Anti-human HVEM (TNFRSF14) antibodies and their uses

Abstract

The present invention discloses antibodies that bind to the extracellular portion of human HVEM on human HVEM-expressing cells, prevent BTLA from binding to HVEM when the antibody binds to the extracellular portion of HVEM, and displace BTLA from binding to the extracellular portion of HVEM. The present invention also discloses the use of such antibodies in combating certain diseases.

Description

[Technical field]

[0001] The present invention relates to the field of antibodies and uses of such antibodies. The present invention provides, in particular, antibodies that bind to HVEM. The present invention also provides kits and compositions that include anti-HVEM antibodies, as well as therapeutic methods using the antibodies described herein. [Background technology]

[0002] Activation of T cells requires the simultaneous activation of at least two signals, the engagement of the T cell receptor and an additional signal delivered by a costimulatory molecule. Several costimulatory molecules, belonging to the B7 / CD28 and TNF / TNFR families, play important roles in regulating immune responses and improving antitumor immunity. Tumors can escape immune surveillance by generating an immunosuppressive microenvironment, and antitumor T cell responses are dampened by the lack of costimulatory molecules such as PD-L1 / L2 and / or the overexpression of co-inhibitory molecules on the surface of cancer cells. Targeting costimulatory and co-inhibitory pathways represents an attractive therapeutic strategy to enhance antitumor immunity in several human cancers. Clinical trials targeting the co-inhibitory Ig molecules CTLA-4 and PD-1 have already yielded promising results in patients with melanoma, renal cell and prostate cancer, as well as non-Hodgkin's lymphoma, leading to drug approvals such as Yervoy® and Opdivo®.

[0003] There is interest in evaluating the potential role of other costimulatory and co-inhibitory receptor / ligand interactions. One such molecule is herpes virus entry mediator (HVEM / CD270 / TNFRSF14) and its ligands. The interaction between HVEM and its ligands is more complex than, for example, PD-1 / PD-L1, as there is evidence of bidirectional signaling. HVEM is a molecular switch between stimulatory and inhibitory signaling upon interaction with its ligands, which include BTLA (B and T lymphocyte attenuator), CD160, LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) and TNFβ / LTα (tumor necrosis factor β / lymphotoxin α). HVEM and its ligands play a role in the physiopathology of immune regulation. In the present invention, it was shown that dysregulation of this network contributes to various diseases.

[0004] HVEM was first discovered as a coreceptor for glycoprotein D of herpes simplex virus 1, which allows the virus to enter cells. HVEM is widely expressed in tissues, with the highest expression levels found in the lung, kidney, and liver. HVEM has also been found to be expressed on T cells, B cells, NK cells, and myeloid cells. Notably, HVEM expression is upregulated in some cancers. HVEM is known to interact with BTLA, CD160, LIGHT, and TNFβ. Most of the ligands, and HVEM itself, can be expressed on either side of the immune synapse. CD160 has been found to be expressed in NK cells, NKT cells, and T cells, while BTLA is highly expressed in activated T cells and resting B cells, as well as less expressed in naive T cells, NK cells, dendritic cells (DCs), and macrophages. LIGHT has been found to be expressed by immature DCs, granulocytes, monocytes and activated T cells, whereas TNFβ is expressed in B and T cells and NK cells.

[0005] Without being bound by theory, it is believed that engagement of BTLA and CD160 on T lymphocytes by HVEM provides a co-inhibitory signal to T lymphocytes via BTLA and CD160, and ligation of HVEM on T lymphocytes by LIGHT and TNFβ delivers a co-stimulatory signal via HVEM. These interactions are bidirectional. HVEM induces inhibitory signals in T cells after interaction with BTLA and CD160 on T cells, whereas both BTLA and CD160 act as activating ligands for HVEM, resulting in NFκB activation. Furthermore, LIGHT delivers co-stimulatory signals to T cells when it interacts with HVEM expressed on T cells, and HVEM is also involved in transmitting co-stimulatory signals to T cells when it interacts with LIGHT expressed by T cells. However, LIGHT does not contain obvious signaling motifs, and its mechanism of signaling is not fully defined.

[0006] When LIGHT and / or TNFβ, BTLA and / or CD160 simultaneously interact with HVEM, the net effect is an inhibitory signal for T cell activation. Many tumors (e.g., melanoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer) overexpress HVEM. Thus, the therapeutic potential of inhibitory interactions between HVEM on cancer cells and BTLA / CD160 on T cells may be important. Therapeutic blocking This may enhance anti-tumor T cell responses while leaving LIGHT-mediated signaling intact in HVEM-expressing T cells. Antibodies capable of interfering with the binding of BTLA to HVEM have been described (WO2014 / 184360A1). In one embodiment, the present invention provides a method for interfering with the binding of BTLA to HVEM. blocking and take the BTLA bound to the HVEM. except These and other antibodies are the subject of the present disclosure. Summary of the Invention

[0007] In one aspect, the disclosure provides an antibody that binds to an extracellular portion of herpesvirus entry mediator (HVEM) on an HVEM-expressing cell, wherein the antibody binds to the extracellular portion of HVEM. did Provided is an antibody that, at times, prevents the binding of B and T lymphocyte attenuator (BTLA) to HVEM. In one embodiment, the antibody removes BTLA bound to the extracellular portion of HVEM. Except obtain.

[0008] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0009] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 24 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 25 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0010] In one aspect, the disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42-44 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45-47 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0011] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 40 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 41 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0012] In one aspect, the disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18-20 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21-23 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0013] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0014] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 34-36 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 37-39 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0015] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 32 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 33 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0016] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 50-52 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53-55 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0017] In one aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 48 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 49 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0018] In one aspect, the present disclosure provides a nucleic acid molecule(s) encoding an antibody disclosed herein or an antigen-binding fragment disclosed herein. Further provided are nucleic acids encoding the variable regions disclosed herein.

[0019] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule described herein. In one aspect, the present disclosure provides a cell comprising the antibody, nucleic acid molecule, and / or vector disclosed herein. Preferably, the host cell is a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell. More preferably, the cell is a human cell. Preferably, the host cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NSO cell, or a PER-C6™ cell.

[0020] In one aspect, the disclosure provides a method for producing an antibody disclosed herein. The method includes harvesting the antibody. Preferably, the antibody is produced using a cell and harvested from the cell. Preferably, the cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NSO cell, or a PER-C6™ cell. Preferably, the antibody is produced synthetically.

[0021] In one aspect, the present disclosure provides a pharmaceutical composition comprising the disclosed antibody or its antigen-binding fragment, nucleic acid, and / or cell.Preferably, the composition or antibody or its antigen-binding fragment disclosed herein is for use in the manufacture of a medicament.Preferably, the medicament is for the treatment and / or prevention of cancer and immune-related disorders.

[0022] In one aspect, the present disclosure provides a method for the treatment of cancer and immune-related disorders in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0023] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof for use in the treatment of cancer and immune-related disorders.

[0024] In one aspect, the disclosure provides a method for modulating HVEM signaling activity, comprising contacting an HVEM-expressing cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0025] In one aspect, the disclosure provides a method for increasing an immune response in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0026] In one aspect, the disclosure provides a method for reducing tumor growth in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure describes antibodies that bind to the extracellular portion of human HVEM on HVEM-expressing cells and to soluble HVEM. The antibodies described herein are characterized in that the antibodies bind to the extracellular portion of human HVEM. did Sometimes, it is useful to prevent the binding of BTLA and CD160 to HVEM. Several antibodies that bind to HVEM have been generated. Antibodies that specifically bind to HVEM are known in the art. For example, the antibody eBio HVEM-122 (eBiosciences) is commercially available and is referenced in Example 2. Antibodies that can interfere with the binding of BTLA to HVEM have been described (WO2014 / 184360A1). The present invention relates to a method for preventing the binding of BTLA to HVEM. blocking Then, take the BTLA once bound to the HVEM. except An antibody is provided.

[0028] In one aspect, the disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, the antibody binding to the extracellular portion of HVEM. did The present invention prevents the binding of BTLA to HVEM and removes BTLA once bound to the extracellular portion of HVEM. except An antibody is provided.

[0029] The term "antibody" typically refers to an immunoglobulin molecule consisting of two identical polypeptide chain pairs, each pair having one "heavy" (H) chain and one "light" (L) chain. Human light chains are classified as kappa (κ) and lambda (λ). Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region of IgD, IgG, and IgA consists of three domains, CH1, CH2, and CH3, while the heavy chain constant region of IgM and IgE consists of four domains, CH1, CH2, CH3, and CH4. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The light and heavy chain variable regions together form the antibody binding site and define the specificity for the epitope. Various methods are known in the art for assigning amino acids to regions or domains in an antibody. Well-known methods include the Kabat method and the Chothia method (Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991); Chothia et al. Conformations of immunoglobulin hypervariable regions in Nature 1989;342(6252):877-83).The assignment of amino acids to each region or domain of the present disclosure follows the Kabat definition.

[0030] The term "antibody" includes murine, humanized, deimmunized human and chimeric antibodies, as well as antibodies that are multimeric forms of antibodies, such as dimers, trimers, or higher multimers of monomeric antibodies. Antibodies also include monospecific, bispecific or multispecific antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity. It also includes antibodies that are linked or attached to non-antibody moieties. Furthermore, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes monoclonal antibodies, recombinant antibodies, and polyclonal antibodies. The present invention provides antibodies as described herein. Furthermore, the present invention provides portions, derivatives, and / or analogs of the antibodies disclosed herein. The portions, derivatives, and / or analogs retain the antigen-binding properties of the antibody in kind, not necessarily in amount. Non-limiting examples of portions and / or derivatives include portions of antibodies that are antigen-binding portions, typically containing one or more variable domains of the antibody. Non-limiting examples are the various Fab fragments. The portions can also be so-called single domain antibody fragments. Single domain antibody fragments (sdAbs, called nanobodies by their developer Ablynx) are antibody fragments that have a single monomeric variable antibody domain. Like whole antibodies, they can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibody fragments are much smaller than typical antibodies (150-160 kDa), which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25 kDa, two variable regions, one light chain and one heavy chain). Single domain antibodies themselves are not much smaller than regular antibodies (which are typically 90-100 kDa). Single domain antibody fragments are engineered from heavy chain antibodies found mainly in camelids, and these are called VHH fragments (Nanobodies®). Some fish also have heavy chain only antibodies (IgNAR, "immunoglobulin neoantigen receptor"), from which single domain antibody fragments called VNAR fragments can be obtained.An alternative approach is to split the dimeric variable domain from common immunoglobulin G (IgG) of human or mouse origin into monomers. Most of the research on single domain antibodies is currently based on the heavy chain variable domain, but nanobodies derived from the light chain have also been shown to specifically bind target epitopes. Non-limiting examples of antibody moieties contain the heavy and / or light chain variable domains of an antibody or its equivalent. Non-limiting examples of such moieties are VHH, human domain antibodies (dAbs) and unibodies. Preferred antibody moieties or derivatives have at least the heavy and light chain variable domains of the antibodies described herein. Non-limiting examples of derivatives or moieties are F(ab) fragments and single chain Fv fragments. Functional moieties of bispecific antibodies include the antigen-binding moieties of the bispecific antibodies, or derivatives and / or analogues of the binding moieties.

[0031] A "single-chain antibody" (scFv) has a single polypeptide chain that includes a VL domain linked to a VH domain, and the VL domain and the VH domain are paired to form a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). A "diabody" has two chains, each chain includes a heavy chain variable region connected to a light chain variable region on the same polypeptide chain connected by a short peptide linker, and the two regions on the same chain do not pair with each other, but pair with the complementary domain on the other chain to form a bispecific molecule. Methods for preparing diabodies are known in the art (see, for example, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak RJ et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies that correspond to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, for example, U.S. Patent Nos. 6,291,158, 6,582,915, 6,593,081, WO04 / 003019 and WO03 / 002609). Nanobodies are derived from the heavy chains of antibodies. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent No. 6,765,087, U.S. Patent No. 6,838,254, WO06 / 079372). Unibodies have one light chain and one heavy chain of an IgG4 antibody. Unibodies can be made by removing the hinge region of an IgG4 antibody. Further details of unibodies and their preparation methods can be found in WO2007 / 059782.The list of analogs to antibodies is growing every year. The current extensive knowledge of the variable domain sequences and 3D structures of many different antibodies allows the skilled artisan to convert the antibodies of the invention into one or other antibody analogs, portions or derivatives.

[0032] In addition to the binding molecule, the molecules of the invention may further comprise moieties to increase the in vivo half-life of the molecule, such as polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran. Such additional moieties may be conjugated to or otherwise combined with the binding moiety using methods well known in the art.

[0033] Also provided are chimeric antigen receptors (CARs) that include the variable domains of the antibodies described herein. CARs are engineered receptors that combine new specificities (typically antigen-binding portions of antibodies or derivatives thereof) with immune cells against target cells. The receptors are called chimeric because they are fusions of portions from different sources (T-lymphocytes genetically engineered to express one or more chimeric antigen receptors (CARs, see, for example, Eshhar, U.S. Pat. No. 7,741,465; Eshhar, U.S. Patent Application Publication No. 2012 / 0093842). In some embodiments, the antibodies disclosed herein can be coupled to active compounds, such as toxins. Additionally, the disclosed antibodies or antigen-binding fragments can be coupled to labels, such as fluorescent proteins, chemical labels, organic dyes, colored particles, or enzymes. The antibodies disclosed herein can be coupled to drugs to form antibody-drug conjugates (ADCs). The present invention provides antibody analogs, antibody portions, and antibody derivatives, even when the molecules of the antibody analogs, antibody portions, and antibody derivatives are coupled or incorporated into other molecules.

[0034] In some embodiments, the antibodies disclosed herein are chimeric antibodies.

[0035] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from two different species, such as human and mouse, typically a combination of mouse variable regions (from heavy and light chains) and human constant regions (from heavy and light chains). Non-limiting examples of generating such chimeric antibodies are described in the Examples (e.g., Example 5). In this chimeric antibody, the mouse IgG1 / kappa constant region is exchanged with a human IgG / kappa constant domain. In some embodiments, the antibody disclosed herein is a humanized antibody. The term "humanized antibody" refers to an antibody that contains some or all of the CDRs from a non-human animal antibody, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody into human framework sequences, followed by back-substitution of certain human framework residues for the corresponding mouse residues from the source antibody. The term "deimmunized antibody" also refers to an antibody of non-human origin, typically in one or more variable regions, from which one or more epitopes with a high propensity to constitute human T-cell and / or B-cell epitopes have been removed in order to reduce immunogenicity. The amino acid sequence of epitope can be completely or partially removed.However, typically, the amino acid sequence is modified by replacing one or more of the amino acids that constitute epitope with one or more other amino acids, thereby changing the amino acid sequence to a sequence that does not constitute human T cell and / or B cell epitope.In some cases, the amino acid is replaced by the amino acid that exists at the corresponding position in the corresponding human variable heavy chain or variable light chain.

[0036] In some embodiments, the antibody disclosed herein is a human antibody. The term "human antibody" refers to an antibody that consists of the amino acid sequence of only human immunoglobulin sequences. A human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be prepared in various ways known in the art. Chimeric, humanized, deimmunized, and human antibodies are within the scope of the present invention.

[0037] The antibody that binds to human HVEM binds to HVEM under conditions that are normally used for antibody binding. When the antibody and human HVEM are contacted with each other under conditions suitable for antibody binding, the antibody binds to human HVEM. The antibody binds to the membrane-bound human HVEM expressed on HEK293F cells, while the antibody does not significantly bind to HEK293F cells that do not express human HVEM on the cell membrane. The binding of the antibody to human HVEM-expressing cells can be detected by methods known to those skilled in the art. For example, by using a secondary antibody carrying a fluorescent label, flow cytometry (FACS) is used to measure the labeled cells.

[0038] HVEM, also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14) and CD270, is a human cell surface receptor of the TNF receptor (tumor necrosis factor) superfamily. In humans, the protein is encoded by the TNFRSF14 gene. HVEM can engage with at least four different ligands, TNFSF members LIGHT (TNFSF14) and TNFβ / LTα (tumor necrosis factor β / lymphotoxin α), as well as immunoglobulin superfamily members B and T lymphocyte attenuator (BTLA) and CD160. For the reference sequence of human HVEM, see SEQ ID NO: 1 (Swiss-Prot number Q92956.3, aa1-283). Reference is made only to identify the HVEM gene / protein. It is not intended to limit HVEM described herein to a particular sequence of database entries. Natural variants of HVEM capable of binding to BTLA, CD160, LIGHT and TNFβ and capable of binding to the antibodies described herein are within the scope of the present invention. Recombinant human HVEM is also within the scope of the present invention if it is capable of binding to BTLA, CD160, LIGHT and TNFβ and capable of binding to the antibodies described herein.

[0039] HVEM is widely expressed in tissues, with the highest expression level in lung, kidney, and liver, and is found to be expressed in T cells, B cells, NK cells, and bone marrow cells. HVEM expression is found to be upregulated in some cancers. The term "HVEM-expressing cells" refers to cells that express HVEM. Exemplary cells are T cells, B cells, NK cells, and bone marrow cells. BTLA and / or CD160 binding to HVEM can have an inhibitory effect, while LIGHT and / or TNFβ binding to HVEM can have a stimulatory effect.

[0040] The term "extracellular" literally means outside the cell. The term "extracellular portion" refers to the portion of the molecule that is outside the cell membrane. This portion of the molecule may be available for interaction with other molecules outside the cell. HVEM has an extracellular portion defined by a cysteine-rich feature. The extracellular domain of HVEM contains four cysteine-rich domains, namely, CRD1-CRD4, and a linker. Without being bound by theory, it is believed that the interaction between BTLA and CD160 and HVEM occurs through CRD1 of HVEM, while the interaction between LIGHT and TNFβ and HVEM occurs through CRD2 and CRD3 of HVEM.

[0041] In one embodiment, the antibody of the invention binds to CRD1 of HVEM.

[0042] HVEM is present on the cell surface of most hematopoietic cell lineages, including T cells, B cells, NK cells, and bone marrow cells. HVEM expression has been found to be downregulated in T cells and B cells upon activation, and upregulated in some cancers. Non-limiting examples include melanoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer. The term "human HVEM-expressing cell" refers to cells that express human HVEM. Exemplary cells are T cells, B cells, NK cells, and bone marrow cells.

[0043] The term "preventing binding" refers to the ability of a protein to be bound by an antibody. did Sometimes, this refers to the ability of the antibody or antigen-binding fragment thereof to prevent the binding of a ligand to the protein. When reference is made to "preventing binding," this refers to "preventing binding." blocking do" (blocking) It can also be interpreted as: No Ligand binding is said to be prevented if binding of the ligand is prevented by more than 70% (>) compared to binding of the ligand in the presence of antibody. Ligand binding is said to be partially prevented if binding of the ligand is prevented by more than 30% (≧) but not more than 70% (≦) compared to binding of the ligand in the absence of antibody. Ligand binding is said to be unaffected if binding of the ligand is prevented by less than 30% (<) compared to binding of the ligand in the absence of antibody. Ligand binding is said to be enhanced if binding of the ligand is increased by more than 30% (>) compared to binding of the ligand in the absence of antibody.

[0044] The antibodies or antigen-binding fragments disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, they prevent BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells. Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0045] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, and does not prevent or partially prevents LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0046] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. didSometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, and partially prevents LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0047] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, but does not prevent LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. An exemplary antibody is 11H7.

[0048] In further embodiments, the anti-HVEM antibodies disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, and partially prevents CD160 from binding to the extracellular portion of HVEM on HVEM-expressing cells. Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0049] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, and partially prevents CD160 and LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0050] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. didSometimes, it prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells, partially prevents CD160 from binding to the extracellular portion of HVEM on HVEM-expressing cells, and does not prevent LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. An exemplary antibody is 11H7.

[0051] Typically, the above-mentioned anti-HVEM antibody or antigen-binding fragment thereof binds to the CRD1 domain of the HVEM protein.

[0052] Binding of BTLA, CD160 and LIGHT to HVEM is preferably measured by the method described in the Examples. An exemplary method is described, for example, in Example 6b, and the results are shown in Figures 6A-D. Preferably, HEK293F cells transfected with full-length HVEM are used. Preferably, the cells stably express full-length HVEM on the plasma membrane. HVEM-expressing HEK293F cells are used to test the test antibody. The cells are incubated with the anti-HVEM antibody of interest. After washing, the cells are incubated with biotin-labeled or His-tagged human BTLA, CD160, or LIGHT. After washing, the label or tag is detected with fluorescently labeled streptavidin or anti-His antibody. Binding of BTLA, CD160 or LIGHT to HVEM expressed on the cells can be measured by detecting fluorescence using a flow cytometer (FACS). The ability to prevent binding is then determined by comparing the percentage of BTLA, CD160 or LIGHT bound to HVEM in the presence of an anti-HVEM antibody to the percentage bound in the presence of a control antibody that does not bind HVEM. Less binding of BTLA, CD160 or LIGHT to HVEM indicates a strong inhibition of the antibody. blocking Demonstrate ability.

[0053] " except " (to displace)The term refers to the ability of a first entity to remove a second entity from its location, whereby the second entity is replaced by the first entity. More than (>) 70% of the ligand bound to the extracellular portion of HVEM is removed compared to the presence of the ligand in the absence of antibody. Excluded If the ligand is Removed More than 30% (≥ 100%) and up to 70% (≤ 100%) of the ligand bound to the extracellular portion of HVEM was removed compared to the presence of the ligand in the absence of antibody. Excluded When the ligand is partially removed, Excluded Less than 30% (<) of the ligand bound to the extracellular portion of HVEM was removed compared to the presence of the ligand in the absence of antibody. Excluded If the ligand is Excluded Binding of a ligand is said to be enhanced if binding of the ligand is increased by more than (>) 30% compared to the presence of the ligand in the absence of antibody.

[0054] The antibodies or antigen-binding fragments disclosed herein prevent the binding of BTLA to the extracellular portion of HVEM on HVEM-expressing cells. The antibodies preferably remove BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Removed Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0055] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and does not prevent or partially prevents LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0056] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0057] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did At times, the antibody prevents binding of BTLA to the extracellular portion of HVEM on HVEM-expressing cells and does not prevent binding of LIGHT to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded An exemplary antibody is 11H7.

[0058] In further embodiments, the anti-HVEM antibodies disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents CD160 from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. except Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0059] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents CD160 and LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0060] In further embodiments, the anti-HVEM antibodies or antigen-binding fragments thereof disclosed herein are characterized in that the antibodies bind to the extracellular portion of HVEM. did Sometimes, the antibody prevents binding of BTLA to the extracellular portion of HVEM on HVEM-expressing cells, partially prevents binding of CD160 to the extracellular portion of HVEM on HVEM-expressing cells, and does not prevent binding of LIGHT to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded An exemplary antibody is 11H7.

[0061] Typically, the above-mentioned anti-HVEM antibody or antigen-binding fragment thereof binds to the CRD1 domain of the HVEM protein.

[0062] The anti-HVEM antibodies disclosed herein are capable of binding to ligands bound to the extracellular portion of HVEM. exceptTo analyze whether the antibody has the ability, the skilled person can use several known suitable assays. One of the suitable methods is disclosed in the Examples section. The assay is described in detail, for example, in Example 6c. Preferably, HEK293F cells transfected with full-length HVEM are used. Preferably, the cells stably express full-length HVEM on the cell membrane. The cells are incubated with a soluble ligand (e.g., biotin-labeled or His-tagged BTLA, CD160 or LIGHT). The cells are then incubated with an antibody of the present invention that binds to the extracellular portion of HVEM. After washing, the ligand bound to the cells can be detected using fluorescently labeled streptavidin or anti-His antibody, and after washing, the fluorescent signal of the antibody bound to the ligand can be detected using a flow cytometer (FACS). The amount of ligand bound to the extracellular portion of HVEM indicates the ability of the anti-HVEM antibody to displace the ligand bound to the extracellular portion of HVEM of the antibody. A lower fluorescence signal of the ligand indicates its ability to displace a stronger anti-HVEM antibody. A preferred method is described in the Examples, the results of which are shown, for example, in Figures 7A-D. except Percentages are typically given as a percentage compared to the binding of ligand to HVEM in the presence of a non-specific antibody under otherwise identical conditions. Excluding can be measured using metabolically active cells (e.g., incubated overnight at 37° C.) or using metabolically inactive cells (e.g., incubated at 4° C. in the presence of sodium azide).

[0063] Without being bound by theory, it is believed that T cell activation is inhibited upon interaction of BTLA and / or CD160 with HVEM, even in the presence of LIGHT and / or TNFβ. The antibodies disclosed herein are useful for targeting cells expressing HVEM. Binding of the antibodies disclosed herein is useful for targeting cells expressing BTLA. Except and preferably at least partially removes CD160 bound to the extracellular portion of HVEM. exceptHowever, LIGHT bound to the extracellular portion of HVEM was not taken up by the Excluded As a result, the ability to prevent the binding of BTLA to CD160 and the ability to uptake BTLA Except Preferably, CD160 is partially removed. except This ability suppresses the inhibitory effects of BTLA and CD160 on T cell activation. Cells bound to the antibodies disclosed herein are available to respond to other stimuli, such as LIGHT or TNFβ binding to HVEM.

[0064] The full-length Macaca fasciculari monkey HVEM protein (Met1-Ser280, NCBI Reference Sequence: XP_005545061.1, see SEQ ID NO:5) has an amino acid sequence similar to human HVEM, and shows 82% homology to the human HVEM protein (Met1-His283, Swiss-Prot No. Q92956.3, see SEQ ID NO:1). The predicted amino acid sequence of the extracellular domain of cynomolgus monkey HVEM (i.e., Leu39-Val203, NCBI Reference Sequence: XP_005545061.1) shows 87% homology to the amino acid sequence of the extracellular domain of the human HVEM protein (i.e., Leu39-Val202, Swiss-Prot No. Q92956.3). Substitution of amino acids of human HVEM with corresponding amino acids of cynomolgus monkey HVEM can be used to test the specificity and cross-specificity of the antibody. In one embodiment, the antibody of the present invention binds to human HVEM and is cross-specific to cynomolgus monkey (Macaca fascicularis) monkey HVEM. Exemplary antibodies are 45H6, 11H7, 48H6 and 49G4. Without being bound by theory, it is believed that such antibodies are particularly suitable for binding to human HVEM carrying mutations. Furthermore, such antibodies are suitable for toxicity testing.

[0065] One aspect of the present disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31.

[0066] In a further aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain region having the amino acid sequence of SEQ ID NO: 25 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in the framework regions of the light chain and / or heavy chain variable region. Preferably, the antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24, and a light chain variable region having the amino acid sequence of SEQ ID NO: 25. An exemplary antibody having these characteristics is 45H6.

[0067] One aspect of the present disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42 to 44 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42 to 44, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47.

[0068] In a further aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 40 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain region having the amino acid sequence of SEQ ID NO: 41 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in the framework regions of the light chain and / or heavy chain variable region. Preferably, the antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 40, and a light chain variable region having the amino acid sequence of SEQ ID NO: 41. An exemplary antibody having these characteristics is 11H7.

[0069] One aspect of the present disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18-20 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21-23 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18-20, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21-23.

[0070] In a further aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain region having the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in the framework regions of the light chain and / or heavy chain variable region. Preferably, the antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16, and a light chain variable region having the amino acid sequence of SEQ ID NO: 17. An exemplary antibody having these characteristics is 36H12.

[0071] One aspect of the present disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 34 to 36 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 37 to 39 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 34 to 36, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 37 to 39.

[0072] In a further aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 32 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain region having the amino acid sequence of SEQ ID NO: 33 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in the framework regions of the light chain and / or heavy chain variable region. Preferably, the antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 32, and a light chain variable region having the amino acid sequence of SEQ ID NO: 33. An exemplary antibody having these characteristics is 48H6.

[0073] One aspect of the present disclosure provides an antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 50 to 52 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53 to 55 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody that binds to an extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 50 to 52, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53 to 55.

[0074] In a further aspect, the disclosure provides an antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 48 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain region having the amino acid sequence of SEQ ID NO: 49 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in the framework regions of the light chain and / or heavy chain variable region. Preferably, the antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 48, and a light chain variable region having the amino acid sequence of SEQ ID NO: 49. An exemplary antibody having these characteristics is 49G4.

[0075] In one embodiment, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did In some cases, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. except Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0076] In a further embodiment, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0077] In a further embodiment, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did At times, the antibody prevents binding of BTLA to the extracellular portion of HVEM on HVEM-expressing cells and does not prevent binding of LIGHT to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded An exemplary antibody is 11H7.

[0078] In a further embodiment, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents CD160 from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. except Exemplary antibodies are 45H6, 11H7, 36H12, 48H6, and 49G4.

[0079] In a further embodiment, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did Sometimes, the antibody prevents BTLA from binding to the extracellular portion of HVEM on HVEM-expressing cells and partially prevents CD160 and LIGHT from binding to the extracellular portion of HVEM on HVEM-expressing cells. Preferably, the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded Exemplary antibodies are 45H6, 36H12, 48H6, and 49G4.

[0080] In a further aspect, an anti-HVEM antibody or antigen-binding fragment referred to herein by a sequence is an antibody that binds to the extracellular portion of HVEM. did Sometimes, the antibody prevents binding of BTLA to the extracellular portion of HVEM on HVEM-expressing cells, partially prevents binding of CD160 to the extracellular portion of HVEM on HVEM-expressing cells, and does not prevent binding of LIGHT to the extracellular portion of HVEM on HVEM-expressing cells, and the antibody removes BTLA bound to the extracellular portion of HVEM on HVEM-expressing cells. Except The CD160 bound to the extracellular portion of HVEM on HVEM-expressing cells was partially removed. Except Uptake of LIGHT bound to the extracellular portion of HVEM on HVEM-expressing cells Excluded An exemplary antibody is 11H7.

[0081] Typically, the anti-HVEM antibodies or antigen-binding fragments referred to herein by their sequences bind to the CRD1 domain of the HVEM protein.

[0082] The anti-HVEM antibody or antigen-binding fragment thereof of the present disclosure preferably comprises a heavy chain variable region and a light chain variable region as described herein. Such an antibody has good characteristics. Of course, it is possible to generate variants of such original antibodies by modifying one or more amino acids therein. Many of such variants will behave more or less similarly when compared to the original. Such variants are also included in the scope of the present disclosure.

[0083] Variants may have amino acid substitutions, insertions, deletions, or additions with respect to the sequence of the original antibody. An amino acid substitution is the exchange of an amino acid with another amino acid. Preferably, an amino acid is pre-placed by an amino acid with similar chemical properties, which is often called a conservative substitution. An amino acid deletion results in the deletion of one or more amino acids forming a sequence. An amino acid insertion results in one or more additional amino acids in a sequence. An amino acid addition results in one or more amino acids at the beginning or end of an amino acid sequence.

[0084] A non-limiting example of such a modification is an antibody containing pyroglutamate instead of glutamate. Other non-limiting examples of such modifications are the insertion, deletion, inversion and / or substitution of one or more amino acids when compared to the original antibody. Preferably, the amino acid substitution, insertion, deletion or addition is outside the CDRs of the variable domain. To Preferably, the amino acid substitutions, insertions, deletions, or additions are in the framework regions of the variable region and / or in the constant region of the antibody. The HVEM binding of the variants can be tested as described herein.

[0085] In some embodiments, the constant region of the antibody of the invention is the constant region of an IgG, IgA, IgD, IgE, or IgM antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. The constant region may contain modifications such as amino acid substitutions to confer specific properties to the constant region. For example, mutations in the IgG4 hinge region to make the antibody more stable against half-molecule exchange. Other modifications affect the half-life of the antibody, add or remove glycosylation sites, improve production, improve the homogeneity of the antibody product produced in large-scale fermenters, etc.

[0086] The antibodies of the invention are preferably murine IgG1, human IgG1 mutated in the constant region to reduce or prevent complement activation or Fc receptor interaction, or human IgG4, or human IgG4 mutated to prevent half-molecule exchange with other IgG4 molecules.

[0087] Some variation in the CDRs (CDR1-CDR2-CDR3) of the antibodies disclosed herein is permitted. Typically, about 0-2 amino acid substitutions, insertions, deletions, or additions are permitted in one CDR. In many cases, more than two amino acid changes are permitted.

[0088] The antibodies of the present invention may have a heavy chain CDR1, CDR2, or CDR3 that has 0 to 5, preferably 0 to 2, and more preferably 2, 1, or 0 amino acid substitutions, insertions, deletions, or additions relative to the naturally occurring heavy chain CDR1, CDR2, or CDR3.

[0089] Such antibodies may have a light chain CDR1, CDR2, or CDR3 that has 0 to 5, preferably 0 to 2, more preferably 0 to 1, and more preferably 0 amino acid substitutions, insertions, deletions, or additions relative to the naturally occurring light chain CDR1, CDR2, or CDR3.

[0090] Some variation in the variable regions of the antibodies disclosed herein is tolerated. Typically, about 0-10 amino acid substitutions are tolerated in the variable chain. Often, more than 10 amino acid changes are tolerated.

[0091] The antibodies of the present invention may have a heavy chain variable region that has 0 to 15, preferably 0 to 10, more preferably 0 to 5, more preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions, insertions, deletions, or additions relative to a naturally occurring variable heavy chain.

[0092] Such antibodies may have a light chain variable region that has 0 to 15, preferably 0 to 10, more preferably 0 to 5, and more preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions, insertions, deletions, or additions relative to a naturally occurring light chain variable region.

[0093] Some variation in the constant regions of the antibodies disclosed herein is tolerated. Typically, about 0-10 amino acid substitutions are tolerated in the constant region. Often, more than 10 amino acid changes are tolerated. The antibodies of the present invention may have a heavy chain constant region (CH1-CH2-CH3) with 0-15, preferably 0-10, more preferably 0-5, more preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions with respect to the naturally occurring heavy chain constant region (CH1-CH2-CH3). Such antibodies may have a light chain constant region with 0-5, preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions with respect to the naturally occurring light chain constant region.

[0094] Some variation in IgG4 occurs naturally and / or is tolerated without altering the immunological properties of the resulting antibody. Antibodies with IgG4 constant regions or mutated IgG1 constant regions will have at least most of the pharmacological properties of antibodies, but will not bind complement and therefore will not induce in vivo depletion of cells to which they bind. Preferably, the constant region is a human antibody constant region (chimera).

[0095] Preferably, the constant region is a region that is complement activation deficient, preferably a human IgG4 constant region or a mutated human IgG1 constant region.

[0096] The HVEM binding by the antibodies and antigen-binding fragments thereof disclosed herein can be confirmed by several suitable assays known to those skilled in the art. Such assays include, for example, affinity assays such as Western blots, radioimmunoassays, FACS, and ELISA (enzyme-linked immunosorbent assays). Examples (e.g., Examples 2c and 6a) provide detailed descriptions of some of the many assays that can be used to measure HVEM binding, and methods for determining the relative binding affinity of antibodies to human HVEM.

[0097] In a further aspect, the present disclosure provides a nucleic acid molecule(s) encoding the antibody disclosed herein or the antigen-binding fragment disclosed herein. Further provided is a nucleic acid molecule encoding the variable region disclosed herein. The nucleic acid used in the present disclosure is typically, but not exclusively, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Based on the genetic code, a person skilled in the art can determine the nucleic acid sequence encoding the antibody variant disclosed herein. Based on the degeneracy of the genetic code, 64 codons can be used to encode 20 amino acids and a translation termination signal. As known to a person skilled in the art, codon usage bias in different organisms can affect gene expression levels. Depending on the organism in which the desired nucleic acid is expressed, various computational tools are available to a person skilled in the art to optimize codon usage.

[0098] In a further aspect, the present disclosure provides a vector comprising the nucleic acid sequence molecule described herein. The term "vector" as used herein refers to a nucleic acid molecule, such as a plasmid, a bacteriophage, or an animal virus, capable of introducing a heterologous nucleic acid sequence into a host cell. A vector according to the present invention allows the expression or production of an antibody of the present invention encoded by a heterologous nucleic acid sequence in a host cell. Vectors used according to the present invention are derived, for example, from animal viruses, examples of which include, but are not limited to, vaccinia virus (including attenuated derivatives such as modified vaccinia virus Ankara, MVA), Newcastle Disease Virus (NDV), adenovirus or retrovirus. A vector according to the present invention preferably comprises an expression cassette comprising a promoter suitable for initiating transcription of an antibody according to the present invention in a selected host cell. Examples of promoters suitable for expression of a polypeptide according to the present invention in a eukaryotic host cell include, but are not limited to, β-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus-derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV) and Simian virus 40 (SV40) promoters for mammalian hosts.

[0099] When the nucleic acid molecule(s) disclosed herein are expressed in the cell, the cell may produce an antibody according to the disclosure. Thus, in one embodiment, a cell is provided that comprises an antibody, a nucleic acid molecule(s), and / or a vector according to the disclosure. The host cell may be a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell. The cell is preferably an animal cell, preferably a mammalian cell, and most preferably a human cell. Examples of mammalian cell lines suitable as host cells include hybridoma cells, Chinese Hamster Ovary (CHO) cells, NSO cells, or PER-C6™ cells. A suitable cell for the purposes of the disclosure is any cell that can contain and preferably produce the antibody and / or the nucleic acid. The disclosure further encompasses a cell culture comprising the cell.

[0100] The term "host cell" refers to a cell into which an expression vector has been introduced. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in successive generations, either due to environmental influences or mutations, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell."

[0101] The antibodies disclosed herein may be produced by any method known to those skilled in the art. In a preferred embodiment, the antibodies are produced using cells, preferably the cells are hybridoma cells, CHO cells, NS0 cells, or PER-C6™ cells. In a particular preferred embodiment, the cells are CHO cells, preferably the cells are cultured in serum-free medium. This includes harvesting the antibodies from the culture. The antibodies are preferably purified from the medium, preferably the antibodies are affinity purified. Alternatively, the antibodies may be synthetically produced.

[0102] Various institutions and companies have developed cell lines for large-scale production of antibodies, for example for clinical use. These cells are also used for other purposes, such as for the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, preferred embodiments of the present disclosure provide for the use of cell lines developed for the large-scale production of said antibodies.

[0103] The antibodies according to the present invention exhibit several activities that can be advantageously used in therapeutic and non-therapeutic uses. In particular, the antibodies according to the present invention are useful for treating individuals. Preferably, the antibodies according to the present invention are useful for treating immune-related diseases or for preventing immune-related diseases. In a preferred embodiment, the antibodies according to the present invention are useful for treating cancer. In some embodiments, the antibodies according to the present invention are preferably used in therapy, preferably human therapy. In some embodiments, the antibodies disclosed herein can be used for research purposes, for example in in vitro experiments, cell culture, organotypic culture and in vivo models.

[0104] Methods are also described for the treatment of cancer, examples of which include melanoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer.

[0105] The present invention provides a method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of the antibody disclosed herein.A method is further provided for preparing a medicament for treating a subject suffering from cancer.The present disclosure describes a method for preventing the inhibition of T cell activation by preventing the binding of BTLA and HVEM, and CD160 and HVEM.

[0106] The present invention provides a method for treating a subject suffering from inflammatory disease, comprising administering to the subject a therapeutically effective amount of the antibody disclosed herein.A method is further provided for preparing a medicament for treating a subject suffering from inflammatory disease.The present disclosure describes a method for preventing the inhibition of T cell activation by preventing the binding of BTLA and HVEM, and CD160 and HVEM.

[0107] The present disclosure further includes pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, or nucleic acids encoding the same, or cells comprising the antibodies or antigen-binding fragments thereof disclosed herein, or nucleic acids encoding the same. Pharmaceutical compositions are provided that comprise the polypeptides according to the present invention, or pharma-ceutically acceptable salts thereof, and at least one pharma-ceutically acceptable carrier, diluent, and / or excipient. Such compositions are particularly suitable for use as medicaments. The compositions may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. The dose and schedule of the selected formulation may be determined by standard procedures well known by those skilled in the art. Such procedures include extrapolating and extrapolating the dosing schedule to form an animal model, and then determining the optimal dosage in a human clinical dose range study. The dosage in the pharmaceutical composition varies depending on several factors, such as the desired release and pharmacological characteristics.

[0108] One embodiment of the present disclosure provides a pharmaceutical composition as described herein for use as a prophylaxis or in the treatment of cancer and / or immune-related disorders.

[0109] Methods are further provided for modulating HVEM signaling activity. The term "modulating" refers to the activity of modulating the output signal of the system. The output signal or output can be modulated so that an inhibitory output signal becomes stimulatory, or vice versa. The present invention provides a method for modulating HVEM signaling, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein. Without being bound by theory, it is believed that HVEM delivers a co-inhibitory signal to T cells expressing BTLA or CD160. Meanwhile, LIGHT and TNFβ deliver a co-stimulatory signal to T cells when interacting with HVEM expressed on T cells. When LIGHT and / or TNFβ, BTLA and / or CD160 interact simultaneously with HVEM, the net result is an inhibitory signal for T cell activation. This interaction is bidirectional. HVEM induces an inhibitory signal in T cells after interacting with BTLA and CD160 on T cells, while both BTLA and CD160 act as activating ligands for HVEM, resulting in NFκB activation. Furthermore, LIGHT delivers costimulatory signals to T cells when it interacts with HVEM expressed on T cells, and HVEM has also been implicated in transmitting costimulatory signals to T cells when it interacts with LIGHT expressed by T cells, however, LIGHT does not contain obvious signaling motifs and its signaling mechanism has not been fully defined.

[0110] The costimulatory and costenulatory signaling relayed by HVEM and BTLA in T cells can be measured in the levels of NFkB or NFAT, and the release of IL-2, TNFα, and IFNγ. Methods for measuring the levels of NFAT are known in the art. An exemplary method is described, for example, in Example 3c, the results of which are shown in Figures 4A-B. Methods for measuring the levels of NFkB are known in the art. An exemplary method is described, for example, in Example 6f, the results of which are shown in Figures 8A-C. Methods for measuring the levels of IL-2, TNFα, and IFNγ are known in the art. An exemplary method is described, for example, in Example 6k, the results of which are shown in Figures 11A-C.

[0111] In one aspect, the disclosure provides a method for modulating HVEM signaling activity, comprising contacting an HVEM-expressing cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0112] In one aspect, the disclosure provides a method for increasing an immune response in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0113] In one aspect, the disclosure provides a method for reducing tumor growth in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, or vector disclosed herein.

[0114] As used herein, a "subject" is a human or an animal. Subjects include, but are not limited to, mammals such as humans, pigs, ferrets, seals, rabbits, cats, dogs, cows, and horses, and birds such as chickens, ducks, geese, and turkeys. In a preferred embodiment of the present invention, the subject is a mammal. In a particularly preferred embodiment, the subject is a human.

[0115] The term "antigen-binding fragment" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (i.e., human HVEM) that the antibody binds. The term "antigen-binding fragment" also encompasses portions of an antibody that are part of a larger molecule formed by non-covalent or covalent association, or that have one or more additional molecular entities. Examples of additional molecular entities include amino acids, peptides, or proteins, such as the streptavidin core region, which can be used to create tetrameric scFv molecules (Kipriyanov et al. Hum Antibodies Hybridomas 1995;6(3):93-101). Exemplary antigen-binding fragments are the VH and / or VL of an antibody. Antigen-binding fragments include Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, and other antigen-recognizing immunoglobulin fragments. In some examples, the term "antibody" as used herein can be understood to include antigen-binding fragments thereof.

[0116] The term "human antibody" refers to an antibody that consists of amino acid sequences of human immunoglobulin sequences only. A human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be prepared in a variety of ways known in the art.

[0117] The term "epitope" refers to a portion of an antigen that can specifically bind to an antibody or T-cell receptor or otherwise interact with a molecule. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes generally consist of chemically active surface groups of molecules, such as amino acids, carbohydrates, or sugar side chains. Epitopes can be "linear" or "non-linear / conformational." Once a desired epitope is determined (e.g., by epitope mapping), antibodies can be generated against that epitope. The generation and characterization of antibodies can also provide information about the desired epitope. From this information, it is then possible to screen antibodies for those that bind to the same epitope, for example, by performing cross-competition studies to find antibodies that competitively bind with each other, i.e., antibodies that compete for binding to the antigen.

[0118] As used herein, "comprise" and its conjugations are used in their open-ended sense, meaning that the items following the word are included, but not excluding items not specifically mentioned. In addition, the verb "consisting of" may be replaced with "consisting essentially of." This means that the compounds or ancillary compounds defined herein may contain additional components other than those specifically identified, which additional components do not alter the inherent characteristics of the invention.

[0119] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0120] The word "approximately" or "about" when used in connection with a numerical value (eg, approximately 10, about 10), preferably means that the value may be 1% more or less than the given value by 10.

[0121] As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to an individual susceptible to infection (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors) prior to the onset of symptoms. Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0122] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having all or any combination of the described features.

[0123] All patent and literature references cited herein are incorporated by reference in their entirety.

[0124] The present invention will be further described in the following examples, which do not limit the scope of the invention but merely serve to clarify it. [Brief description of the drawings]

[0125] [Figure 1] Flow cytometry binding characteristics of mouse anti-human HVEM antibodies to membrane-bound full-length human HVEM, membrane-bound human HVEM deleted for CRD1, or membrane-bound full-length cynomolgus HVEM on HEK293F cells. The dashed line represents background (i.e., no binding of mouse anti-human HVEM antibody). [Diagram 2]Effect of mouse anti-human HVEM antibodies on the binding of (A) soluble human BTLA and (B) soluble human LIGHT to membrane-bound full-length human HVEM on HEK293F cells. The dashed line represents the negative control (i.e., ligand / receptor binding without addition of mouse anti-human antibody or addition of mouse IgG1 negative isotype control = 100% binding of ligand to the HVEM receptor). [Diagram 3] (A) Effect of mouse anti-human HVEM antibody on NFκB signaling in membrane-bound human HVEM-expressing cells. Soluble human LIGHT ligand was included as a reference. (B) Soluble human LIGHT in membrane-bound human HVEM-expressing cells.

number

number

number

[0126] Example 1. Generation of mouse anti-human HVEM monoclonal antibodies (a) Generation of Sf9 insect cells and HEK293F cells expressing surface human HVEM The cDNA encoding the human HVEM protein (Swiss-Prot number Q92956.3, see SEQ ID NO: 1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO: 2). This cDNA was subcloned into the baculovirus transcription plasmid pVL1393 (BD ​​Transfection Kit, Catalog No. 560129, BD Biosciences). The transcription plasmid pVL1393 containing the cDNA encoding human HVEM was then transfected into Sf9 insect cells (Spodoptera frugiperda) using the baculoCOMPLETE all-in-one kit (Oxford Expression Technologies) and incubated at 27 °C for 4-5 days. After this transfection step, the supernatant was collected, stored at 4 °C, and used to infect more Sf9 insect cells for virus amplification. For this purpose, Sf9 insect cells were transfected with the amplified recombinant baculovirus and then incubated at 27 °C for 3 to 5 days. These Sf9 insect cells were harvested, washed with sterile PBS, and diluted with 20.0 × 10 6 Cell lysates were obtained by aliquoting cells / mL and storing at −80° C. Prior to storage, human HVEM surface expression was confirmed on transfected Sf9 insect cells using a 1:20 diluted phycoerythrin (PE)-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience) and flow cytometry.

[0127] A cDNA encoding the human HVEM protein (Swiss-Prot number Q92956.3, see SEQ ID NO: 1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO: 2). The cDNA was subcloned in an expression plasmid derived from pcDNA3.1. The full-length human HVEM plasmid was transfected into FreeStyle™ 293F cells (Life Technologies) using the FreeStyle™ 293 Expression System (Life Technologies). Stable human full-length HVEM transfected HEK293F cells (clone number 128) were selected using 125 μg / mL G418 (Gibco). These HEK293F cells were harvested, washed with sterile PBS, and diluted with 19.0 × 10 6 Cell lysates were obtained by aliquoting cells / mL and storing at −80° C. Prior to storage, human HVEM surface expression was confirmed on transfected HEK293F cells using a 1:20 diluted phycoerythrin (PE)-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience) and flow cytometry.

[0128] (b) Immunization and generation of mouse anti-human HVEM monoclonal antibodies Two immunization protocols were applied. During the first immunization protocol, BALB / c mice (female, 6-8 weeks old, Charles River Laboratories) were

number

[0129] During the second immunization protocol, BALB / c mice (female, 6-8 weeks old, Charles River Laboratories) were

number

[0130] (c) Screening for the presence of mouse anti-human HVEM monoclonal antibodies Starting 13 days after fusion, supernatants from growing hybridomas were screened for the presence of mouse anti-human HVEM antibodies (i.e., "high affinity" IgG as opposed to "low affinity" IgM) using an ELISA with soluble recombinant C-terminally polyhistidine-tagged human HVEM (rhuHVEM, Sino Biological) as the target protein. For this purpose, rhuHVEM was coated at 1 μg / mL (50 ng / 50 μL / well) in PBS using half-section flat-bottom 96-well EIA plates (Corning) for 16-24 h at 4-8 °C. After extensive washing with PBS / 0.05%, w / v Tween 20, the plates were incubated with PBS / 0.05% Tween 20 / 1%, w / v bovine serum albumin (BSA, Roche) for 1 h at room temperature. blocking Plates were then incubated with 50 μL of undiluted hybridoma supernatant / well for 1 h at room temperature. After extensive washing with PBS / 0.05% Tween 20, antibody binding was determined using a horseradish peroxidase-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) diluted 1:5000 for 1 h at room temperature, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After addition of 1 M H2SO4, antibody binding was measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (model iMark, BioRad).

[0131] From day 13 post-fusion, supernatants from growing hybridomas were also screened for mouse anti-human HVEM antibody (i.e., "high affinity" IgG as opposed to "low affinity" IgM) production using a cell-based ELISA with membrane-bound human HVEM as the target protein. To this end, HEK293F cells stably transfected with human full-length HVEM (clone no. 128, see Example 1a above) were cultured at 2×10 6 Viable cells / mL (0.1×10 6Half-area flat-bottom 96-well EIA plates (Corning) were coated with 50 μL of undiluted hybridoma supernatant / well for 16–24 h at 4–8 °C. Untransfected (i.e., negative for membrane-bound human HVEM expression) wild-type (WT) HEK293F coated cells were run in parallel as a negative control. After extensive washing with PBS / 0.05%, w / v Tween 20, plates were blocked with PBS / 0.05% Tween 20 / 1%, w / v BSA (Roche) for 1 h at room temperature. Plates were subsequently incubated with 50 μL of undiluted hybridoma supernatant / well for 1 h at room temperature. After extensive washing with PBS / 0.05% Tween 20, antibody binding was determined using a horseradish peroxidase-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) diluted 1:5000 for 1 h at room temperature, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After addition of 1 M H2SO4, antibody binding was measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (model iMark, BioRad).

[0132] From day 13 post-fusion, supernatants from growing hybridomas were also screened for mouse anti-human HVEM antibody (i.e., "high affinity" IgG as opposed to "low affinity" IgM) production using FACS with membrane-bound human HVEM as the target protein. To this end, HEK293F cells stably transfected with human full-length HVEM (clone no. 128, see Example 1a above) were incubated with 50 μg / mL of human IgG (which has potential Fcγ receptors) for 1 h. blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were then incubated at 4° C. for 10 min at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL of undiluted hybridoma supernatant / tube) were incubated for 30 min at 4°C. Non-transfected (i.e., negative for membrane-bound human HVEM expression) WT HEK293F cells were run in parallel as a negative control to determine antibody specificity. After extensive washing with PBS / BSA / NaN3, the cells were subsequently incubated with a PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) diluted 1:200 for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Antibody binding was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0133] Triple HVEM positive (i.e., rhuHVEM+ by ELISA, membrane HVEM+ HEK293F cells by ELISA, and membrane HVEM+ HEK293F cells by FACS) hybridomas were expanded and cryopreserved. Supernatants from these triple HVEM positive hybridomas showed no reactivity with non-transfected (i.e., negative for membrane-bound human HVEM expression) WT HEK293F cells. Using this approach, 18 anti-human HVEM-specific antibody-producing hybridomas were obtained. Mouse antibodies were purified from these anti-human HVEM-specific antibody-producing hybridoma supernatants using a Protein G column (GE Healthcare). Heavy and light chains were typed for isotype class using the IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit (Roche). The supernatants and / or purified antibodies from these anti-human HVEM specific antibody producing hybridomas were then tested for their effect on human HVEM ligand (i.e. human BTLA and human LIGHT) binding to membrane-bound human HVEM, for binding to membrane-bound human HVEM with a deletion of cysteine-rich domain 1 (CRD1), and for cross-reactivity to membrane-bound cynomolgus monkey HVEM, as described in Example 2. In addition, a selection of these purified antibodies from these anti-human HVEM specific antibody producing hybridomas were tested for their effect on NFκB signaling in membrane human HVEM expressing cells, their effect on soluble human LIGHT-induced NFκB signaling in membrane human HVEM expressing cells, and their effect on membrane human BTLA / human HVEM-mediated inhibition of TCR-induced NFAT signaling in membrane human BTLA / human TCR expressing cells, as described in Example 3.

[0134] Example 2. Flow cytometric characterization of mouse anti-human HVEM monoclonal antibodies (a) Binding of mouse anti-human HVEM antibodies to membrane-bound full-length human HVEM and membrane-bound human HVEM lacking CRD1. To analyze the fine specificity of mouse anti-human HVEM antibodies, the location of the epitopes recognized by the generated mouse anti-human HVEM antibodies was determined by domain mapping. The ability of mouse anti-human HVEM antibodies to bind to CRD1-cleaved human HVEM expressed on the surface of (HEK-derived) 293F cells was determined by FACS analysis.

[0135] Based on the literature (Swiss-Prot number Q92956.3, Montgomery et al. Cell 1996;87:427-436, Hsu et al. J Biochem Chem 1997,272:13471-13474, Naismith et al. Trends Biochem Sci 1998;23:74-79, Carfi et al. Molecul Cell 2001;8:169-179, Bodmer et al. Trends Biochem Sci 2002;27:19-26, Compaan et al. J Biochem Chem 2005;280:39553-39561), cysteine-rich domains (CRDs) in the extracellular region of human HVEM have been identified and are coded as CRD1, CRD2, CRD3, and (truncated) CRD4. CRDs contain topologically distinct types of modules, called A-modules and B-modules. A-modules are C-type structures and B-modules are S-type structures. A typical CRD is usually composed of A1-B2 or A2-B1 modules (or different pairs of modules such as A1-B1) with six conserved cysteine ​​residues, and the numbers indicate the number of disulfide bridges in each module. Human HVEM-CRD1 contains the A1-B2 module (42-75, see SEQ ID NO: 1), human HVEM-CRD2 contains the A1-B2 module (78-119, see SEQ ID NO: 1), human HVEM-CRD3 contains the A2 module and the non-canonical (reminiscent of the B1-module) B0 module (121-162, see SEQ ID NO: 1), and human truncated HVEM-CRD4 contains only the A1 module (165-179, see SEQ ID NO: 1). Two different human HVEM constructs were generated and expressed.(1) a full-length human HVEM construct, starting from the N-terminal CRD1 (i.e., the CRD1A1-B2 module covers amino acids 42-75, see SEQ ID NO:1), thus designated as "full length" and including amino acids 1-283 (see SEQ ID NO:1), as well as (2) a "CRD1 truncated" construct, starting from the N-terminal CRD2 (i.e., the CRD2 A1-B2 module covers amino acids 22-63, see SEQ ID NO:3), including amino acids 20-227 linked to the mouse Ig signal peptide amino acids 1-19 (see SEQ ID NO:3). A cDNA (Swiss-Prot number Q92956.3) encoding the CRD1 truncated human HVEM protein was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO:4). This cDNA was subcloned in an expression plasmid derived from pcDNA3.1.

[0136] The generation of human "full-length" HVEM-transfected HEK293F cells (clone no. 128) is described in Example 1a. FreeStyle™ 293F cells (Invitrogen) were transiently transfected with the "CRD1-truncated" variant of human HVEM using the FreeStyle™ 293 Expression System (Invitrogen). After 72 hours, surface human HVEM expression on the transfected cells was analyzed by FACS analysis. For this purpose, stable human full-length HVEM-transfected HEK293F cells and transiently human "CRD1-truncated" HVEM-transfected HEK293F cells were incubated with 50 μg / mL of human IgG (which has a potential Fcγ receptor) for 1 h. blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were then incubated at 4° C. for 10 min at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL of undiluted hybridoma supernatant / tube) were incubated for 30 min at 4°C. A 1:20 phycoerythrin (PE)-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience) was run as a positive control antibody. After extensive washing with PBS / BSA / NaN3, the cells were subsequently incubated for 30 min at 4°C with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Antibody binding was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0137] As shown in Figure 1, all 18 tested mouse anti-human HVEM antibodies recognized full-length human HVEM on transfected HEK293F cells, but the majority of these mouse anti-human HVEM antibodies (15 / 18) did not show binding to "CRD1-cleaved" human HVEM on transfected HEK293F cells. In contrast, mouse anti-human HVEM antibody numbers 38G10, 39B9, and 47E10 recognized "CRD1-cleaved" human HVEM on transfected HEK293F cells.

[0138] These results demonstrated that mouse anti-human HVEM antibodies Nos. 38G10, 39B9, and 47E10 appear to recognize linear and / or non-linear / conformational epitopes in CRD2, CRD3, CRD4, and / or the "linker" fragment (aa sequence 124-146, see SEQ ID NO:3) of the extracellular domain of human HVEM, whereas all other generated mouse anti-human HVEM antibodies (15 / 18) appear to recognize linear and / or non-linear / conformational epitopes in CRD1 of the extracellular domain of human HVEM.

[0139] (b) Binding of mouse anti-human HVEM antibodies to membrane-bound cynomolgus HVEM To analyze the multispecies cross-reactivity of mouse anti-human HVEM antibodies, the ability of mouse anti-human HVEM antibodies to bind to cynomolgus monkey HVEM expressed on the surface of (HEK-derived) 293F cells was determined by FACS analysis.

[0140] A cDNA encoding the cynomolgus monkey HVEM protein (SEQ ID NO: 5, see NCBI reference sequence XP_005545061.1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (SEQ ID NO: 6). This cDNA was subcloned into an expression plasmid derived from pcDNA3.1.

[0141] The generation of human "full-length" HVEM-transfected HEK293F cells (clone no. 128) is described in Example 1a. FreeStyle™ 293F cells (Invitrogen) were transiently transfected with cynomolgus (full-length) HVEM using the FreeStyle™ 293 Expression System (Invitrogen). After 72 hours, surface cynomolgus HVEM expression on the transfected cells was analyzed by FACS analysis. For this purpose, stable human full-length HVEM-transfected HEK293F cells and transiently cynomolgus full-length HVEM-transfected HEK293F cells were incubated with 50 μg / mL of human IgG (containing a potential Fcγ receptor) and 100 μg / mL of human IgG (containing a potential Fcγ receptor). blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were then incubated at 4° C. for 10 min at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL of undiluted hybridoma supernatant / tube) were incubated for 30 min at 4°C. A 1:20 phycoerythrin (PE)-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience) was run as a positive control antibody. After extensive washing with PBS / BSA / NaN3, the cells were subsequently incubated for 30 min at 4°C with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Antibody binding was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0142] As shown in Figure 1, all 18 tested mouse anti-human HVEM antibodies recognized full-length human HVEM on transfected HEK293F cells, and the majority of these mouse anti-human HVEM antibodies (14 / 18) showed varying degrees of cross-reactivity to full-length cynomolgus monkey HVEM on transfected HEK293F cells. In contrast, mouse anti-human HVEM antibodies nos. 36H12, 37D11, 41F11, and 49A11 did not recognize full-length cynomolgus monkey HVEM on transfected HEK293F cells.

[0143] These results demonstrated that the majority of mouse anti-human HVEM antibodies generated (14 / 18) appear to recognize linear and / or non-linear / conformational epitopes in CRD1, CRD2, CRD3, CRD4, and / or the "linker" fragment (aa sequence 180-203, see SEQ ID NO:5) of the extracellular domain of full-length cynomolgus HVEM.

[0144] The predicted amino acid sequence of the full-length cynomolgus HVEM protein (Met1-Ser280, NCBI Reference Sequence: XP_005545061.1) shows 82% homology with the amino acid sequence of the human HVEM protein (Met1-His283, Swiss-Prot number Q92956.3), and the predicted amino acid sequence of the extracellular domain of cynomolgus HVEM (i.e., Leu39-Val203, NCBI Reference Sequence: XP_005545061.1) shows 87% homology with the amino acid sequence of the extracellular domain human HVEM protein (i.e., Leu39-Val202, Swiss-Prot number Q92956.3). These results demonstrated that the majority (14 / 18) of the mouse anti-human HVEM monoclonal antibodies generated cross-reacted with the homologous cynomolgus HVEM on transfected HEK293F cells.

[0145] (c) Effect of mouse anti-human HVEM antibodies on the binding of human BTLA and human LIGHT to membrane-bound human HVEM. Extracellular HVEM has two spatial ligand-binding regions (Cai et al. Immunol Rev 2009;229:244-258, Steinberg et al. Immunol Rev 2011;244:169-187, Pasero et al. Curr Opin Pharmacol 2012;12:478-485), one region for canonical ligands belonging to the TNF superfamily (i.e., LIGHT and TNFβ), and another region for non-canonical ligands belonging to the Ig superfamily (i.e., BTLA and CD160). Mutational analysis and molecular modeling revealed that BTLA and CD160 interact with CRD1, whereas LIGHT and TNFβ binding reside in CRD2 and CRD3 on the opposite face of HVEM.

[0146] To analyze the effect of mouse anti-human HVEM antibodies on the binding of human BTLA and human LIGHT to membrane-bound human HVEM, the ability of mouse anti-human HVEM antibodies to sterically interfere with the interaction of human BTLA and human LIGHT to human full-length HVEM expressed on the surface of (HEK-derived) 293F cells was determined by FACS analysis.

[0147] The generation of human "full-length" HVEM-transfected HEK293F cells (clone no. 128) is described in Example 1a. The binding of soluble human BTLA, soluble human CD160, and soluble human LIGHT on surface human HVEM-transfected cells was analyzed by FACS analysis. To this end, stable human full-length HVEM-transfected HEK293F cells were incubated with 50 μg / mL of human IgG (containing potential Fcγ receptors) and incubated for 1 h at 4°C for 1 h. blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were incubated at 4° C. for 10 minutes at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL of 10 μg / mL / tube of protein G purified anti-HVEM antibody or 10 μg / mL / tube of negative control mouse IgG1 (BD Biosciences) were incubated for 30 min at 4° C. After this (i.e., without washing), the cells were incubated with 1 μg / mL of soluble human BTLA-human Fcγ fusion protein (Sino Biological Inc) or 0.1 μg / mL of soluble His-tagged human LIGHT (R&D Systems) in PBS / BSA / NaN3 for 30 min at 4° C. After extensive washing with PBS / BSA / NaN3, the cells were incubated with 10 μg / mL of biotinylated mouse anti-human IgG Fcγ specific antibody (BTLA detection, Southern Biotech) or with 5 μg / mL of biotinylated mouse anti-His antibody (LIGHT detection, R&D Systems) for 30 min at 4° C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (Jackson ImmunoResearch) for 30 min at 4° C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4° C. Binding of ligands BTLA and LIGHT to membrane human HVEM was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0148] As shown in Figure 2 and Table 1, four types of mouse anti-human HVEM antibodies were found. (Type 1 antibodies, 6 / 18) blocking (Type 2 antibodies, 3 / 18) BTLA / HVEM interaction blocking and LIGHT / HVEM interaction blocking (Type 3 antibodies, 4 / 18) BTLA / HVEM interaction blocking without LIGHT / HVEM interaction blocking and (type 4 antibodies, 5 / 18) BTLA / HVEM and LIGHT / HVEM interactions. blocking do.

[0149] [Table 1]

[0150] These results suggest that human ligands with multiple characteristics (i.e., mouse anti-human HVEM antibodies type 1-4 (see above)) blocking and non-ligand blocking A panel of mouse anti-human HVEM antibodies was generated and demonstrated. Notably, antibodies that bind to CRD1, such as (36H12, 45H6), inhibited the binding of sBTLA or sCD160. blocking (52D3) or not. The CRD1 antibody 52D3 actually appears to enhance ligand binding. In addition, it clearly demonstrates the distinct functional activities of CRD1-targeting antibodies.

[0151] Example 3. Biological characterization of mouse anti-human HVEM monoclonal antibodies (a) Effect of mouse anti-human HVEM antibody on NFκB signaling in human HVEM-expressing cells HVEM signaling can induce the activation of NFκB in multiple HVEM-expressing cells from the immune system, such as T and B lymphocytes and dendritic cells, turning on several genes important for their cellular functions. Ligation of HVEM on T lymphocytes by LIGHT provides a positive co-stimulatory signal, which leads to T lymphocyte survival, proliferation, and differentiation, as well as IFNγ secretion (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235). Ligation of HVEM on B lymphocytes by LIGHT co-stimulates CD40L-mediated proliferation and antibody secretion, thereby enhancing humoral responses (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235). Ligation of HVEM on immature dendritic cells by LIGHT costimulates CD40L-mediated maturation, cytokine secretion (IL-12, IL-6, and TNF-α), and priming of specific antitumor CTLs and production of IFN-γ (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235).

[0152] To analyze the effect of mouse anti-human HVEM antibodies on membrane-bound human HVEM-mediated NFκB signaling, NFκB response element-luciferase (RE-luc) human HVEM bioassay reporter cells (HEK293, Promega) were used to test the ability of mouse anti-human HVEM antibodies to activate HVEM-mediated NFκB signaling.

[0153] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 35,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with or without mouse anti-human HVEM antibodies at 0.0015-10 μg / mL (3-fold dilution steps). Titered (i.e., 0, 0.0015-10 μg / mL (3-fold dilution steps)) soluble His-tagged human LIGHT (R&D Systems) was run in parallel for reference purposes. After 6 h of incubation at 37°C / 5% CO2, luciferase production in human HVEM-expressing NFκB-NFκB-RE-luc cells was measured using the Bio-Glo™ Luciferase Assay System (Promega) in a luminometer.

[0154] As shown in FIG. 3A and Table 2, several tested (7 / 12) mouse anti-human HVEM antibodies induced dose-dependent NFκB activation in human HVEM-expressing NFκB-RE-luc cells (i.e., compared to soluble LIGHT-mediated NFκB induction) to various degrees (rank order, nos. 48H6>36H12>29C2>8H5=45H6=49G4>52D3), demonstrating their agonistic activity. Control soluble human LIGHT also showed dose-dependent NFκB activation in these human HVEM-expressing NFκB-RE-luc cells. In contrast, mouse anti-human HVEM antibodies nos. 11H7, 41F11, 43E10, 47E10, and 49A11 did not show agonistic activity in human HVEM-expressing NFκB-RE-luc cells (i.e., compared to soluble LIGHT-mediated NFκB induction). Interestingly, these tested mouse anti-human HVEM antibodies stereochemically characterized the soluble human LIGHT / human HVEM interaction. blocking There appeared to be a relationship between the ability of these compounds to inhibit NF-κB expression (see Example 2c) and their agonistic activity (i.e., NFκB induction) on membrane human HVEM-expressing cells (see Table 2).

[0155] [Table 2]

[0156] These results suggest that human LIGHT / human HVEM interaction blocking It was demonstrated that the mouse anti-human HVEM antibody (see Example 2c) can mimic soluble human LIGHT / human HVEM-mediated NKκB signal transduction.Notably, it has been shown that soluble human LIGHT is much less potent than membrane-bound LIGHT expressing cells in activating human HVEM expressed on cells (Cheung et al.PNAS2009;106:6244-6249).

[0157] (b) Effect of mouse anti-human HVEM antibody on soluble human LIGHT-induced NFκB signaling in membrane human HVEM-expressing cells To analyze the effect of mouse anti-human HVEM antibodies on soluble human LIGHT-induced NFκB signaling in membrane human HVEM-expressing cells, NFκB-RE-luc human HVEM bioassay reporter cells (HEK293, Promega) were used to assay the effect of mouse anti-human HVEM antibodies interfering with soluble human LIGHT / membrane HVEM-mediated NFκB signaling (e.g., blocking The ability of the compounds to exert an inhibitory or inhibitory effect (additive or synergistic effect) was examined.

[0158] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 35,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with or without mouse anti-human HVEM antibody at 0.0015–10 μg / mL (3-fold dilution steps) in combination with 0.3 μg / mL soluble His-tagged human LIGHT (R&D Systems) (

number

[0159] As shown in Figure 3B and Table 3, a very weak agonist but a moderate LIGHT / HVEM interaction blocker (See Table 2) Mouse anti-human HVEM antibody no. 52D3 was able to weakly but dose-dependently inhibit soluble human LIGHT-mediated NFκB activation in human HVEM-expressing NFκB-RE-luc cells. Surprisingly, the non-agonist and non-LIGHT / HVEM interaction blocker (See Table 2) Mouse anti-human HVEM antibody No. 49A11 was also able to weakly but dose-dependently inhibit soluble human LIGHT-mediated NFκB activation in human HVEM-expressing NFκB-RE-luc cells. In addition, it was a non-agonist and inhibited the LIGHT / HVEM interaction. blocker (See Table 2) Mouse anti-human HVEM antibodies no. 11H7, 41F11, 43E10, and 47E10 showed no effect on soluble human LIGHT-mediated NFκB activation in human HVEM-expressing NFκB-RE-luc cells, whereas they were weak / moderate / strong agonists and weak / moderate / strong LIGHT / HVEM interactions. blocker (See Table 2) Mouse anti-human HVEM antibodies nos. 8H5, 29C2, 36H12, 45H6, 48H6, and 49G4 showed possible additive, but not synergistic, effects on soluble human LIGHT-mediated NFκB activation in human HVEM-expressing NFκB-RE-luc cells.

[0160] [Table 3]

[0161] These results suggest that human LIGHT / human HVEM interaction blockingMouse anti-human HVEM antibody (non-LIGHT / HVEM interaction) blocker Mouse anti-human HVEM antibody No. 49A11 (see also Example 2c) inhibited soluble human LIGHT / human HVEM-mediated NKκB signaling. blocking or mimicking it.

[0162] (c) Effect of mouse anti-human HVEM antibodies on membrane human BTLA / membrane human HVEM-mediated inhibition of TCR-induced NFAT signaling in T cells expressing membrane human BTLA / membrane human TCR. As mentioned above, ligation of HVEM on T lymphocytes by LIGHT delivers a positive co-stimulatory signal via HVEM, whereas engagement of BTLA on T lymphocytes by HVEM provides a negative co-inhibitory signal to T lymphocytes via BTLA (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235). This BTLA / HVEM pathway downregulates TCR-mediated signaling in both CD4 and CD8 T lymphocytes, resulting in reduced T lymphocyte proliferation and cytokine production. Engagement of BTLA on B lymphocytes by HVEM reduces activation of signaling molecules downstream of the BCR, attenuating B cell proliferation (Vendel et al. Journal Immunology 2009;182:1509-1517). Unlike PD-1 and CTLA4, BTLA is not expressed on regulatory T (Treg) lymphocytes (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235).

[0163] To analyze the effect of mouse anti-human HVEM antibodies on membrane human BTLA / membrane human HVEM-mediated inhibition of membrane human TCR-induced NFAT signaling, we used NFAT-response element-luciferase (RE-luc) human BTLA / HVEM Blockade BioassayWe have used a combination of (1) CHO-K1 activator cells (artificial antigen presenting cells) expressing membrane human HVEM and proprietary membrane human TCR activator, and (2) NFAT-RE-luc Jurkat effector T cells expressing membrane human BTLA and membrane human TCR, Promega, to demonstrate BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling. blocking The ability of mouse anti-human HVEM antibodies to

[0164] This BTLA / HVEM Blockade Bioassay In this study, NFAT-RE-luc Jurkat effector T cells expressing membrane human BTLA and membrane human TCR are used as effector cells, and CHO-K1 activator cells expressing membrane human HVEM and membrane human TCR activator are used as artificial antigen presenting cells. When these two cells are co-cultured, the TCR complex on the effector cells is activated by the artificial antigen presenting cells expressing the TCR activator, resulting in expression of the NFAT luciferase reporter. However, BTLA and HVEM ligation prevents TCR activation and suppresses NFAT-responsive luciferase activity. This inhibition is blocking This can be specifically reversed by exposure to anti-HVEM antibodies. Neutralizing anti-HVEM antibodies block the BTLA / HVEM interaction. blocking , promoting T cell activation (i.e., "releasing the brake") and resulting in reactivation of the NFAT-responsive luciferase reporter (see Figure 4A).

[0165] Briefly, CHO-K1 activator cells expressing human HVEM and human TCR activators were seeded at 40,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with or without mouse anti-human HVEM antibodies at 0.0015-10 μg / mL (3-fold dilution steps). NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR were then added at 50,000 cells / well. After 6 h of incubation at 37°C / 5% CO2, luciferase production in NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR was measured using the Bio-Glo™ Luciferase Assay System (Promega) in a luminometer.

[0166] As shown in Figure 4B and Table 4, several tested (8 / 12) mouse anti-human HVEM antibodies inhibited BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling in NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR in a dose-dependent manner to various degrees. blocking (rank order, nos. 45H6>49G4>36H12>11H7>8H5=41F11=48H6=49A11). In contrast, mouse anti-human HVEM antibodies nos. 29C2, 43E10, 47E10, and 52D3 showed no effect on BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling in NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR. Interestingly, none of these tested mouse anti-human HVEM antibodies stereochemically inhibited the soluble human BTLA / human HVEM interaction. blocking (see Example 2c) and NFAT-RE-luc human BTLA / HVEM Blockade Bioassay Those in blocking There appeared to be a relationship between the ability of BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling to be increased by TCR-induced NFAT signaling (i.e., abolition of BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling) (see Table 4).

[0167] [Table 4]

[0168] These results suggest that human BTLA / human HVEM interaction blocking Mouse anti-human HVEM antibodies (see Example 2c) inhibited human BTLA / human HVEM-mediated inhibition of TCR-induced NFAT signaling. blocking It has been demonstrated that this is possible.

[0169] Example 4. BTLA / HVEM blocking Molecular and genetic characterization of mouse anti-human HVEM monoclonal antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 BTLA / HVEM blocking Hybridoma cells producing mouse anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 were washed with PBS and diluted to 5 × 10 6 The cells were aliquoted into microvials containing the cells and stored as pellets at -80°C. These cell pellets were used to isolate RNA by using the RNeasy Mini Isolation Kit (QIAGEN). The RNA concentration was determined (A260nm) and the RNA was stored at -80°C. cDNA was synthesized from 1 μg of RNA by reverse transcriptase using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) and stored at -80°C. Based on the isotype mouse IgG1 / kappa, primers as shown in Table 5 were used to synthesize BTLA / HVEM blocking It was designed to amplify the variable (V) regions of mouse anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4.

[0170] [Table 5]

[0171] Primers 383, 387, and 389 are sense primers designed to anneal with the signal peptide of the light chain of mouse antibody, and primer 394 is an antisense primer that anneals with the constant region of mouse kappa light chain. Primers 404, 407, 408, and 409 are sense primers that anneal with the signal peptide of the heavy chain of mouse antibody, and primer 416 is an antisense primer that anneals with the constant region of mouse IgG1 heavy chain. Various PCRs were performed using the primer combinations shown in Table 5. The generated PCR products were subcloned into pCR™-Blunt II-TOPO® vector. The cloned inserts were then sequenced.

[0172] A total of eight and four informative sequences of the amino acid sequence of mouse anti-human HVEM antibody 36H12 were obtained from the heavy and light chain sequencing reactions, respectively. Based on this information, consensus amino acid sequences of the VH and VL regions of mouse anti-human HVEM antibody 36H12 were determined and are set forth in SEQ ID NOs: 16 and 17, respectively. The amino acid sequences of the CDRs of the VH and VL regions of mouse anti-human HVEM antibody 36H12 are set forth in SEQ ID NOs: 18-20 and 21-23, respectively.

[0173] A total of four source sequences of mouse anti-human HVEM antibody 45H6 were obtained from the sequence reactions of both the heavy and light chains. Based on this information, the consensus amino acid sequences of the VH and VL regions of mouse anti-human HVEM antibody 45H6 were determined and are set forth in SEQ ID NOs: 24 and 25, respectively. The amino acid sequences of the CDRs of the VH and VL regions of mouse anti-human HVEM antibody 45H6 are set forth in SEQ ID NOs: 26-28 and 29-31, respectively.

[0174] A total of five and four informative sequences of mouse anti-human HVEM antibody 48H6 were obtained from the heavy and light chain sequence reactions, respectively. Based on this information, the consensus amino acid sequences of the VH and VL regions of mouse anti-human HVEM antibody 48H6 were determined and are set forth in SEQ ID NOs: 32 and 33, respectively. The amino acid sequences of the CDRs of the VH and VL regions of mouse anti-human HVEM antibody 48H6 are set forth in SEQ ID NOs: 34-36 and 37-39, respectively.

[0175] A total of 9 and 3 informative sequences of mouse anti-human HVEM antibody 11H7 were obtained from the heavy and light chain sequence reactions, respectively. Based on this information, the consensus amino acid sequences of the VH and VL regions of mouse anti-human HVEM antibody 11H7 were determined and are set forth in SEQ ID NOs: 40 and 41, respectively. The amino acid sequences of the CDRs of the VH and VL regions of mouse anti-human HVEM antibody 11H7 are set forth in SEQ ID NOs: 42-44 and 45-47, respectively.

[0176] A total of five and three informative sequences of mouse anti-human HVEM antibody 49G4 were obtained from the heavy and light chain sequence reactions, respectively. Based on this information, the consensus amino acid sequences of the VH and VL regions of mouse anti-human HVEM antibody 49G4 were determined and are set forth in SEQ ID NOs: 48 and 49, respectively. The amino acid sequences of the CDRs of the VH and VL regions of mouse anti-human HVEM antibody 49G4 are set forth in SEQ ID NOs: 50-52 and 53-55, respectively.

[0177] Example 5. BTLA / HVEM blocking Generation of chimeric mouse / human IgG4 / kappa (i.e., replacing the mouse constant IgG1 / kappa region with the human constant IgG4 / kappa region) anti-human HVEM monoclonal antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 BTLA / HVEM blockingBased on the determined mouse V regions of the mouse anti-human HVEM antibody (see Example 4 above), a design was made to generate a chimeric mouse / human anti-human HVEM antibody version. For this purpose, cDNA sequences optimized for mammalian expression were selected from SEQ ID NO:56 (encoding chimeric mouse / human heavy IgG4 chain 36H12), SEQ ID NO:57 (encoding chimeric mouse / human heavy IgG4 chain 45H6), SEQ ID NO:58 (encoding chimeric mouse / human heavy IgG4 chain 48H6), SEQ ID NO:59 (encoding chimeric mouse / human heavy IgG4 chain 11H7), and SEQ ID NO:60 (encoding chimeric mouse / human heavy IgG4 chain 49G4), as well as SEQ ID NO:61 (encoding chimeric mouse / human light kappa chain 36H12). H12), SEQ ID NO:62 (encoding chimeric mouse / human light kappa chain 45H6), SEQ ID NO:63 (encoding chimeric mouse / human light kappa chain 48H6), and SEQ ID NO:64 (encoding chimeric mouse / human light kappa chain 11H7), and SEQ ID NO:65 (encoding chimeric mouse / human light kappa chain 49G4) were ordered from GENEART (Regensburg, Germany), which encoded either a human signal peptide followed by a mouse VH chain linked to a human stabilized IgG4 constant region (i.e., S239P, according to Angal et al in Mol. Immunol., Vol. 30, No. 1, pp. 105-108, 1993), or followed by a mouse VL chain linked to a human kappa constant region. The generated cDNAs were subcloned into pcDNA3.1-derived expression plasmids using appropriate restriction enzymes. The chimeric antibodies were then transiently expressed in 293-F cells (Invitrogen) using the FreeStyle™ 293 Expression System (Invitrogen). The expressed chimeric anti-human HVEM antibodies were purified from the supernatant using a conventional affinity chromatography Protein A column. LPS levels were then measured using an LAL chromogenic endpoint assay (Hycult Biotech), and all of our purified chimeric mouse / human anti-human HVEM antibodies (i.e., 36H12, 45H6, 48H6, 11H7, and 49G4) contained <0.0005EU LPS / μg chimeric IgG.

[0178] For chimeric amino acid sequences, see SEQ ID NO:66 (chimeric mouse / human heavy IgG4 chain 36H12), SEQ ID NO:67 (chimeric mouse / human heavy IgG4 chain 45H6), SEQ ID NO:68 (chimeric mouse / human heavy IgG4 chain 48H6), SEQ ID NO:69 (chimeric mouse / human heavy IgG4 chain 11H7), and SEQ ID NO:70 (chimeric mouse / human heavy IgG4 chain 49G4), as well as SEQ ID NO:71 (chimeric mouse / human light κ chain 36H12), SEQ ID NO:72 (chimeric mouse / human light κ chain 45H6), SEQ ID NO:73 (chimeric mouse / human light κ chain 48H6), SEQ ID NO:74 (chimeric mouse / human light κ chain 11H7), and SEQ ID NO:75 (chimeric mouse / human light κ chain 49G4).

[0179] Example 6. BTLA / HVEM blocking Binding and biological characterization of chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 (a) BTLA / HVEM on membrane-bound human HVEM blocking Relative binding affinities of chimeric mouse / human anti-human HVEM antibodies Purified BTLA / HVEM for human HVEM blocking FACS analysis was used to determine the relative binding affinities of chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4.

[0180] For this purpose, HEK293F cells stably transfected with human full-length HVEM (clone no. 128, see Example 1(a) above) were incubated with 50 μg / mL of human IgG (containing potential Fcγ receptors) blocking 10 × 10 cells were cultured in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3) supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (Sigma). 6 The cells were incubated at 4° C. for 10 minutes at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6 These cells were cultured in 100 μL of titrated purified BTLA / HVEM (in PBS / BSA / NaN3). blocking The cells were incubated with or without chimeric mouse / human anti-human HVEM antibodies / tube for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were incubated with 1:200 diluted PE-conjugated goat anti-human IgG Fcγ specific antibody (Jackson ImmunoResearch) for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 4% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. The binding (geo-mean fluorescence intensity) of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 on membrane human HVEM was measured using a flow cytometer (FACSCalibur, BD Biosciences). For comparison, purified BTLA / HVEM blocking Mouse anti-human HVEM antibody counterparts nos. 36H12, 45H6, 48H6, 11H7 and 49G4 were run in parallel and their binding monitored as described in Example 2(a).

[0181] As shown in Figure 5, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, 48H6, 11H7, and 49G4 bound to membrane human HVEM in a dose-dependent manner. Based on their binding profiles, the following relative affinity ranking was found (from high affinity to low affinity): 45H6=49G4>36H12=48H6>11H7. For comparison, their BTLA / HVEM blockingThe mouse anti-human HVEM antibody counterparts nos. 36H12, 45H6, 48H6, 11H7, and 49G4 also showed dose-dependent binding to membrane human HVEM and demonstrated very similar relative affinity rankings, i.e., 45H6=49G4>36H12=48H6>11H7. More specifically, chimeric mouse / human anti-human HVEM antibody numbers 45H6, 49G4, 36H12, 48H6, and 11H7 provided the following relative affinities (i.e., half-maximal binding EC50) of 306, 312, 433, 472, and 630 ng / mL, respectively, and the corresponding mouse anti-human HVEM antibody numbers 45H6, 49G4, 36H12, 48H6, and 11H7 provided relative affinities of 260, 266, 430, 356, and 532 ng / mL, respectively, which indicates that the chimeric mouse / human anti-human HVEM antibody numbers 45H6, 49G4, 36H12, 48H6, and 11H7 provided relative affinities of 260, 266, 430, 356, and 532 ng / mL, respectively, indicating that the chimeric mouse / human anti-human HVEM antibody numbers 45H6, 49G4, 36H12, 48H6, and 11H7 provided relative affinities of 260, 266, 430, 356, and 532 ng / mL, respectively, indicates ... blocking We showed that chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 appear to remain unchanged.

[0182] b) BTLA / HVEM binding of human BTLA, human CD160, human LIGHT, and human TNFβ to membrane-bound human HVEM blocking Effect of chimeric mouse / human anti-human HVEM antibody Extracellular HVEM has two spatial ligand-binding regions (Cai et al. Immunol Rev 2009;229:244-258, Steinberg et al. Immunol Rev 2011;244:169-187, Pasero et al. Curr Opin Pharmacol 2012;12:478-485), one region for canonical ligands belonging to the TNF superfamily (i.e., LIGHT and TNFβ), and another region for non-canonical ligands belonging to the Ig superfamily (i.e., BTLA and CD160). Mutational analysis and molecular modeling revealed that BTLA and CD160 interact with CRD1, whereas LIGHT and TNFβ binding reside in CRD2 and CRD3 on the opposite face of HVEM.

[0183] Purified BTLA / HVEM for binding of human BTLA, human CD160, human LIGHT, and human TNFβ to membrane-bound human HVEM blocking To analyze the effects of chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4, the ability of the chimeric mouse / human anti-human HVEM antibodies to sterically interfere with the interaction of human BTLA, human CD160, human LIGHT, and human TNFβ (also called LTα) with human full-length HVEM expressed on the surface of 293F (HEK-derived) cells was determined by FACS analysis.

[0184] The generation of human "full-length" HVEM-transfected HEK293F cells (clone no. 128) is described in Example 1a. The binding of soluble human BTLA, soluble human CD160, soluble human LIGHT, and soluble human TNFβ on the surface of human HVEM-transfected cells was analyzed by FACS analysis. To this end, stable human full-length HVEM-transfected HEK293F cells were incubated with 50 μg / mL of human IgG (which has a potential Fcγ receptor) and incubated for 1 h at 4°C for 1 h. blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were incubated at 4° C. for 10 minutes at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL of purified chimeric mouse / human anti-HVEM antibody at 10 μg / mL / tube or human IgG4 / κ (Sigma) negative isotype control at 10 μg / mL / tube or without them for 30 min at 4° C. After this (i.e., without washing), the cells were subsequently incubated with 1 μg / mL soluble biotinylated human BTLA (Sino Biological Inc), 10 μg / mL soluble His-tagged human CD160 (Sino Biological Inc), 1 μg / mL soluble His-tagged human LIGHT (Sino Biological Inc), or 0.1 μg / mL soluble biotinylated human TNFβ (Sino Biological Inc) in PBS / BSA / NaN3 for 30 min at 4° C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (detects BTLA and TNFβ, Jackson ImmunoResearch) or biotinylated mouse anti-His antibody (detects CD160 and LIGHT, R&D Systems) at 10 μg / mL for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (detects CD160 and LIGHT, Jackson ImmunoResearch) for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 4% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Binding of ligands BTLA, CD160, LIGHT and TNFβ on membrane human HVEM was measured using a flow cytometer (model FACSCalibur, BD Biosciences). For comparison, purified BTLA / HVEM at 10 μg / mL / tube blocking Mouse anti-human HVEM antibody counterparts numbers 36H12, 45H6, 48H6, 11H7, and 49G4 and mouse IgG1 (BD Biosciences) negative isotype control at 10 μg / mL / tube were run in parallel.

[0185] As shown in Figure 6A, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 prevented binding of human BTLA to membrane human HVEM (i.e., >95% blocking ), their BTLA / HVEM blocking The antibody was comparable (i.e., >95% identical) to the mouse anti-human HVEM antibody counterparts 36H12, 45H6, 48H6, 11H7, and 49G4. blocking ).

[0186] As shown in Figure 6B, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 partially prevented binding of human CD160 to membrane human HVEM (i.e.

number

number

[0187] As shown in Figure 6C, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, and 49G4 partially prevented binding of human LIGHT to membrane human HVEM (i.e.

number

number

number

number

[0188] As shown in Figure 6D, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 inhibited binding of human TNFβ to membrane human HVEM to various degrees (in the order, nos. 36H12=45H6=48H6 (i.e., >94%) Blocking )>49G4 (i.e., >80% Blocking )>11H7 (i.e., >55% Blocking )) (partially) prevents those BTLA / HVEM blocking Mouse anti-human HVEM antibody counterparts nos. 36H12, 45H6, 48H6, 11H7, and 49G4 (rank order, nos. 36H12=45H6=48H6 (i.e., >95% Blocking )>49G4 (i.e., >85% Blocking )>11H7 (i.e., >60% Blocking ) was equivalent.

[0189] c) Uptake of prebound human BTLA, human CD160, human LIGHT, and human TNFβ from membrane-bound human HVEM. Excluding BTLA / HVEM blocking Effect of chimeric mouse / human anti-human HVEM antibody Purified BTLA / HVEM blockingChimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 removed prebound human BTLA, human CD160, human LIGHT, and human TNFβ from membrane-bound human HVEM. except To analyze whether the uptake of human BTLA, human CD160, human LIGHT, and human TNFβ (also called LTα) from human full-length HVEM expressed on the surface of (HEK-derived) 293F cells could be achieved, we Excluding The effect of chimeric mouse / human anti-human HVEM antibodies on IL-1 was determined by FACS analysis.

[0190] The generation of human "full-length" HVEM-transfected HEK293F cells (clone no. 128) is described in Example 1a. Uptake of prebound soluble human BTLA, soluble human CD160, soluble human LIGHT, and soluble human TNFβ from cells transfected with surface human HVEM. Excluding To this end, stable human full-length HVEM-transfected HEK293F cells were incubated with 50 μg / mL of human IgG (containing capable Fcγ receptors). blocking The cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN3 supplemented with 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3). 6 The cells were incubated at 4° C. for 10 minutes at 4° C. Then, 10 μL / tube (i.e., 0.1×10 6These cells (100 μL / tube) were incubated in PBS / BSA / NaN3 with or without 50 μL of soluble biotinylated human BTLA-human Fcγ fusion protein (Sino Biological Inc) at 2 μg / mL / tube, with soluble His-tagged human CD160 (Sino Biological Inc) at 20 μg / mL / tube, with soluble His-tagged human LIGHT (Sino Biological Inc) at 2 μg / mL / tube, or with soluble biotinylated human TNFβ (Sino Biological Inc) at 0.2 μg / mL / tube, for 30 minutes at 4° C. After this (i.e., without washing), the cells were subsequently incubated with 50 μL of purified chimeric mouse / human anti-HVEM antibody at 20 μg / mL / tube or human IgG4 / κ (Sigma) negative isotype control at 20 μg / mL / tube for 30 minutes at 4° C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (detects BTLA and TNFβ, Jackson ImmunoResearch) or biotinylated mouse anti-His antibody (detects CD160 and LIGHT, R&D Systems) at 10 μg / mL for 30 minutes at 4° C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (detects BTLA and TNFβ, Jackson ImmunoResearch) or biotinylated mouse anti-His antibody (detects CD160 and LIGHT, R&D Systems) at 10 μg / mL for 30 minutes at 4° C. After extensive washing with PBS / BSA / NaN3, cells were incubated with 1:200 diluted PE-conjugated streptavidin (detects CD160 and LIGHT, Jackson ImmunoResearch) for 30 minutes at 4° C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 4% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C.Residual binding of ligands BTLA, CD160, LIGHT and TNFβ on membrane human HVEM was measured using a flow cytometer (model FACSCalibur, BD Biosciences) and, for comparison, purified BTLA / HVEM at 10 μg / mL / tube. blocking Mouse anti-human HVEM antibody counterparts numbers 36H12, 45H6, 48H6, 11H7, and 49G4 and mouse IgG1 (BD Biosciences) negative isotype control at 10 μg / mL / tube were run in parallel.

[0191] As shown in Figure 7A, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 remove prebound human BTLA from membrane human HVEM. Except (i.e., >95% success rate) Except ).

[0192] As shown in Figure 7B, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 partially removed prebound human CD160 from membrane human HVEM. Except (i.e.,

number

[0193] As shown in Figure 7C, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, and 49G4 remove prebound human LIGHT from membrane human HVEM. Excluded (i.e., <20% of Except In contrast, BTLA / HVEM blocking The chimeric mouse / human anti-human HVEM antibody #11H7 removes prebound human LIGHT from membrane human HVEM. ExcludedHowever, surprisingly, it appeared to enhance or stabilize pre-bound human LIGHT to membrane human HVEM (i.e.

number

[0194] As shown in Figure 7D, BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, and 49G4 were identified to various degrees (rank order, nos. 36H12=45H6=48H6 (i.e., >90% capture). Except )>49G4 (i.e., >55% of the Except )) Prebound human TNFβ was (partially) removed from human HVEM membranes. Except In contrast, BTLA / HVEM blocking The chimeric mouse / human anti-human HVEM antibody No. 11H7 removes prebound human TNFβ from membrane human HVEM. Excluded There appeared to be no evidence of Except ).

[0195] (d) BTLA / HVEM on NFκB signaling in membrane human HVEM-expressing cells blocking Effect of chimeric mouse / human anti-human HVEM antibody To analyze the effects of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 on membrane-bound human HVEM-mediated NFκB signaling, NFκB-response element-luciferase (RE-luc) human HVEM bioassay reporter cells (HEK293, Promega) were used to test the ability of mouse anti-human HVEM antibodies to activate HVEM-mediated NFκB signaling.

[0196] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 35,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with or without chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 at 0.0015-10 μg / mL (3-fold dilution steps). Titered (i.e., 0, 0.0015-10 μg / mL (3-fold dilution steps)) soluble His-tagged human LIGHT (R&D Systems) were run in parallel for reference purposes. After 6 h incubation at 37° C. / 5% CO2, luciferase production in human HVEM-expressing NFκB-NFκB-RE-luc cells was measured using the Bio-Glo™ Luciferase Assay System (Promega) in a luminometer.

[0197] As shown in Figure 8A, only the chimeric mouse / human anti-human HVEM antibody No. 48H6 induced weak dose-dependent NFκB activation in NFκB-RE-luc cells expressing human HVEM (i.e., compared to soluble LIGHT-mediated NFκB induction), whereas the chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, 11H7, and 49G4 showed no or very weak agonistic activity in NFκB-RE-luc cells expressing human HVEM (i.e., compared to soluble LIGHT-mediated NFκB induction). Control soluble human LIGHT also showed dose-dependent NFκB activation in these human HVEM-expressing NFκB-RE-luc cells. Notably, soluble human LIGHT has been shown to be much less potent at activating human HVEM expressed on cells than membrane-bound LIGHT-expressing cells (Cheung et al. PNAS 2009;106:6244-6249).

[0198] BTLA / HVEM versus membrane-bound HVEM blockingThe binding affinity of the chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, 48H6, 11H7, and 49G4 appeared to remain unchanged during the chimerization process (see Example 6a), whereas the binding affinity of the BTLA / HVEM antibodies with their human constant IgG4 Fc tails was not altered during the chimerization process (see Example 6a). blocking It was surprising that the chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, 48H6, and 49G4 did not or could only weakly mimic soluble human LIGHT / human HVEM-mediated NKκB signaling, in contrast to their mouse anti-human HVEM antibody IgG1 counterparts (see Example 3a), which clearly exhibited NFκB signaling activity.

[0199] (e) Crosslinking BTLA / HVEM on NFκB signaling in membrane human HVEM-expressing cells. blocking Effect of chimeric mouse / human anti-human HVEM antibody It is well known that cross-linking of antibodies against human CD40 and OX40 / CD134 (both members of the TNF receptor superfamily like human HVEM / CD270) can enhance their agonist activity (i.e., mimicking the mediated effects of CO40L and OX40L, respectively) upon binding to membrane-bound CD40- and OX40-expressing cells (Xu et al. Cancer Cell 2018;33:664-675, Zhang et al. J Biol Chem 2016;291:27134-27146). BTLA / HVEM blockingDue to the surprising results described above with chimeric mouse / human anti-human HVEM antibody numbers 36H12, 45H6, 48H6, and 49G4 (i.e., they did not or could only weakly mimic soluble human LIGHT / human HVEM-mediated NKκB signaling, in contrast to their mouse anti-human HVEM antibody counterparts that clearly demonstrated NFκB signaling activity (see Examples 6b and 3a, respectively)), the extent of antibody aggregation of (1) the chimeric mouse / human and (2) fully mouse versions of anti-human HVEM antibody numbers 36H12, 45H6, 48H6, 11H7, and 49G4 was determined using size exclusion chromatography analysis to demonstrate the following: (1) 2.3%, 0.7%, 1.2%, 1.6%, and 9.4% aggregation for chimeric mouse / human anti-human HVEM antibody numbers 36H12, 45H6, 48H6, 11H7, and 49G4, respectively, and (2) 36.3%, 25.8%, 19.9%, 12.4%, and 14.8% aggregation for fully mouse anti-human HVEM antibody numbers 36H12, 45H6, 48H6, 11H7, and 49G4, respectively. The relatively high degree of antibody aggregation in mouse anti-human HVEM antibody nos. 36H12, 45H6, 48H6, and 49G4 preparations strongly suggested that the agonist activity of mouse anti-human HVEM antibody nos. 36H12, 45H6, 48H6, and 49G4 in NFκB response element-luciferase (RE-luc) human HVEM bioassay reporter cells was an artifactual effect caused by antibody aggregation (i.e., mimicking the antibody cross-linking effect). To prove this hypothesis, we performed a CT scan of non-cross-linked and cross-linked BTLA / HVEM blocking Chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 (which have relatively low levels of antibody aggregation) were examined using NFκB-response element-luciferase (RE-luc) human HVEM bioassay reporter cells.

[0200] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 35,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with or without chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 at 0.016-10 μg / mL (5-fold dilution steps), which were pretreated with or without 10 μg / mL cross-linking goat anti-human IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 15-30 min at room temperature. Titrated (i.e., 0, 0.016-10 μg / mL (5-fold dilution steps)) soluble His-tagged human LIGHT (Sino Biological Inc) was run in parallel for reference purposes. After 6 h incubation at 37 °C / 5% CO2, luciferase production in human HVEM-expressing NFκB-NFκB-RE-luc cells was measured using the Bio-Glo™ Luciferase Assay System (Promega) in a luminometer.

[0201] As shown in FIG. 8B, only the non-crosslinked chimeric mouse / human anti-human HVEM antibody No. 48H6 induced a weak dose-dependent NFκB activation in NFκB-RE-luc cells expressing human HVEM (i.e., compared to soluble LIGHT-mediated NFκB induction), whereas the non-crosslinked chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, 11H7, and 49G4 showed no or very weak agonistic activity in NFκB-RE-luc cells expressing human HVEM (i.e., compared to soluble LIGHT-mediated NFκB induction). In contrast, all tested crosslinked chimeric mouse / human mouse anti-human HVEM antibodies induced a dose-dependent NFκB activation in human HVEM expressing NFκB-RE-luc cells (i.e., compared to soluble LIGHT-mediated NFκB induction). Control soluble human LIGHT also showed dose-dependent NFκB activation in these human HVEM-expressing NFκB-RE-luc cells. Notably, soluble human LIGHT was shown to be much less potent than membrane-bound LIGHT expressing cells in activating human HVEM expressed on cells (Cheung et al. PNAS 2009; 106: 6244-6249).

[0202] These results demonstrate that non-crosslinked chimeric mouse / human anti-human HVEM antibody numbers 36H12, 45H6, 48H6, 11H7, and 49G4 (which have relatively low antibody aggregation) did not mimic or could only weakly mimic soluble human LIGHT / human HVEM-mediated NKκB signaling, while crosslinked chimeric mouse / human anti-human HVEM antibody numbers 36H12, 45H6, 48H6, 11H7, and 49G4 could mimic soluble human LIGHT / human HVEM-mediated NKκB signaling. Notably, soluble human LIGHT has been shown to be much less potent than membrane-bound LIGHT-expressing cells in activating human HVEM expressed on cells (Cheung et al.PNAS2009;106:6244-6249).

[0203] (f) BTLA / HVEM on soluble human LIGHT-induced NFκB signaling in membrane human HVEM-expressing cells blocking Effect of chimeric mouse / human anti-human HVEM antibody To analyze the effect of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 on soluble human LIGHT-induced NFκB signaling in membrane human HVEM-expressing cells, NFκB-RE-luc human HVEM bioassay reporter cells (HEK293, Promega) were used to assay the effect of mouse anti-human HVEM antibodies interfering with soluble human LIGHT / membrane HVEM-mediated NFκB signaling (e.g., blocking The ability of the compounds to exert an inhibitory or inhibitory effect (additive or synergistic effect) was examined.

[0204] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 35,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with 0.3 μg / mL soluble His-tagged human LIGHT (Sino Biological Inc) with or without 0.0015-10 μg / mL (3-fold dilution steps) of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 (

number

[0205] As shown in Figure 8C, chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 showed no effect on soluble human LIGHT-mediated NFκB activation in NFκB-RE-luc cells expressing human HVEM.

[0206] These results demonstrated that chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 were unable to affect soluble human LIGHT / human HVEM-mediated NKκB signaling.

[0207] (g) BTLA / HVEM on soluble human TNFβ-induced NFκB signaling in membrane human HVEM-expressing cells blocking Effect of chimeric mouse / human anti-human HVEM antibody TNFβ / LTα has been reported to have weak binding to HVEM, and its exact functional role in the HVEM pathway remains unclear (Cai et al. Immunol Rev 2009;229:244-258), but there is general agreement that the TNFβ / HVEM pathway (along with the LIGHT / HVEM pathway) provides a costimulatory signal that leads to enhanced immune responses (Cai et al. Immunol Rev 2009;229:244-258, Steinberg et al. Immunol Rev 2011;244:169-187, Pasero et al. Curr Opin Pharmacol 2012;12:478-485, Del Rio et al. Am J Transplant 2013;13:541-551, Schaer et al. J Immunother Cancer 2014;2:7).

[0208] To analyze the effect of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 on soluble human TNFβ-induced NFκB signaling in membrane human HVEM-expressing cells, NFκB-RE-luc human HVEM bioassay reporter cells (HEK293, Promega) were used to assay the effect of mouse anti-human HVEM antibodies interfering with soluble human TNFβ / membrane HVEM-mediated NFκB signaling (e.g., blocking The ability of the compounds to exert an inhibitory or inhibitory effect (additive or synergistic effect) was examined.

[0209] Briefly, human HVEM-expressing NFκB-RE-luc cells were seeded at 32,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, the cells were washed and subsequently incubated with 0.05 μg / mL soluble recombinant human TNFβ (Sino Biological) with or without 0.0015-10 μg / mL (3-fold dilution steps) of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 (

number

[0210] As shown in Figure 9B, chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 showed no effect on soluble human TNFβ-mediated NFκB activation in NFκB-RE-luc cells, which expressed relatively low levels of membrane-bound human HVEM (i.e., signal-to-noise ratio of <5 at 1:20 using PE-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience)). We found that chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 prevented binding of soluble human TNFβ to and / or removed pre-bound soluble human TNFβ from HEK293F cells clone no. 128. Except This observation was surprising because the IL-16 cells expressed relatively high levels of membrane-bound human HVEM (i.e., overexpressed, using a PE-conjugated mouse anti-human HVEM antibody (clone eBioHVEM-122, eBioscience) at 1:20) (see Example 5(b) and Example 5(c), respectively).

number

[0211] These results demonstrated that when membrane-bound human HVEM and membrane-bound human TNFR1 were coexpressed at relatively low levels on cells, chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 were unable to affect soluble human TNFβ-mediated NKκB signaling.

[0212] (h) BTLA / HVEM for membrane human BTLA / membrane human HVEM-mediated inhibition of TCR-induced NFAT signaling in T cells expressing membrane human BTLA / membrane human TCR blocking Effect of chimeric mouse / human anti-human HVEM antibody To analyze the effect of chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 on membrane human BTLA / membrane human HVEM-mediated inhibition of membrane human TCR-induced NFAT signaling, we used NFAT-response element-luciferase (RE-luc) human BTLA / HVEM Blockade Bioassay (see Example 3(c) above) to demonstrate BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling. blocking The ability of mouse anti-human HVEM antibodies to

[0213] Briefly, CHO-K1 activator cells expressing human HVEM and human TCR activators were seeded at 40,000 cells / well in flat-bottom TC-treated white solid 96-well plates (Corning) and incubated overnight at 37°C / 5% CO2. The next day, these cells were washed and subsequently incubated with or without chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 at 0.0015–10 μg / mL (3-fold dilution steps). NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR were then added at 50,000 cells / well. After 6 h incubation at 37° C. / 5% CO2, luciferase production in NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR was measured in a luminometer using the Bio-Glo™ Luciferase Assay System (Promega).

[0214] As shown in Figure 10, all tested chimeric mouse / human anti-human HVEM antibodies inhibited BTLA / HVEM-mediated inhibition of TCR-induced NFAT signaling in NFAT-RE-luc Jurkat effector T cells expressing human BTLA and human TCR to various degrees (in this order: 45H6>49G4>11H7>36H12>>48H6) in a dose-dependent manner. blocking did.

[0215] These results demonstrate that chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 inhibit human BTLA / human HVEM-mediated inhibition of TCR-induced NFAT signaling. blocking It has been demonstrated that this is possible.

[0216] (k) BTLA / HVEM for membrane human BTLA / membrane human HVEM-mediated inhibition of TCR-induced cytokine release from primary naive human T cells expressing membrane human BTLA / membrane human TCR blocking Effect of chimeric mouse / human anti-human HVEM antibody As mentioned above, ligation of HVEM on T lymphocytes by LIGHT delivers a positive co-stimulatory signal via HVEM, whereas engagement of BTLA on T lymphocytes by HVEM provides a negative co-inhibitory signal to T lymphocytes via BTLA (Del Rio et al. Journal Leukocyte Biology 2010;87:223-235). This BTLA / HVEM pathway downregulates TCR-mediated signaling in both CD4 and CD8 T lymphocytes, resulting in decreased T lymphocyte proliferation and cytokine production.

[0217] To analyze the effects of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 on membrane-BTLA / membrane-human HVEM-mediated inhibition of membrane-human TCR-induced cytokine release, (1) we performed immunization with 100% chimeric mouse / human anti-human HVEM antibodies as previously described (Chen et al. Front Immunol. 2017;8:793, artificial antigen presenting cells) stable human full-length HVEM-transfected HEK293F cells transiently transfected with membrane-bound anti-human CD3 (OKT3) single-chain variable fragment (scFv) TCR activator with minor modifications (clone no. 128, see Example 1a above), and (2) co-cultures of primary human naive T cells expressing membrane human BTLA and membrane human TCR complexes (responder cells) were used to test the ability of mouse anti-human HVEM antibodies to attenuate / reverse BTLA / HVEM-mediated inhibition of TCR-induced cytokine release (for the principle of the assay, see also Figure 4A, except that CHO-K1 artificial antigen presenting cells expressing human HVEM and Jurkat effector T cells expressing human BTLA were replaced with HEK293F artificial antigen presenting cells expressing human HVEM and primary human naive T cells expressing human BTLA, respectively).

[0218] Briefly, a cDNA encoding a membrane-bound anti-human CD3 (OKT3) scFv TCR activator protein (SEQ ID NO: 76) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO: 77). The cDNA was subcloned into a pcDNA3.1-derived expression plasmid. The anti-human CD3 (OKT3) scFv TCR activator protein plasmid was transfected into stable human full-length HVEM-transfected HEK293F cells (clone no. 128, see Example 1a above) using the FreeStyle™ 293 Expression System (Life Technologies). After 2 days, the HEK293F cells were harvested and cultured at 1.0×10 in RPMI-1640 culture medium (Gibco) containing 10% fetal bovine serum (Capricorn) and 50 μg / mL gentamicin (Gibco). 6 Prior to co-culture, anti-human CD3(OKT3)scFvTCR activator protein surface expression on HEK293F cells expressing transiently transfected human HVEM (i.e., used as artificial antigen presenting cells) was confirmed using a 1:200 diluted PE-conjugated goat anti-human IgG Fcγ-specific antibody (Jackson ImmunoResearch) and flow cytometry.

[0219] Human peripheral blood mononuclear cells (PBMCs) from healthy donors (informed consent) were isolated by density centrifugation on Lymhoprep™ (1.077 g / mL, Nycomed). Human T lymphocytes (i.e., CD4 and CD8) were subsequently enriched from this PBMC fraction using the Dynabeads™ Untouched™ Human T Cells Kit (Invitrogen) and cultured at 1.0 × 10 in RPMI-1640 culture medium (Gibco) containing 10% fetal bovine serum (Capricorn) and 50 μg / mL gentamicin (Gibco). 6Prior to co-culture, human BTLA surface expression on enriched human naive T lymphocytes (i.e., used as responder cells) was confirmed using a 1:20 diluted PE-conjugated mouse anti-human BTLA-specific antibody (BD Biosciences) and flow cytometry.

[0220] 1.0×10 6 Artificial antigen-presenting HEK293F cells expressing human HVEM at 1000 cells / mL were pretreated with 40 µg / mL chimeric mouse / human anti-human HVEM antibodies nos. 36H12, 45H6, 48H6, 11H7, and 49G4 for 15-30 min at room temperature. In parallel, 40 µg / mL of human IgG4 / κ (Sigma) was run as a negative isotype control. After this (i.e., without washing), these chimeric mouse / human anti-human HVEM antibody pretreated artificial antigen-presenting HEK293F cells and enriched human naive T lymphocytes were co-cultured in a 1:1 ratio (i.e., 50,000 T cells / 50,000 artificial antigen-presenting cells / 200 μL / well) in flat-bottom TC-treated clear 96-well plates (Corning) in the presence of 20 μg / mL of chimeric mouse / human anti-human HVEM antibodies no. 36H12, 45H6, 48H6, 11H7, and 49G4 or 20 μg / mL of human IgG4 / κ (Sigma) negative isotype control, with or without 0.5 μg / mL of costimulatory mouse anti-CD28 antibody (clone CD28.2, BD Biosciences) for 2 days at 37°C / 5% CO2. After 2 days of culture, the supernatants were harvested and frozen at -80°C until use.

[0221] The release of human IL-2, human TNFα, and human IFNγ in these supernatants from primary human naive T lymphocytes was assayed using in-house developed conventional sandwich ELISAs, i.e., (I) for IL-2 ELISA, a combination of a rat anti-human IL-2 monoclonal coating antibody (clone MQ1-17H12, eBioscience), a titrated rhuIL-2 standard (PeproTech), and a biotinylated rabbit anti-human IL-2 polyclonal detection antibody (eBioscience) was used, and (II) for TNFα ELISA, a combination of a mouse anti-human TNFα monoclonal antibody coating ( (III) for the IFNγ ELISA, it was determined using a combination of a mouse anti-human IFNγ monoclonal coating antibody (clone NIB42, eBioscience), a titrated rhuIFNγ standard (PeproTech), and a biotinylated mouse anti-human IFNγ monoclonal detection antibody (clone 4S.B3, eBioscience).

[0222] Supernatants from artificial antigen-presenting HEK293F cells or enriched human naive T lymphocytes cultured separately for 2 days (i.e., 50,000 artificial antigen-presenting cells / 200 µL / well or 50,000 T cells / 200 µL / well) but not in co-culture, show no measurable human IL-2, human TNFα, or human IFNγ levels.

[0223] As shown in Figure 11A, all tested chimeric mouse / human anti-human HVEM antibodies attenuated / reversed to various degrees (rank order, numbers 45H6>11H7>36H12>49G4>>48H6) BTLA / HVEM-mediated inhibition of TCR / CD28-induced IL-2 release from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from all 6 healthy donors. In addition, chimeric mouse / human anti-human HVEM antibodies numbers 36H12, 45H6, 11H7, and 49G4 attenuated / reversed to various degrees (rank order, numbers 45H6>11H7>36H12>49G4) BTLA / HVEM-mediated inhibition of TCR-induced IL-2 release (if any) from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from 2 / 6 healthy donors (i.e., donors A and H).

[0224] As shown in Figure 11B, chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 attenuated / reversed to various degrees (rank order, Nos. 45H6>11H7>36H12) BTLA / HVEM-mediated inhibition of TCR / CD28-induced TNFα release from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from all 6 healthy donors. In addition, chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 attenuated / reversed to various degrees (rank order, Nos. 45H6>11H7=36H12) BTLA / HVEM-mediated inhibition of TCR-induced TNFα release (if any) from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from 4 / 6 healthy donors (i.e., donors A, D, H, and K).

[0225] As shown in Figure 11C, chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 attenuated / reversed to various degrees (rank order, Nos. 45H6>11H7>36H12) BTLA / HVEM-mediated inhibition of TCR / CD28-induced IFNγ release from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from all six healthy donors. In addition, chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 attenuated / reversed to various degrees (rank order, Nos. 45H6=11H7>36H12) BTLA / HVEM-mediated inhibition of TCR-induced IFNγ release (if any) from enriched co-cultured human BTLA-expressing primary human naive T lymphocytes from 4 / 6 healthy donors (i.e., donors A, D, H, and K).

[0226] These results demonstrated that chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 were able to attenuate / reverse human BTLA / human HVEM-mediated inhibition of TCR / CD28-induced IL-2, TNFα, and IFNγ release from primary naive human T cells expressing human BTLA. In addition, these results demonstrated that chimeric mouse / human anti-human HVEM antibodies Nos. 36H12, 45H6, and 11H7 were able to attenuate / reverse human BTLA / human HVEM-mediated inhibition of TCR-induced IL-2, TNFα, and IFNγ release (if any) from primary naive human T cells expressing human BTLA.

[0227] Array Description SEQ ID NO:1 Amino acid sequence Human HVEM (Swiss-Prot number Q92956.3, aa1-283) 1 MEPPGDWGPP PWRSTPKTDV LRLVLYLTFL GAPCYAPALP SCKEDEYPVG SECCPKCSPG 61 YRVKEACGEL TGTVCEPCPP GTYIAHLNGL SKCLQCQMCD PAMGLRASRN CSRTENAVCG 121 CSPGHFCIVQ DGDHCAACRA YATSSPGQRV QKGGTESQDT LCQNCPPGTF SPNGTLEECQ 181 HQTKCSWLVT KAGAGTSSSH WVWWFLSGSL VIVIVCSTVG LIICVKRRKP RGDVVKVIVS 241 VQRKRQEAEG EATVIEALQA PPDVTTVAVE ETIPSFTGRS PNH It contains a signal peptide (aa sequence 1-38), an extracellular domain (aa sequence 39-202, including CRD1 (aa sequence 42-75), CRD2 (aa sequence 78-119), CRD3 (aa sequence 121-162), and a truncated CRD4 (aa sequence 165-179), a "linker" (aa sequence 180-202)), a transmembrane domain (aa sequence 203-223), and a cytoplasmic domain (aa sequence 224-283). SEQ ID NO:2 cDNA sequence encoding human HVEM protein (optimized for mammalian expression) 1 ATGGAGCCCC CTGGCGATTG GGGACCTCCA CCTTGGAGAA GCACCCCCAA GACCGACGTG 61 CTGCGGCTGG TGCTGTACCT GACCTTTCTG GGCGCTCCCT GTTACGCCCC TGCCCTGCCT 121 AGCTGCAAAG AGGACGAGTA CCCTGTGGGC AGCGAGTGCT GCCCTAAGTG CAGCCCTGGC 181 TACAGAGTGA AAGAGGCCTG CGGCGAGCTG ACCGGCACCG TGTGTGAACC TTGTCCCCCT 241 GGCACCTATA TCGCCCACCT GAACGGCCTG AGCAAGTGCC TGCAGTGCCA GATGTGCGAC 301 CCCGCTATGG GCCTGAGAGC CAGCAGAAAC TGCAGCCGGA CCGAGAATGC CGTGTGCGGC 361 TGTTCTCCTG GCCACTTCTG CATCGTGCAG GACGGCGATC ACTGCGCCGC CTGTAGAGCC 421 TACGCCACAT CTAGCCCAGG CCAGAGAGTG CAGAAGGGCG GCACCGAGAG CCAGGATACC 481 CTGTGCCAGA ATTGCCCTCC CGGCACCTTC AGCCCCAACG GCACACTGGA AGAGTGCCAG 541 CACCAGACCA AGTGCAGCTG GCTCGTGACC AAAGCCGGCG CTGGCACAAG CAGCTCTCAC 601 TGGGTGTGGT GGTTTCTGAG CGGCAGCCTC GTGATCGTGA TTGTGTGCAG CACCGTGGGC 661 CTGATCATCT GCGTGAAGCG GAGAAAGCCC AGAGGCGACG TCGTGAAAGT GATCGTGTCC 721 GTGCAGCGGA AGCGGCAGGA AGCCGAAGGC GAGGCCACAG TGATTGAGGC CCTGCAGGCT 781 CCCCCTGACG TGACAACAGT GGCCGTGGAA GAGACAATCC CCAGCTTCAC CGGCAGATCC 841 CCCAACCAC SEQ ID NO: 3 Amino acid sequence of CRD1-truncated human HVEM 1 MEWSGVFMFL LSVTAGVHSE PCPPGTYIAH LNGLSKCLQC QMCDPAMGLR ASRNCSRTEN 61 AVCGCSPGHF CIVQDGDHCA ACRAYATSSP GQRVQKGGTE SQDTLCQNCP PGTFSPNGTL 121 EECQHQTKCS WLVTKAGAGT SSSHWVWWFL SGSLVIVIVC STVGLIICVK RRKPRGDVVK 181 VIVSVQRKRQ EAEGEATVIE ALQAPPDVTT VAVEETIPSF TGRSPNH Mouse Ig signal peptide (aa sequence 1-19), extracellular domain (aa sequence 20-146, including CRD2 (aa sequence 22-63), CRD3 (aa sequence 65-106), and truncated CRD4 (aa sequence 109-123), a "linker" fragment (aa sequence 124-146)), transmembrane domain (aa sequence 147-167), and cytoplasmic domain (aa sequence 168-227). SEQ ID NO:4 cDNA sequence encoding the CRD1-truncated human HVEM protein (optimized for mammalian expression) 1 ATGGAGTGGA GCGGCGTGTT CATGTTCCTG CTGAGCGTGA CAGCCGGCGT GCACAGCGAA 61 CCTTGTCCCC CTGGCACCTA TATCGCCCAC CTGAACGGCC TGAGCAAGTG CCTGCAGTGC 121 CAGATGTGCG ACCCCGCTAT GGGCCTGAGA GCCAGCAGAA ACTGCAGCCG GACCGAGAAT 181 GCCGTGTGCG GCTGTTCTCC TGGCCACTTC TGCATCGTGC AGGACGGCGA TCACTGCGCC 241 GCCTGTAGAG CCTACGCCAC ATCTAGCCCT GGCCAGAGAG TGCAGAAGGG CGGCACCGAG 301 AGCCAGGATA CCCTGTGCCA GAATTGCCCT CCCGGCACCT TCAGCCCCAA CGGCACACTG 361 GAAGAGTGCC AGCACCAGAC CAAGTGCAGC TGGCTCGTGA CCAAAGCCGG CGCTGGCACA 421 AGCAGCTCTC ACTGGGTGTG GTGGTTTCTG AGCGGCAGCC TCGTGATCGT GATTGTGTGC 481 AGCACCGTGG GCCTGATCAT CTGCGTGAAG CGGAGAAAGC CCAGAGGCGA CGTCGTGAAA 541 GTGATCGTGT CCGTGCAGCG GAAGCGGCAG GAAGCCGAAG GCGAGGCCAC AGTGATTGAG 601 GCCCTGCAGG CTCCCCCTGA CGTGACAACA GTGGCCGTGG AAGAGACAAT CCCCAGCTTC 661 ACCGGCAGAT CCCCCAACCA C SEQ ID NO:5 Amino acid sequence: Cynomolgus monkey HVEM (NCBI reference sequence: XP_005545061.1, aa1-280) 1 MEPPGGWGSP PRRPAPKADI LTLVLYLTFL GSPCYAPALP SCKEDEYPVG SECCPKCGPG 61 FHVRQACGEQ TGTVCEPCSP GTYIAHFNGL SKCLQCQMCD PAMGLRTSRN CSTTANALCG 121 CSPGHFCIIQ DGDHCAACRA YATSSPGQRV QKGGTESQDT LCQNCPPGTF SSNGTLEECQ 181 HGTKCSKWLV TEAGPGTSSF RWVWWLLSGT LIVIIVGLIL GLIYVKRRKS RGDVVKVIVS 241 VQRKRQEAEG EAIVTEALQA PPDITTVAVE ETEPAFTGRS SEQ ID NO:6 cDNA sequence encoding cynomolgus monkey HVEM protein (optimized for mammalian expression) 1 ATGGAGCCTC CAGGCGGATG GGGATCTCCC CCAAGAAGGC CTGCCCCCAA GGCCGATATC 61 CTGACCCTGG TGCTGTACCT GACCTTCCTG GGCAGCCCTT GTTACGCCCC TGCCCTGCCT 121 AGCTGCAAAG AGGACGAGTA CCCTGTGGGC AGCGAGTGCT GCCCTAAGTG CGGCCCTGGA 181 TTTCATGTGC GGCAGGCCTG TGGCGAGCAG ACCGGCACAG TGTGCGAGCC TTGTAGCCCC 241 GGCACCTATA TCGCCCACTT CAACGGCCTG AGCAAGTGCC TGCAGTGCCA GATGTGCGAC 301 CCCGCTATGG GCCTGCGGAC CAGCAGAAAT TGCAGCACCA CCGCCAATGC CCTGTGCGGC 361 TGTTCTCCTG GCCACTTCTG CATATTTCAG GACGGCGACC ACTGCGCCGC CTGCAGAGCC 421 TATGCCACAT CTAGCCCTGG CCAGCGGGTG CAGAAGGGCG GAACAGAGTC TCAGGACACC 481 CTGTGCCAGA ACTGCCCCCC TGGCACCTTC AGCAGCAACG GCACCCTGGA AGAGTGCCAG 541 CACGGCACCA AGTGCAGCAA GTGGCTCGTG ACAGAGGCCG GACCTGGCAC CAGCAGCTTC 601 AGATGGGTGT GGTGGCTGCT GAGCGGCACA CTGATCGTGA TCATCGTGGG CCTGATCCTG 661 GGACTGATCT ACGTGAAGCG GCGGAAGTCC AGAGGCGACG TCGTGAAAGT GATCGTGTCC 721 GTGCAGCGGA AGAGACAGGA AGCCGAGGGC GAGGCCATTG TGACCGAAGC CCTGCAGGCC 781 CCTCCCGACA TTACAACCGT GGCCGTGGAA GAAACCGAGC CCGCCTTTAC CGGCAGATCC sequence no. 7 PCR primers ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG SEQ ID NO:8 PCR primers ATGGATTTWCAGGTGCAGATTWTCAGCTTC SEQ ID NO:9 PCR primers ATGGGCWTCAAAGATGGAGTCACA SEQ ID NO:10 PCR primers ACTGGATGGTGGGAAGATGG SEQ ID NO:11 PCR primers ATGAAATGCAGCTGGGGCATSTTCTTC SEQ ID NO:12 PCR primers ATGRACTTTGGGYTCAGCTTGRTTT SEQ ID NO:13 PCR primers ATGGGACTCCAGGCTCAATTTAGTTTTCCTT SEQ ID NO:14 PCR primers ATGGCTTGTCYTTRGSGCTRCTCTTCTGC SEQ ID NO:15 PCR primers CAGTGGATAGACAGATGGGGG SEQ ID NO:16 Consensus amino acid sequence of the heavy chain variable region of mouse anti-human HVEM antibody 36H12 1 EVQLQQSGAG LVKPGASVKL SCTASGFNIK DTYMHWVRQR PEQGLEWIGR IDPATANTKY 61 DPKFQGKATL TTDTSSNTAY LQLSSLTSED TAVYYCVTYG YDVSWFAYWG LGALVTVSA SEQ ID NO:17 Consensus amino acid sequence of the light chain variable region of mouse anti-human HVEM antibody 36H12 1 DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGITYLEW YLQKPGQSPK LLIYKVSNRF 61 SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP LTFGAGTKLE LK Complementarity determining regions (CDRs) of mouse anti-human HVEM antibody 36H12: SEQ ID NOs: 18 to 23 SEQ ID NO:18 Amino acid sequence of 36H12 heavy chain CDR1 DTYMH SEQ ID NO:19 Amino acid sequence of 36H12 heavy chain CDR2 RIDPATANTKYDPKFQG SEQ ID NO:20 Amino acid sequence of the heavy chain CDR3 of 36H12 YGYDVSWFAY SEQ ID NO:21 Amino acid sequence of the light chain CDR1 of 36H12 RSSQSIVHSNGITYLE SEQ ID NO:22 Amino acid sequence of the light chain CDR2 of 36H12 KVSNRFS SEQ ID NO:23 Amino acid sequence of the light chain CDR3 of 36H12 FQGSHVPLT SEQ ID NO:24 Consensus amino acid sequence of the heavy chain variable region of mouse anti-human HVEM antibody 45H6 1 DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY ISSGNSNIYY 61 VDTVKGRFTI SRDNPKNTLF LQMTSLRSED TAMYYCARKR AYGDYSGFSM DYWGQGTSVT 121 VSS SEQ ID NO:25 Consensus amino acid sequence of the light chain variable region of mouse anti-human HVEM antibody 45H6 1 DIVMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKP GQSPKALIYS ASYRYSGVPD 61 RFTGSGSGTD FTLTISNVQS EDLAEYFCQQ YNKFPLTFGG GTKLEIK Complementarity determining regions (CDRs) of mouse anti-human HVEM antibody 45H6: SEQ ID NO:26-31 SEQ ID NO:26 Amino acid sequence of heavy chain CDR1 of 45H6 SFGMH SEQ ID NO:27 Amino acid sequence of 45H6 heavy chain CDR2 YISSGNSNIYYVDTVKG SEQ ID NO:28 Amino acid sequence of the heavy chain CDR3 of 45H6 KRAYGDYSGFSMDY SEQ ID NO:29 Amino acid sequence of the light chain CDR1 of 45H6 KASQNVDTNVA SEQ ID NO:30 Amino acid sequence of the light chain CDR2 of 45H6 SASYRYS SEQ ID NO:31 Amino acid sequence of the light chain CDR3 of 45H6 QQYNKFPLT SEQ ID NO:32 Consensus amino acid sequence of the heavy chain variable region of mouse anti-human HVEM antibody 48H6 1 EVKLVESGGG LVKPGGSLKL SCAASGFTFS GYAMSWVRQT PEKRLEWVAS ISSGGSTYYP 61 DSVKGRFTIP RDDARNILYL QMSSLRSEDT AIYYCARGGH GSSYVYWGQG TTLTVSS SEQ ID NO:33 Consensus amino acid sequence of the light chain variable region of mouse anti-human HVEM antibody 48H6 1 DIVMSQSPSS LAVSVGEKVT MSCKSSQSLL YSSNQKNYLA WYQQKPGQSP KLLIYWASTR 61 ESGVPDRFTG SGSGTDFTLT ISSVKAEDLA VYYCHQYYSY PLTFGAGTKL ELN Complementarity determining regions (CDRs) of mouse anti-human HVEM antibody 48H6: SEQ ID NOs: 34 to 39 SEQ ID NO:34 Amino acid sequence of 48H6 heavy chain CDR1 GYAMS SEQ ID NO:35 Amino acid sequence of 48H6 heavy chain CDR2 SISSGGSTYYPDSVKG SEQ ID NO:36 Amino acid sequence of 48H6 heavy chain CDR3 GGHGSSYVY SEQ ID NO:37 Amino acid sequence of 48H6 light chain CDR1 KSSQSLLYSSNQKNYLA SEQ ID NO:38 Amino acid sequence of the light chain CDR2 of 48H6 WASTRES SEQ ID NO:39 Amino acid sequence of the light chain CDR3 of 48H6 HQYYSYPLT SEQ ID NO:40 Consensus amino acid sequence of the heavy chain variable region of mouse anti-human HVEM antibody 11H7 1 QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT IYGVHWVRQS PGKGLEWLGV IWSGGSTDYN 61 AAFISRLSIS KDNSKSQVFF KMNSLQANDT AIYYCARRDY GSRSFYYAMD YWGQGTSVTV 121SS SEQ ID NO:41 Consensus amino acid sequence of the light chain variable region of mouse anti-human HVEM antibody 11H7 1 EIVLTQSPAL MAASPGEKVT ITCSVSSSIS SSNLHWYQQK SETSPKPWIY GTSNLASGVP 61 VRFSGSGSGT SYSLTISSME AEDAATYYCQ QWSSYPLTFG GGTKLEIK Complementarity determining regions (CDRs) of mouse anti-human HVEM antibody 11H7: SEQ ID NO:42-47 SEQ ID NO:42 Amino acid sequence of 11H7 heavy chain CDR1 IYGVH SEQ ID NO:43 Amino acid sequence of 11H7 heavy chain CDR2 VIWSGGSTDYNAAFIS SEQ ID NO:44 Amino acid sequence of 11H7 heavy chain CDR3 RDYGSRSFYYAMDY SEQ ID NO:45 Amino acid sequence of 11H7 light chain CDR1 SVSSSISSSNLH SEQ ID NO:46 Amino acid sequence of 11H7 light chain CDR2 GTSNLAS SEQ ID NO:47 Amino acid sequence of the light chain CDR3 of 11H7 QQWSSYPLT SEQ ID NO:48 Consensus amino acid sequence of the heavy chain variable region of mouse anti-human HVEM antibody 49G4 1 EVQLQQSGAE LVKPGASVKL SCRASGFNIK DTYMHWVKQR PEQGLEWIGR IDPARGNTKY 61 DPKFQGKATI TADTSSNTAY LQLSSLTSED TAVYYCASAM DYWGQGTSVT VSS SEQ ID NO:49 Consensus amino acid sequence of the light chain variable region of mouse anti-human HVEM antibody 49G4 1 DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW FLQKPGQSPK LLIYKVSNRF 61 SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP LTFGAGTKLE LK Complementarity determining regions (CDRs) of mouse anti-human HVEM antibody 49G4: SEQ ID NO:50-55 SEQ ID NO:50 Amino acid sequence 49G4 heavy chain CDR1 DTYMH SEQ ID NO:51 Heavy chain CDR2 of amino acid sequence 49G4 RIDPARGNTKYDPKFQG SEQ ID NO:52 Heavy chain CDR3 of amino acid sequence 49G4 AMDY SEQ ID NO:53 Light chain CDR1 of amino acid sequence 49G4 RSSQSIVHSNGNTYLE SEQ ID NO:54 Light chain CDR2 of amino acid sequence 49G4 KVSNRFS SEQ ID NO:55 Light chain CDR3 of amino acid sequence 49G4 FQGSHVPLT SEQ ID NO:56 cDNA sequence encoding chimeric mouse VH 36H12 and the human constant heavy IgG4 chain 1 ATGGAGCTGG GCCTGAGCTG GATTTTTCTG CTGGCCATCC TGAAGGGCGT GCAGTGCGAA 61 GTTCAGCTGC AGCAATCTGG CGCCGGACTG GTTAAGCCTG GCGCCTCTGT GAAGCTGAGC 121 TGTACCGCCA GCGGCTTCAA CATCAAGGAC ACCTACATGC ACTGGGTCCG ACAGAGGCCT 181 GAGCAGGGAC TCGAATGGAT CGGCAGAATC GATCCCGCCA CCGCCAACAC CAAATACGAC 241 CCCAAGTTCC AGGGCAAAGC CACACTGACC ACCGACACCA GCAGCAACAC AGCCTACCTG 301 CAGCTGTCTA GCCTGACCAG CGAAGATACC GCCGTGTACT ACTGCGTGAC CTACGGCTAC 361 GATGTGTCTT GGTTTGCCTA CTGGGGACTG GGCGCCCTGG TTACAGTTTC TGCCGCCTCT 421 ACAAAGGGCC CCAGCGTTTT CCCACTGGCT CCCTGTAGCA GAAGCACCAG CGAATCTACA 481 GCCGCTCTGG GCTGCCTGGT CAAGGACTAC TTTCCTGAGC CTGTGACCGT GTCCTGGAAC 541 TCTGGCGCTC TGACATCTGG CGTGCACACC TTTCCAGCCG TGCTGCAAAG CAGCGGCCTG 601 TACTCTCTGA GCAGCGTGGT CACAGTGCCT AGCTCTAGCC TGGGCACCAA GACCTACACC 661 TGTAATGTGG ACCACAAGCC TAGCAACACC AAGGTGGACA AGCGCGTGGA ATCTAAGTAC 721 GGCCCTCCTT GTCCTCCATG TCCTGCACCT GAGTTTCTCG GCGGACCCTC CGTGTTCCTG 781 TTTCCTCCAA AGCCTAAGGA CACCCTGATG ATCAGCAGAA CCCCTGAAGT GACCTGCGTG 841 GTGGTGGACG TTTCCCAAGA GGACCCTGAG GTGCAGTTCA ATTGGTACGT GGACGGCGTG 901 GAAGTGCACA ACGCCAAGAC CAAGCCTAGA GAGGAACAGT TCAACAGCAC CTACAGAGTG 961 GTGTCCGTGC TGACAGTGCT GCACCAGGAT TGGCTGAACG GCAAAGAGTA CAAGTGCAAG 1021 GTGTCCAACA AGGGCCTGCC TAGCAGCATC GAGAAAACCA TCAGCAAGGC CAAGGGCCAG 1081 CCAAGAGAAC CCCAGGTGTA CACACTGCCT CCAAGCCAAG AGGAAATGAC CAAGAACCAG 1141 GTGTCCCTGA CCTGCCTCGT GAAGGGCTTC TACCCTTCCG ATATCGCCGT GGAATGGGAG 1201 AGCAATGGCC AGCCTGAGAA CAACTACAAG ACAACCCCTC CTGTGCTGGA CAGCGACGGC 1261 TCATTCTTCC TGTACAGCAG ACTGACCGTG GACAAGAGCA GATGGCAAGA GGGCAACGTG 1321 TTCTCCTGCA GCGTGATGCA CGAGGCCCTG CACAACCACT ACACCCAGAA GTCTCTGTCC 1381 CTGTCTCTGG GCAAG SEQ ID NO: 57 cDNA sequence encoding chimeric mouse VH 45H6 and human constant heavy IgG4 chain 1 ATGGAGCTGG GCCTGAGCTG GATTTTTCTG CTGGCCATCC TGAAGGGCGT GCAGTGTGAT 61 GTGCAGCTGG TGGAATCTGG CGGAGGACTG GTTCAACCTG GCGGCAGCAG AAAGCTGTCT 121 TGTGCCGCCA GCGGCTTCAC CTTCAGCAGC TTTGGAATGC ACTGGGTCCG ACAGGCCCCT 181 GAGAAAGGCC TTGAGTGGGT CGCCTACATC AGCAGCGGCA ACAGCAACAT CTACTACGTG 241 GACACCGTGA AGGGCAGATT CACCATCTCC AGAGACAACC CCAAGAATAC CCTGTTCCTG 301 CAGATGACCA GCCTGCGGAG CGAGGATACC GCCATGTACT ACTGCGCCCG GAAAAGAGCC 361 TACGGCGACT ACAGCGGCTT TAGCATGGAT TACTGGGGCC AGGGCACCAG CGTGACAGTG 421 TCTAGCGCCT CTACAAAGGG CCCTAGCGTG TTCCCTCTGG CTCCTTGTAG CAGAAGCACC 481 AGCGAGTCTA CAGCCGCTCT GGGCTGTCTG GTCAAGGACT ACTTTCCCGA GCCTGTGACC 541 GTGTCCTGGA ATTCTGGCGC TCTGACAAGC GGCGTGCACA CCTTTCCAGC TGTGCTGCAA 601 AGCAGCGGCC TGTACTCTCT GAGCAGCGTG GTCACAGTGC CTAGCTCTAG CCTGGGCACC 661 AAGACCTACA CCTGTAATGT GGACCACAAG CCTAGCAACA CCAAGGTGGA CAAGCGCGTG 721 GAATCTAAGT ACGGCCCTCC TTGTCCTCCA TGTCCTGCTC CAGAGTTTCT CGGCGGACCC 781 TCCGTGTTCC TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA 841 GTGACCTGCG TGGTGGTGGA CGTTTCCCAA GAGGACCCTG AGGTGCAGTT CAATTGGTAC 901 GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTTCAACAGC 961 ACCTACAGAG TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG 1021 TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCTAGCAGCA TCGAGAAAAC CATCAGCAAG 1081 GCCAAGGGCC AGCCAAGAGA ACCCCAGGTG TACACACTGC CTCCAAGCCA AGAGGAAATG 1141 ACCAAGAACC AGGTGTCCCT GACCTGCCTG GTTAAGGGCT TCTACCCCTC CGATATCGCC 1201 GTGGAATGGG AGAGCAATGG CCAGCCTGAG AACAACTACA AGACCACACC ACCTGTGCTG 1261 GACAGCGACG GCTCATTCTT CCTGTACAGC AGACTGACCG TGGACAAGAG CAGATGGCAA 1321 GAGGGCAACG TGTTCAGCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 1381 AAGTCTCTGA GCCTGTCTCT GGGCAAG SEQ ID NO:58 cDNA sequence encoding chimeric mouse VH 48H6 and human constant heavy IgG4 chain 1 ATGGAGCTGG GCCTGAGCTG GATTTTTCTG CTGGCCATCC TGAAGGGCGT GCAGTGCGAA 61 GTGAAGCTGG TGGAATCTGG CGGCGGACTG GTTAAGCCTG GCGGATCTCT GAAGCTGAGC 121 TGTGCCGCCA GCGGCTTTAC CTTTAGCGGC TACGCCATGA GCTGGGTCCG ACAGACACCC 181 GAGAAGAGAC TGGAATGGGT CGCCAGCATC AGCAGCGGCG GCAGCACATA TTACCCCGAC 241 TCTGTGAAGG GCCGCTTTAC AATCCCCAGA GATGACGCCC GGAACATCCT GTACCTGCAG 301 ATGAGCAGCC TGCGGAGCGA GGATACCGCC ATCTACTATT GTGCCAGAGG CGGCCACGGC 361 ACCAGCTATG TTTATTGGGG CCAGGGCACC ACACTGACCG TGTCTAGGGC CTCTACAAAG 421 GGCCCTAGCG TGTTCCCTCT GGCTCCTTGT COMMUNITY CCAGCGAGTC TACAGCCGCT 481 CTGGGCTGTC TGGTCAAGGA CTACTTTCCC GAGCCTGTGA CAGTGTCCTG GAACTCTGGC 541 GCTCTGACAA GCGGCGTGCA CACATTTCCA GCCGTGCTGC AAAGCAGCGG CCTGTACTCT 601 CTGAGCAGCG TGGTCACAGT GCCTAGCTCT AGCCTGGGCA CCAAGACCTA CACCTGTAAT 661 GTGGACCACA AGCCTAGCAA CACCAAGGTG GACAAGCGCG TGGAATCTAA GTACGGCCCT 721 CCTTGTCCTC CATGTCCTGC ACCTGAGTTT CTCGGCGGAC CCTCCGTGTT CCTGTTTCCT 781 CCAAAGCCTA AGGACACCCT GATGATCAGC AGAACCCCTG AAGTGACCTG CGTGGTGGTG 841 GACGTTTCCC AAGAGGACCC TGAGGTGCAG TTCAATTGGT ACGTGGACGG CGTGGAAGTG 901 CACAACGCCA AGACCAAGCC TAGAGAGGAA CAGTTCAACA GCACCTACAG AGTGGTGTCC 961 GTGCTGACAG TGCTGCACCA GGATTGGCTG AACGGCAAAG AGTACAAGTG CAAGGTGTCC 1021 AACAAGGGCC TGCCTAGCAG CATCGAGAAA ACCATCAGCA AGGCCAAGGG CCAGCCAAGA 1081 GAACCCCAGG TGTACACACT GCCTCCAAGC CAAGAGGAAA TGACCAAGAA CCAGGTGTCC 1141 CTGACCTGCC TCGTGAAGGG CTTCTACCCT TCCGATATCG CCGTGGAATG GGAGAGCAAT 1201 GGCCAGCCTG AGAACAACTA CAAGACAACC CCTCCTGTGC TGGACAGCGA CGGCTCATTC 1261 TTCCTGTACA GCAGGCTGAC CGTGGACAAG AGCAGATGGC AAGAGGGCAA CGTGTTCAGC 1321 TGCAGCGTGA TGCACGAGGC CCTGCACAAC CACTACACCC AGAAGTCTCT GAGCCTGTCT 1381 CTGGGCAAG SEQ ID NO: 59 cDNA sequence encoding chimeric mouse VH 11H7 and human constant heavy IgG4 chain 1 ATGGAGCTGG GCCTGAGCTG GATTTTTCTG CTGGCCATCC TGAAGGGCGT GCAGTGTCAG 61 GTGCAGCTGA AGCAGTCTGG ACCTGGACTG GTGCAGCCTA GCCAGAGCCT GAGCATCACC 121 TGTACCGTGT CCGGCTTCAG CCTGACCATC TATGGCGTGC ACTGGGTCCG ACAGAGCCCT 181 GGCAAAGGAC TGGAATGGCT GGGAGTGATT TGGAGCGGCG GCAGCACCGA TTACAACGCC 241 GCCTTTATCA GCAGACTGAG CATCTCCAAG GACAACAGCA AGAGCCAGGT GTTCTTCAAG 301 ATGAACTCCC TGCAGGCCAA CGACACCGCC ATCTACTACT GCGCCAGAAG AGACTACGGC 361 AGCCGGTCCT TCTACTACGC TATGGACTAT TGGGGCCAGG GCACCAGCGT GACAGTGTCT 421 AGCGCCTCTA CAAAGGGCCC TAGCGTGTTC CCTCTGGCTC CTTGTAGCAG AAGCACCAGC 481 GAGTCTACAG CCGCTCTGGG CTGTCTGGTC AAGGACTACT TTCCCGAGCC AGTGACCGTG 541 TCCTGGAATT CTGGCGCTCT GACAAGCGGC GTGCACACCT TTCCAGCTGT GCTGCAAAGC 601 AGCGGCCTGT ACTCTCTGAG CAGCGTGGTC ACAGTGCCTA GCTCTAGCCT GGGCACCAAG 661 ACCTACACCT GTAATGTGGA CCACAAGCCT AGCAACACCA AGGTGGACAA GCGCGTGGAA 721 TCTAAGTACG GCCCTCCTTG TCCTCCATGT CCTGCTCCAG AGTTTCTCGG CGGACCCTCC 781 GTGTTCCTGT TTCCTCCAAA GCCTAAGGAC ACCCTGATGA TCAGCAGAAC CCCTGAAGTG 841 ACCTGCGTGG TGGTGGACGT TTCCCAAGAG GACCCTGAGG TGCAGTTCAA TTGGTACGTG 901 GACGGCGTGG AAGTGCACAA CGCCAAGACC AAGCCTAGAG AGGAACAGTT CAACAGCACC 961 TACAGAGTGG TGTCCGTGCT GACCGTGCTG CATCAGGATT GGCTGAACGG CAAAGAGTAC 1021 AAGTGCAAGG TGTCCAACAA GGGCCTGCCT AGCAGCATCG AGAAAACCAT CAGCAAGGCC 1081 AAGGGCCAGC CAAGAGAACC CCAGGTGTAC ACACTGCCTC CAAGCCAAGA GGAAATGACC 1141 AAGAATCAGG TGTCCCTGAC CTGCCTCGTG AAGGGCTTCT ACCCTTCCGA TATCGCCGTG 1201 GAATGGGAGA GCAATGGCCA GCCTGAGAAC AACTACAAGA CAACCCCTCC TGTGCTGGAC 1261 AGCGACGGCT CATTCTTCCT GTACTCCAGA CTGACCGTGG ACAAGAGCAG ATGGCAAGAG 1321 GGCAACGTGT TCAGCTGCTC CGTGATGCAC GAGGCCCTGC ACAACCACTA CACCCAGAAG 1381 TCCCTGAGCC TGTCTCTGGG CAAA SEQ ID NO: 60 cDNA sequence encoding chimeric mouse VH 49G4 and human constant heavy IgG4 chain 1 ATGGAGCTGG GCCTGAGCTG GATTTTTCTG CTGGCCATCC TGAAGGGCGT GCAGTGCGAA 61 GTTCAGCTGC AGCAGTCTGG CGCCGAGCTT GTGAAACCTG GCGCCTCTGT GAAGCTGAGC 121 TGTAGAGCCA GCGGCTTCAA CATCAAGGAC ACCTACATGC ACTGGGTCAA GCAGAGGCCT 181 GAGCAGGGCC TCGAATGGAT CGGCAGAATC GATCCCGCCA GAGGCAACAC CAAATACGAC 241 CCCAAGTTCC AGGGCAAAGC CACCATCACC GCCGACACCT CTAGCAACAC AGCCTACCTG 301 CAGCTGTCCA GCCTGACCTC TGAAGATACC GCCGTGTACT ACTGCGCCAG CGCTATGGAT 361 TATTGGGGCC AGGGCACAAG CGTGACCGTG TCTAGCGCCT CTACAAAGGG CCCTAGCGTG 421 TTCCCACTGG CTCCCTGTAG CAGAAGCACC AGCGAATCTA CAGCCGCTCT GGGCTGCCTG 481 GTCAAGGACT ACTTTCCTGA GCCTGTGACA GTGTCCTGGA ACTCTGGCGC TCTGACAAGC 541 GGCGTGCACA CATTTCCAGC CGTGCTGCAA AGCAGCGGCC TGTACTCTCT GAGCAGCGTG 601 GTCACAGTGC CTAGCTCTAG CCTGGGCACC AAGACCTACA CCTGTAATGT GGACCACAAG 661 CCTTCCAACA CCAAGGTGGA CAAGCGCGTG GAATCTAAGT ACGGCCCTCC TTGTCCTCCA 721 TGTCCTGCAC CTGAGTTTCT CGGCGGACCC TCCGTGTTCC TGTTTCCTCC AAAGCCTAAG 781 GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG TGGTGGTGGA CGTTTCCCAA 841 GAGGACCCTG AGGTGCAGTT CAATTGGTAC GTGGACGGCG TGGAAGTGCA CAACGCCAAG 901 ACCAAGCCTA GAGAGGAACA GTTCAACAGC ACCTACAGAG TGGTGTCCGT GCTGACAGTG 961 CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA AGGTGTCCAA CAAGGGCCTG 1021 CCTAGCAGCA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC AGCCAAGAGA ACCCCAGGTG 1081 TACACACTGC CTCCAAGCCA AGAGGAAATG ACCAAGAACC AGGTGTCCCT GACCTGCCTC 1141 GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG AGAGCAATGG CCAGCCTGAG 1201 AACAACTACA AGACAACCCC TCCTGTGCTG GACTCCGACG GCTCATTCTT CCTGTACAGC 1261 AGACTGACCG TGGACAAGAG CAGATGGCAA GAGGGCAACG TGTTCTCCTG CAGCGTGATG 1321 CACGAAGCCC TGCACAACCA CTACACCCAG AAGTCTCTGT CCCTGTCTCT GGGCAAG SEQ ID NO: 61 cDNA sequence encoding chimeric mouse VL 36H12 and human constant light κ chain 1 ATGGACATGA GAGTTCCCGC TCAGCTGCTG GGACTGCTGC TGCTTTGGTT TCCTGGCGCT 61 AGATGCGACG TGCTGATGAC CCAGACACCT CTGAGCCTGC CTGTGTCTCT GGGAGATCAG 121 GCCAGCATCA GCTGCAGATC CAGCCAGAGC ATCGTGCACA GCAACGGCAT CACCTACCTG 181 GAATGGTATC TGCAGAAGCC CGGACAGAGC CCCAAGCTGC TGATCTACAA GGTGTCCAAC 241 CGGTTCAGCG GCGTGCCCGA TAGATTTTCT GGCAGCGGCT CTGGCACCGA CTTCACCCTG 301 AAGATCTCCA GAGTGGAAGC CGAGGACCTG GGCGTGTACT ACTGCTTCCA AGGCTCTCAC 361 GTGCCCCTGA CATTTGGAGC CGGCACCAAG CTGGAACTGA AGAGAACAGT GGCCGCTCCT 421 AGCGTGTTCA TCTTCCCACC TTCCGACGAG CAGCTGAAAA GCGGCACAGC CTCTGTCGTG 481 TGCCTGCTGA ACAACTTCTA CCCCAGAGAA GCCAAGGTGC AGTGGAAGGT GGACAACGCC 541 CTGCAGAGCG GCAATAGCCA AGAGAGCGTG ACCGAGCAGG ACAGCAAGGA CTCTACCTAC 601 AGCCTGAGCA GCACCCTGAC ACTGAGCAAG GCCGACTACG AGAAGCACAA AGTGTACGCC 661 TGCGAAGTGA CCCACCAGGG CCTTTCTAGC CCTGTGACCA AGAGCTTCAA CCGGGGCGAA 721 TGT SEQ ID NO: 62 cDNA sequence encoding chimeric mouse VL 45H6 and human constant light κ chain 1 ATGGACATGA GAGTTCCCGC TCAGCTGCTG GGACTGCTGC TGCTTTGGTT TCCTGGCGCT 61 AGATGCGACA TCGTGATGAC CCAGAGCCAG AAATTCATGA GCACCAGCGT GGGCGACAGA 121 GTGTCCGTGA CATGTAAAGC CAGCCAGAAC GTGGACACCA ACGTGGCCTG GTATCAGCAG 181 AAGCCTGGAC AGAGCCCCAA GGCTCTGATC TACAGCGCCA GCTACAGATA CAGCGGCGTG 241 CCCGATAGAT TCACAGGCAG CGGCTCTGGC ACCGACTTCA CCCTGACAAT CAGCAACGTG 301 CAGAGCGAGG ACCTGGCCGA GTATTTCTGC CAGCAGTACA ACAAGTTCCC TCTGACCTTC 361 GGCGGAGGCA CCAAGCTGGA AATCAAGAGA ACAGTGGCCG CTCCTAGCGT GTTCATCTTC 421 CCACCTTCCG ACGAGCAGCT GAAAAGCGGC ACAGCCTCTG TCGTGTGCCT GCTGAACAAC 481 TTCTACCCCA GAGAAGCCAA GGTGCAGTGG AAGGTGGACA ACGCCCTGCA GAGCGGCAAT 541 AGCCAAGAGA GCGTGACCGA GCAGGACAGC AAGGACTCTA CCTACAGCCT GAGCAGCACA 601 CTGACCCTGA GCAAGGCCGA CTACGAGAAG CACAAAGTGT ACGCCTGCGA AGTGACCCAC 661 CAGGGCCTTT CTAGCCCTGT GACCAAGAGC TTCAACCGGG GCGAATGT sequence no.63 cDNA sequences encoding chimeric mouse VL 48H6 and human constant light κ chain 1 ATGGACATGA GAGTTCCCGC TCAGCTGCTG GGACTGCTGC TGCTTTGGTT TCCTGGCGCT 61 AGATGCGACA TCGTGATGTC TCAGAGCCCT AGCAGCCTGG CCGTGTCTGT GGGAGAGAAA 121 GTGACCATGA GCTGCAAGAG CAGCCAGAGC CTGCTGTACT CCAGCAACCA GAAGAACTAC 181 CTGGCCTGGT ATCAGCAGAA GCCCGGACAG TCTCCCAAGC TGCTGATCTA CTGGGCCAGC 241 ACCAGAGAAA GCGGCGTGCC CGATAGATTC ACAGGCAGCG GCAGCGGAAC CGACTTCACC 301 CTGACAATCA GCAGCGTGAA GGCCGAGGAC CTGGCTGTGT ACTACTGCCA CCAGTATTAC 361 AGCTACCCTC TGACCTTCGG AGCCGGCACC AAGCTGGAAC TGAACAGAAC AGTGGCCGCT 421 CCTAGCGTGT TCATCTTCCC ACCTTCCGAC GAGCAGCTGA AGTCTGGCAC AGCCTCTGTC 481 GTGTGCCTGC TGAACAACTT CTACCCCAGA GAAGCCAAGG TGCAGTGGAA GGTGGACAAC 541 GCCCTGCAGA GCGGCAATAG CCAAGAGAGC GTGACCGAGC AGGACAGCAA GGACTCTACC 601 TACAGCCTGA GCAGCACACT GACCCTGAGC AAGGCCGACT ACGAGAAGCA CAAAGTGTAC 661 GCCTGCGAAG TGACCCACCA GGGCCTTTCT AGCCCTGTGA CCAAGAGCTT CAACCGGGGC 721 GAATGT Accession No. 64 cDNA sequence encoding chimeric mouse VL 11H7 and human constant light κ chain 1 ATGGACATGA GAGTTCCCGC TCAGCTGCTG GGACTGCTGC TGCTTTGGTT TCCTGGCGCT 61 AGATGCGAGA TCGTGCTGAC ACAGAGCCCT GCTCTGATGG CTGCTTCCCC TGGCGAGAAA 121 GTGACCATCA CCTGTAGCGT GTCCAGCAGC ATCAGCAGCT CCAACCTGCA CTGGTATCAG 181 CAGAAGTCCG AGACAAGCCC CAAGCCTTGG ATCTACGGCA CCAGCAATCT GGCCAGCGGA 241 GTGCCTGTCA GATTTTCTGG CAGCGGCTCT GGCACCAGCT ACAGCCTGAC AATCAGCAGC 301 ATGGAAGCCG AGGATGCCGC CACCTACTAC TGCCAGCAGT GGTCTAGCTA CCCTCTGACC 361 TTTGGCGGAG GCACCAAGCT GGAAATCAAG CGGACAGTGG CCGCTCCTAG CGTGTTCATC 421 TTTCCACCTA GCGACGAGCA GCTGAAGTCT GGCACAGCCT CTGTCGTGTG CCTGCTGAAC 481 AACTTCTACC CCAGAGAAGC CAAGGTGCAG TGGAAGGTGG ACAACGCCCT GCAGAGCGGC 541 AATAGCCAAG AGAGCGTGAC CGAGCAGGAC AGCAAGGACT CCACCTATAG CCTGAGCAGC 601 ACCCTGACAC TGAGCAAGGC CGACTACGAG AAGCACAAAG TGTACGCCTG CGAAGTGACC 661 CACCAGGGCC TTTCTAGCCC TGTGACCAAG AGCTTCAACC GGGGCGAATG T Sequence number 65 cDNA sequence encoding chimeric mouse VL and 49G4 human constant light κ chain 1 ATGGACATGA GAGTTCCCGC TCAGCTGCTG GGACTGCTGC TGCTTTGGTT TCCTGGCGCT 61 AGATGCGACG TGCTGATGAC CCAGACACCT CTGAGCCTGC CTGTGTCTCT GGGAGATCAG 121 GCCAGCATCA GCTGCAGATC CAGCCAGAGC ATCGTGCACA GCAACGGCAA TACCTACCTG 181 GAATGGTTCC TGCAGAAGCC CGGACAGAGC CCCAAGCTGC TGATCTACAA GGTGTCCAAC 241 CGGTTCAGCG GCGTGCCCGA TAGATTTTCT GGCAGCGGCT CTGGCACCGA CTTCACCCTG 301 AAGATCTCCA GAGTGGAAGC CGAGGACCTG GGCGTGTACT ACTGCTTCCA AGGCTCTCAC 361 GTGCCCCTGA CATTTGGAGC CGGCACCAAG CTGGAACTGA AGAGAACAGT GGCCGCTCCT 421 AGCGTGTTCA TCTTCCCACC TTCCGACGAG CAGCTGAAAA GCGGCACAGC CTCTGTCGTG 481 TGCCTGCTGA ACAACTTCTA CCCCAGAGAA GCCAAGGTGC AGTGGAAGGT GGACAACGCC 541 CTGCAGAGCG GCAATAGCCA AGAGAGCGTG ACCGAGCAGG ACAGCAAGGA CTCTACCTAC 601 AGCCTGAGCA GCACCCTGAC ACTGAGCAAG GCCGACTACG AGAAGCACAA AGTGTACGCC 661 TGCGAAGTGA CCCACCAGGG CCTTTCTAGC CCTGTGACCA AGAGCTTCAA CCGGGGCGAA 721 TGT SEQ ID NO:66 Amino acid sequences of chimeric mouse VH 36H12 and the human constant heavy IgG4 chain 1 MELGLSWIFL LAILKGVQCE VQLQQSGAGL VKPGASVKLS CTASGFNIKD TYMHWVRQRP 61 EQGLEWIGRI DPATANTKYD PKFQGKATLT TDTSSNTAYL QLSSLTSEDT AVYYCVTYGY 121 DVSWFAYWGL GALVTVSAAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN 181 SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT CNVDHKPSNT KVDKRVESKY 241 GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV 301 EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ 361 PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 421 SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK SEQ ID NO:67 Amino acid sequences of chimeric mouse VH 45H6 and human constant heavy IgG4 chains 1 MELGLSWIFL LAILKGVQCD VQLVESGGGL VQPGGSRKLS CAASGFTFSS FGMHWVRQAP 61 EKGLEWVAYI SSGNSNIYYV DTVKGRFTIS RDNPKNTLFL QMTSLRSEDT AMYYCARKRA 121 YGDYSGFSMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT 181 VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV 241 ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 301 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 361 AKGQPREPQV YTLPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 421 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK SEQ ID NO:68 Amino acid sequences of chimeric mouse VH 48H6 and human constant heavy IgG4 chains 1 MELGLSWIFL LAILKGVQCE VKLVESGGGL VKPGGSLKLS CAASGFTFSG YAMSWVRQTP 61 EKRLEWVASI SSGGSTYYPD SVKGRFTIPR DDARNILYLQ MSSLRSEDTA IYYCARGGHG 121 SSYVYWGQGT TLTVSSASTK GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG 181 ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTKTYTCN VDHKPSNTKV DKRVESKYGP 241 PCPPCPAPEF LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV 301 HNAKTKPREE QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR 361 EPQVYTLPS QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 421 FLYSRLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK SEQ ID NO:69 Amino acid sequences of chimeric mouse VH 11H7 and human constant heavy IgG4 chains 1 MELGLSWIFL LAILKGVQCQ VQLKQSGPGL VQPSQSLSIT CTVSGFSLTI YGVHWVRQSP 61 GKGLEWLGVI WSGGSTDYNA AFISRLSISK DNSKSQVFFK MNSLQANDTA IYYCARRDYG 121 SRSFYYAMDY WGQGTSVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV 181 SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE 241 SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV 301 DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA 361 KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 421 SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK SEQ ID NO:70 Amino acid sequences of chimeric mouse VH 49G4 and the human constant heavy IgG4 chain 1 MELGLSWIFL LAILKGVQCE VQLQQSGAEL VKPGASVKLS CRASGFNIKD TYMHWVKQRP 61 EQGLEWIGRI DPARGNTKYD PKFQGKATIT ADTSSNTAYL QLSSLTSEDT AVYYCASAMD 121 YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS 181 GVHTFPAVLQ SSGLYSLSSV VTPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP 241 CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK 301 TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV 361 YTLPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS 421 RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK SEQ ID NO:71 Amino acid sequences of chimeric mouse VL 36H12 and human constant light kappa chains 1 MDMRVPAQLL GLLLLWFPGA RCDVLMTQTP LSLPVSLGDQ ASISCRSSQS IVHSNGITYL 61 EWYLQKPGQS PKLLIYKVSN RFSGVPDRFS GSGSGTDFTL KISRVEAEDL GVYYCFQGSH 121 VPLTFGAGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA 181 LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE 241 C SEQ ID NO:72 Amino acid sequences of chimeric mouse VL 45H6 and human constant light kappa chains 1 MDMRVPAQLL GLLLLWFPGA RCDIVMTQSQ KFMSTSVGDR VSVTCKASQN VDTNVAWYQQ 61 KPGQSPKALI YSASYRYSGV PDRFTGSGSG TDFTLTISNV QSEDLAEYFC QQYNKFPLTF 121 GGGTKLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN 181 SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO:73 Amino acid sequences of chimeric mouse VL 48H6 and human constant light kappa chains 1 MDMRVPAQLL GLLLLWFPGA RCDIVMSQSP SSLAVSVGEK VTMSCKSSQS LLYSSNQKNY 61 LAWYQQKPGQ SPKLLIYWAS TRESGVPDRF TGSGSGTDFT LTISSVKAED LAVYYCHQYY 121 SYPLTFGAGT KLELNRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN 181 ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG 241 EC SEQ ID NO:74 Amino acid sequences of chimeric mouse VL 11H7 and human constant light kappa chains 1 MDMRVPAQLL GLLLLWFPGA RCEIVLTQSP ALMAASPGEK VTITCSVSSS ISSSNLHWYQ 61 QKSETSPKPW IYGTSNLASG VPVRFSGSGS GTSYSLTISS MEAEDAATYY CQQWSSYPLT 121 FGGGTKLEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 181 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC SEQ ID NO:75 Amino acid sequences of chimeric mouse VL 49G4 and human constant light kappa chains 1 MDMRVPAQLL GLLLLWFPGA RCDVLMTQTP LSLPVSLGDQ ASISCRSSQS IVHSNGNTYL 61 EWFLQKPGQS PKLLIYKVSN RFSGVPDRFS GSGSGTDFTL KISRVEAEDL GVYYCFQGSH 121 VPLTFGAGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA 181 LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE 241 C SEQ ID NO:76 Amino acid sequence of membrane-bound anti-human CD3 scFv (OKT 3 scFv linked to a signal peptide followed by the CH2-CH3 domains of human IgG1 and the human CD80 cytoplasmic tail) 1 MKWVTFISLL FLFSSAYSQV QLQQSGAELA RPGASVKMSC KASGYTFTRY TMHWVKQRPG 61 QGLEWIGYIN PSRGYTNYNQ KFKDKATLTT DKSSSTAYMQ LSSLTSEDSA VYYCARYYDD 121 HYCLDYWGQG TTLTVSSGGG GSGGGGSGGG GSQIVLTQSP AIMSASPGEK VTMTCSASSS 181 VSYMNWYQQK SGTSPKRWIY DTSKLASGVP AHFRGSGSGT SYSLTISGME AEDAATYYCQ 241 QWSSNPFTFG SGTKLEINRA AAPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 301 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 361 ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 421 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GWAITLISVN 481 GIFVICCLTY CFAPRCRERR RNERLRRESV RPV SEQ ID NO:77 cDNA sequence encoding the membrane-bound anti-human CD3 scFv 1 ATGAAATGGG TCACCTTTAT CTCCCTGCTG TTCCTGTTCT CCAGCGCCTA CTCTCAGGTC 61 CAGTTGCAGC AGTCTGGCGC CGAATTGGCT AGACCTGGCG CCTCCGTGAA GATGTCCTGC 121 AAGGCTTCCG GCTACACCTT CACCAGATAC ACCATGCACT GGGTCAAGCA GAGGCCTGGA 181 CAGGGCCTTG AGTGGATCGG CTACATCAAC CCTTCTCGGG GCTACACCAA CTACAACCAG 241 AAGTTCAAGG ACAAGGCTAC CCTGACAACC GACAAGTCCT CCTCCACCGC CTACATGCAG 301 CTGTCCAGCC TGACCTCTGA GGACTCCGCC GTGTACTACT GTGCCCGGTA CTACGACGAC 361 CACTACTGCC TGGATTATTG GGGCCAGGGC ACCACACTGA CAGTGTCTAG CGGAGGCGGA 421 GGATCTGGTG GTGGTGGATC TGGCGGCGGA GGTTCTCAGA TTGTGCTGAC CCAGTCTCCT 481 GCCATCATGT CCGCTTCTCC CGGCGAGAAA GTGACAATGA CCTGCTCCGC CTCTTCCTCC 541 GTGTCCTACA TGAACTGGTA TCAGCAGAAG TCCGGCACCT CTCCTAAGCG GTGGATCTAC 601 GACACCTCCA AGCTGGCATC TGGCGTGCCC GCTCACTTTA GAGGCTCTGG CTCTGGCACC 661 AGCTACTCCC TGACCATCTC TGGCATGGAA GCCGAGGATG CCGCCACCTA CTATTGCCAG 721 CAGTGGTCTA GCAACCCCTT CACCTTCGGC TCCGGCACCA AGCTGGAAAT CAACAGAGCC 781 GCCGCTCCTT CCGTGTTTCT GTTCCCTCCA AAGCCTAAGG ACACCCTGAT GATCTCTCGG 841 ACCCCTGAAG TGACCTGCGT GGTGGTCGAT GTGTCTCACG AGGACCCAGA AGTGAAGTTC 901 AATTGGTACG TGGACGGCGT GGAAGTGCAC AACGCCAAGA CCAAGCCTAG AGAGGAACAG 961 TACAACTCCA CCTACAGAGT GGTGTCCGTG CTGACCGTGC TGCACCAGGA TTGGCTGAAC 1021 GGCAAAGAGT ACAAGTGCAA GGTGTCCAAC AAGGCTCTGC CCGCTCCTAT CGAAAAGACC 1081 ATCTCCAAGG CCAAGGGCCA GCCTAGGGAA CCCCAGGTTT ACACCCTGCC TCCAAGCCGG 1141 GAAGAGATGA CCAAGAACCA GGTGTCCCTG ACCTGCCTGG TCAAGGGCTT CTACCCTTCC 1201 GATATCGCCG TGGAATGGGA GAGCAATGGC CAGCCTGAGA ACAACTACAA GACCACACCT 1261 CCTGTGCTGG ACTCCGACGG CTCATTCTTC CTGTACAGCA AGCTGACAGT GGACAAGTCC 1321 AGATGGCAGC AGGGCAACGT GTTCTCCTGC TCCGTGATGC ACGAGGCCCT GCACAATCAC 1381 TACACACAGA AGTCCCTGTC TCTGTCCCCT GGCTGGGCTA TCACCCTGAT CTCTGTGAAC 1441 GGCATCTTCG TGATCTGCTG CCTGACCTAC TGCTTCGCCC CTAGATGCAG AGAGCGGCGG 1501 AGAAACGAAC GGCTGCGGAG AGAATCTGTC CGGCCTGTG

[0228] [Section 1] An antibody that binds to the extracellular cysteine-rich domain-1 (CRD1) of herpes virus entry mediator (HVEM) on HVEM-expressing cells, and when the antibody binds to the extracellular portion of HVEM, prevents binding of B- and T-lymphocyte attenuator (BTLA) to HVEM and removes BTLA bound to the extracellular portion of HVEM. [Section 2] The antibody of paragraph 1, wherein the antibody does not remove LIGHT bound to the extracellular portion of HVEM when the antibody binds to the extracellular portion of HVEM. [Section 3] The antibody described in item 1, wherein the antibody partially prevents CD160 from binding to HVEM when the antibody binds to the extracellular portion of HVEM and partially removes CD160 bound to the extracellular portion of HVEM. [Section 4] The antibody according to any one of items 1 to 3, which binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26 to 28 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29 to 31 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. [Section 5] The antibody according to any one of items 1 to 4, which binds to an extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 24 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 25 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. [Section 6] An antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42 to 44 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. [Section 7] An antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 40 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 41 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. [Section 8] An antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18 to 20 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21 to 23 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. [Section 9] An antibody that binds to the extracellular portion of HVEM on an HVEM-expressing cell, comprising a heavy chain region having the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. [Section 10] The antibody of any one of items 1 to 3, 6, and 7, wherein the antibody does not prevent binding of LIGHT to the extracellular portion of HVEM when the antibody binds to the extracellular portion of HVEM. [Section 11] The antibody according to any one of items 1 to 10, wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody, a humanized antibody, and an antigen-binding fragment thereof. [Section 12] One or more nucleic acid molecules comprising a nucleotide sequence encoding the antibody according to any one of items 4 to 10 and 11. [Section 13] A cell comprising one or more nucleic acids according to paragraph 12, the product of which can be assembled into the antibody of any one of paragraphs 1 to 11. [Section 14] The antibody according to any one of items 1 to 11, for use in treating a disease. [Section 15] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the nucleic acid molecule according to any one of items 1 to 12, and a pharma- ceutically acceptable carrier and / or diluent. [Section 16] Item 16. The pharmaceutical composition according to item 15, for use in the prevention or treatment of cancer and / or immune-related disorders. [Section 17] A method for regulating HVEM signaling activity, comprising contacting an HVEM-expressing cell with an antibody or antigen-binding fragment thereof, or a nucleic acid molecule described in any one of items 1 to 12. [Section 18] A method for treating a human or animal suffering from a medical indication, the method comprising administering to the human or animal a therapeutically effective amount of the antibody or antigen-binding fragment thereof, or the nucleic acid molecule according to any one of items 1 to 12. [Section 19] A method for reducing tumor growth in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof, or the nucleic acid molecule according to any one of items 1 to 12.

Claims

1. An antibody, The antibody binds to the extracellular cysteine-rich domain-1 (CRD1) of herpesvirus entry mediator (HVEM) on HVEM-expressing cells, and when the antibody binds to the extracellular portion of HVEM, prevents B- and T-lymphocyte attenuator (BTLA) from binding to HVEM and removes BTLA bound to the extracellular portion of HVEM; the antibody, when bound to the extracellular portion of HVEM, partially prevents CD160 from binding to HVEM and partially removes CD160 bound to the extracellular portion of HVEM; The antibody (a) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18-20, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21-23; (b) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31; (c) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 34-36, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 37-39; (d) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42-44, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45-47; or (e) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 50-52, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53-55. Including, The antibody.

2. The antibody (a) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18-20, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 21-23; (b) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31; or (d) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 42-44, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45-47. The antibody of claim 1, comprising:

3. The antibody (b) a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26-28, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31. The antibody of claim 1 or 2, comprising:

4. The antibody of any one of claims 1 to 3, wherein the antibody does not remove LIGHT bound to the extracellular portion of HVEM when the antibody binds to the extracellular portion of HVEM.

5. An antibody described in any one of claims 1 to 4, wherein the antibody binds to human HVEM.

6. a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 25 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, the amino acid insertions, deletions, or substitutions are outside the CDRs of the heavy and light chain variable regions; The antibody according to any one of claims 1 to 4.

7. a heavy chain variable region having the amino acid sequence of SEQ ID NO: 40 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 41 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, the amino acid insertions, deletions, or substitutions are outside the CDRs of the heavy and light chain variable regions; The antibody according to any one of claims 1 to 2 or 4 to 5.

8. a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, the amino acid insertions, deletions, or substitutions are outside the CDRs of the heavy and light chain variable regions; The antibody according to any one of claims 1 to 2 or 4 to 5.

9. The antibody of any one of claims 1 to 2, 4 to 8, wherein the antibody does not prevent binding of LIGHT to the extracellular portion of HVEM when the antibody binds to the extracellular portion of HVEM.

10. The antibody of any one of claims 1 to 9, wherein the antibody is selected from a monoclonal antibody, a humanized antibody, and an antigen-binding fragment thereof.

11. One or more nucleic acid molecules comprising a nucleotide sequence encoding the antibody of any one of claims 1 to 10.

12. A cell comprising one or more nucleic acid molecules of claim 11, wherein the product of said one or more nucleic acid molecules is assembled into an antibody of any one of claims 1 to 10.

13. An antibody according to any one of claims 1 to 10 for use in the treatment of a disease.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or one or more nucleic acid molecules according to claim 11, and a pharmaceutically acceptable carrier and / or diluent.

15. 15. The pharmaceutical composition of claim 14 for use as a prophylaxis or in the treatment of cancer and / or immune-related disorders.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use in regulating HVEM signaling activity.

17. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, for use in the treatment of cancer and / or immune-related disorders.

18. A therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, used to reduce tumor growth in a subject in need thereof.

Images