleviating BTLA-induced T cell suppression, the mAbs of the invention also bind HVEM on the T cells themselves. Since the mAbs increase NF-kB-induced transcription via HVEM signaling, direct binding of the mAbs can directly increase T cell activation. To test this, TILs were stimulated with mAbs or isotype controls - both alone and in the presence of autologous melanoma cells, and the activation of T cells was monitored ( Fig. 12A ). The percentage of activated T cells increased in the presence of the mAb, both in the presence and absence of melanoma cells. This suggests that binding of the antibody to HVEM on T cells does induce activation on its own. Thus, the antibody has at least a dual effect, blocking the immune surveillance escape mechanism of cancer cells while also directly activating T cells.

[0200] Signaling through HVEM was also tested by measuring T cell clustering. Cell clustering is a hallmark of activated T cells. TILs from two different melanoma patients were incubated with mAbs of the invention or hIgG4 isotype controls for 20 hours and then the number of clusters was measured. For both samples tested, mAbs of the invention significantly increased T cell clustering ( Fig. 12B When MAB356 was tested, no changes in T cell clustering were observed compared to the mIgG1 control ( Fig. 12C ). This suggests that while binding of the mAbs of the present invention to HVEM on T cells induces signaling and low-level activation, binding of the commercially available MAB356 does not.

[0201] The activation of PBMCs in the presence of melanoma cells was also measured. As before, PBMCs expressed MART-1 specific TCRs to target melanoma cells. For these non-autologous cells as well, mAbs greatly increased activation, as measured by 41BB expression on the cells ( Fig.12D ).

[0202] Cytokine release syndrome is always a problem when administering immune checkpoint inhibitors. As shown above, the mAbs of the present invention are able to induce mild activation of TILs even in the absence of cancer cells. To further elucidate the extent of this activation, a cytokine release assay was performed. Whole blood was collected from 10 healthy donors and the mAbs of the present invention were tested at concentrations of 0.1, 1, 10 and 100 ug / ml. After 24 hours, the samples were measured for the expression of five cytokines: IL-6, IL-8, IL-10, IENγ and TNFα. Two commercially available anti-cancer antibodies, Erbitux (EGFR inhibitor) and Lemtrada (anti-CD52), were used as controls. The results of the analysis are summarized in Fig.13 Unlike Lemtrada, which induced a robust cytokine response, the mAbs of the present invention produced only a mild response comparable to Erbitux.

[0203] Example 7: In vivo cancer treatment using mAbs of the invention.

[0204] Human melanoma cells were implanted subcutaneously in 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, hIL-2 was administered subcutaneously for five consecutive days while TILs were administered, then twice a week. On day 18, then twice a week, mice were intravenously administered hIgG4 isotype control (black circles), mAbs of the present invention (white squares), anti-PD1 (black triangles), or a combination of the two antibodies (black diamonds). Treatment with the mAbs of the present invention resulted in a 31% tumor growth inhibition (TGI) compared to treatment with the isotype control, while treatment with anti-PD1 resulted in only a 11% TGI. The combined treatment had a synergistic effect resulting in a TGI of 48% ( Fig.14 ). No adverse side effects were observed in any of the mice. These results demonstrate that the antibodies of the present invention are effective for in vivo treatment of cancer, and that combined treatment with anti-PD1 antibodies has a synergistic effect of enhancing treatment.

[0205] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. Sequence Listing <110> 4C Biomedical Tehahsumo Medical Research Infrastructure and Services <120> Anti-HVEM antibodies and uses thereof <130> 4CB-P-001-PCT <150> 62 / 965,921 <151> 2020-01-26 <150> 62 / 839,841 <151> 2019-04-29 <160> 16 <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic <400> 1 Ser Tyr Ala Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic <400> 2 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic <400> 3 Ala Pro Gly Asp Tyr Thr Ala Tyr Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic <400> 4 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic <400> 5 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 6 Gln Gln Tyr Gly Ser Ser Pro Pro Tyr Thr 1 5 10 <210> 7 <211> 120 <212> PRT <213> Artificial <220> <223> Synthetic <400> 7 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Pro Gly Asp Tyr Thr Ala Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 108 <212> PRT <213> Artificial <220> <223> synthetic <400> 8 Glu Leu Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 466 <212> PRT <213> Artificial <220> <223> Synthetic <400> 9 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Ala Pro Gly Asp Tyr Thr Ala Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 450 455 460 Gly Lys 465 <210> 10 <211> 234 <212> PRT <213> Artificial <220> <223> Synthetic <400> 10 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 35 40 45 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser 100 105 110 Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 11 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic <400> 11 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic <400> 12 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 13 <211> 13 <212> PRT <213> Artificial <220> <223> Synthetic <400> 13 Ala Lys Ala Pro Gly Asp Tyr Thr Ala Tyr Phe Asp Tyr 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial <220> <223> Synthetic <400> 14 Gln Ser Val Ser Ser Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial <220> <223> Synthetic <400> 15 Gly Ala Ser 1 <210> 16 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 16 Gln Gln Tyr Gly Ser Ser Pro Pro Tyr Thr 1 5 10

Claims

1. An antibody or antigen-binding fragment thereof that binds to HVEM and inhibits the interaction between said HVEM and BTLA, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 consists of the amino acid sequence set forth in SEQ ID NO:1 (SYAMS), CDR-H2 consists of the amino acid sequence set forth in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 consists of the amino acid sequence set forth in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 consists of the amino acid sequence set forth in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 consists of the amino acid sequence set forth in SEQ ID NO:5 (GASSRAT), and CDR-L3 consists of the amino acid sequence set forth in SEQ ID NO:6 (QQYGSSPPYT).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7), and the light chain variable region comprises the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8).

3. The antibody or antigen-binding fragment thereof according to claim 2, comprising: a heavy chain comprising the sequence MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 9); and a light chain comprising the sequence MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10).

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which upon binding activates downstream signaling through the HVEM.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the downstream signaling through the HVEM comprises nuclear factor activated B-cell kappa-light-chain enhancer (NF-kB)-mediated transcription. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a single-chain antibody (scFv).

7. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which comprises IgG2 or IgG4.

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

9. The pharmaceutical composition of claim 8, for use in the manufacture of a medicament for treating HVEM-positive cancer or precancerous lesions.

10. A combination of a pharmaceutical composition according to claim 8 and an anti-PD-1 / PD-L1 blocker for use in the manufacture of a medicament for treating HVEM-positive cancer or precancerous lesions.

11. A combination of the pharmaceutical composition according to claim 8 and an anti-PD-1 blocking antibody or an anti-PD-L1 blocking antibody for use in the manufacture of a medicament for treating HVEM-positive cancer or precancerous lesions.

12. An ex vivo method for determining the suitability of a subject to be treated with a pharmaceutical composition comprising an antibody or antigen-binding fragment of any one of claims 1-3, the method comprising receiving 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 treatment.

13. The method of claim 12, wherein positive expression of HVEM comprises elevated levels of HVEM compared to a healthy sample or a predetermined threshold.

14. A method for detecting HVEM in a sample, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, thereby detecting HVEM.

15. A kit comprising the pharmaceutical composition of claim 8 and at least one of the following: a. Anti-PD-1 / PD-L1 blockers; b. a label indicating that the pharmaceutical composition of the present invention is used together with an anti-PD-1 / PD-L1 blocking agent; and c. A second detection molecule for detecting the at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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