BTLA antibody

Abstract

This invention provides novel and useful agents capable of modulating human B and T lymphocyte attenuation factors (BTLA). [Solution] The present invention generally relates to antibodies or antigen-binding fragments that bind to BTLA, and to the use thereof. More specifically, the present invention relates to agonist antibodies that bind to human BTLA and modulate its activity, and to the use thereof in treating inflammatory, autoimmune, and proliferative diseases and disorders.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of UK Application No. 1820554.2, filed on Dec. 17, 2018, which is incorporated herein by reference in its entirety.

[0002] Field of the Invention The present invention generally relates to antibodies or antigen-binding fragments that bind to human B and T lymphocyte attenuator (BTLA), and their use. More specifically, the present invention relates to agonist antibodies that bind to human BTLA and modulate its activity, and their use in treating inflammatory, autoimmune and proliferative diseases and disorders.

Background Art

[0003] The immune system must achieve a balance between the destruction of pathogens or cells with dangerous mutations, as well as the tolerance of healthy self-tissues and harmless symbiotic organisms. To facilitate this balance, the activity of immune cells is affected by the integration of signals from a number of stimulatory and inhibitory receptors that adapt the cells to their environment. These surface-expressed receptors present attractive targets for the therapeutic modulation of immune responses. Many human diseases result from abnormal or undesirable activation of the immune system, including autoimmune diseases, graft rejection and graft-versus-host disease. Agonist agents capable of inducing signaling through inhibitory receptors have been able to attenuate these undesirable immune responses.

[0004] B and T lymphocyte attenuation factor (BTLA; also known as CD272) is an inhibitory member of the receptor CD28 family, which also includes CD28, CTLA-4, ICOS, and PD-1 (Watanabe et al., Nat Immunol. 4:670-679, 2003). BTLA is widely expressed throughout the immune system on both myelocytes and lymphocytes (Han et al., J Immunol. 172:5931-9, 2004). After binding to its ligand, herpesvirus entry mediator (HVEM), BTLA replenishes its cytoplasmic domain with phosphatases SHP-1 and SHP-2 (Sedy et al., Nat Immunol. 6:90-8, 2005), which then inhibits the activating receptor signaling cascade. Mice lacking the intact BTLA gene exhibit hyperproliferative B and T cell responses in vitro, higher titers to DNP-KLH immunization, and increased sensitivity to EAEs (Watanabe et al., Nat. Immunol, 4:670-679, 2003). When aged BTLA knockout mice are observed until they spontaneously develop autoantibodies, they develop autoimmune hepatitis-like disease, with inflammatory cells infiltrating numerous organs (Oya et al., Arthritis Rheum 58:2498-2510, 2008). This evidence suggests that BTLA inhibitory receptors play a crucial role in maintaining immune homeostasis and inhibiting autoimmunity. Furthermore, HVEM-BTLA signaling is involved in the regulation of mucosal inflammation and infectious immunity (Shui et al., J Leukoc Biol. 89:517-523, 2011).

[0005] A therapeutic agent capable of regulating BTLA function and inhibiting autoreactive lymphocytes in the context of autoimmune disorders would be highly desirable. Previous studies have shown that monoclonal antibodies that bind to mouse BTLA can act as agonists, inducing receptor-mediated signaling and inhibiting immune cell responses. In the presence of agonist anti-BTLA antibodies (mAbs), anti-CD3 and anti-CD28 activated T cells... The cells show reduced IL-2 production and proliferation (Kreig et al., J. Immunol., 175, 6420-6472, 2005).

[0006] Furthermore, anti-mouse BTLA agonist antibodies have been shown to induce remission of graft-versus-host disease in mouse models (Sakoda et al., Blood. 117:2506-2514; Albring et al., J Exp Med. 207:2551-9, 2010). Agonist antibodies targeting the human BTLA receptor have been shown to inhibit T cell responses ex vivo (see Otsuki et al., Biochem Biophys Res Commun 344:1121-7, 2006; and WO2011 / 014438), but these have not yet been translated into clinical use. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2011 / 014438 [Non-patent literature]

[0008] [Non-Patent Document 1] Watanabe et al., Nat Immunol. 4:670-679, 2003. [Non-Patent Document 2] Han et al., J Immunol. 172:5931-9, 2004. [Non-Patent Document 3] Sedy et al., Nat Immunol. 6:90-8, 2005. [Non-Patent Document 4] Oya et al., Arthritis Rheum 58: 2498-2510, 2008 [Non-Patent Document 5] Shui et al., J Leukoc Biol. 89:517-523, 2011 [Non-Patent Document 6] Kreig et al., J. Immunol., 175, 6420-6472, 2005. [Non-Patent Document 7] Sakoda et al., Blood. 117:2506-2514 [Non-Patent Document 8] Albring et al., J Exp Med. 207:2551-9, 2010 [Non-Patent Document 9] Otsuki et al., Biochem Biophys Res Commun 344:1121-7, 2006 [Overview of the project] [Problems that the invention aims to solve]

[0009] In this field, there is a need to discover novel and useful agents, such as antibodies or antigen-binding antibody fragments, that can modulate BTLA. [Means for solving the problem]

[0010] The present invention relates to an anti-human BTLA agonist antibody or antibody fragment having one or more desirable properties in human subjects, including high binding affinity, high agonist strength, high agonist efficacy, excellent pharmacokinetics, and low antigenicity. The present invention also relates to the use of the antibody or antibody fragment of the present invention in the treatment of diseases.

[0011] According to a first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to BTLA, wherein the antibody is identified in Table 1 and described herein as follows: 11.5.1, 2.8.6, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, The present invention provides an antibody or its antigen-binding fragment having a heavy chain and / or light chain containing at least one complementarity-determining region (CDR) that is present to an antibody selected from the group consisting of 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, 6.2, and 831.

[0012] In accordance with a second aspect of the present invention, an isolated nucleic acid is provided, comprising a nucleotide sequence encoding a heavy chain polypeptide or a light chain polypeptide of the isolated antibody or its antigen-binding fragment according to a first aspect of the present invention.

[0013] In accordance with a third aspect of the present invention, a vector comprising the nucleic acid of the second aspect of the present invention is provided. In accordance with the fourth aspect of the present invention, a host cell is provided comprising a nucleic acid sequence according to the second aspect of the present invention or a vector according to the third aspect of the present invention.

[0014] A method for producing an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, according to the fifth aspect of the present invention, comprising the steps of culturing a host cell according to the fourth aspect of the present invention under conditions for the production of the antibody or antigen-binding fragment, and optionally isolating and / or purifying the antibody or antigen-binding fragment.

[0015] In accordance with the sixth aspect of the present invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or one produced according to the fifth aspect of the present invention.

[0016] A method for preparing a pharmaceutical composition according to a seventh aspect of the present invention is provided, comprising the step of incorporating an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, or one produced according to a fifth aspect of the present invention, into a composition comprising at least one further component. In a particular embodiment, the at least one further component is a pharmaceutically acceptable excipient.

[0017] In accordance with the eighth aspect of the present invention, a method for treating a BTLA-related disease in a patient is provided, comprising the step of administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment of the first aspect of the present invention or a pharmaceutical composition of the sixth aspect of the present invention. [Brief explanation of the drawing]

[0018] [Figure 1] Binding of antibodies to soluble and cell-expressed human and cynomolgus monkey BTLA. (a) Surface plasmon resonance (SPR) binding curves for the extracellular domain of soluble monomeric human BTLA injected at increasing concentrations onto fixed anti-BTLA antibodies; the graph shows the SPR signal after reference and blank subtraction. (b) Association and dissociation rates for binding to human or cynomolgus monkey BTLA, calculated by curve fitting using BiaEvaluation software. (c) Binding of antibody 2.8.6 to human BTLA or cynomolgus monkey BTLA-expressing Jurkat cell lines, compared to isotype control antibodies. (d) EC50 for antibody binding to transfection cell lines, calculated by nonlinear curve fitting using GraphPad Prism software. [Figure 2] (a) Blockade of ligand binding by anti-BTLA antibody was evaluated by SPR. Human BTLA extracellular domains were immobilized on a sensor chip. Human HVEM was injected to confirm binding, and then completely dissociated. Next, a saturated concentration of anti-BTLA antibody was injected, followed immediately by a second injection of HVEM. (b) Equilibrium binding of HVEM after antibody injection was expressed as a percentage of HVEM binding before antibody injection. BTLA saturation in clone 11.5.1 blocked subsequent ligand binding, but not in clone 2.8.6. [Figure 3]Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfected with a BTLA construct in a nicistronic vector also expressing GFP were stained with Pacific blue-conjugated anti-BTLA antibody. Clone 11.5.1 binds to cells transfected with the wild-type receptor (top), but not to cells transfected with BTLA containing the Y39R mutation (bottom). (b) For clones 2.8.6 and 11.5.1, binding to each BTLA mutant construct is expressed as the percentage of binding to wild-type BTLA. (c) Mutations Y39R and K41E, which selectively exclude binding of clone 11.5.1, are mapped onto the crystal structure of human BTLA (black residues). Residues crucial for ligand HVEM binding are highlighted in gray. [Figure 4] (a) Crystal structure of the human BTLA extracellular domain complexed with the Fab' fragment of clone 2.8.6. Residues on BTLA embedded at the interface are highlighted in black. (b) The epitope of antibody 2.8.6 is shown in relation to the HVEM binding site (gray residue) (black residue). [Figure 5] (a) Strategy for generating chimeric BTLA genes in humanized BTLA mice. A human genomic DNA section from the start of exon 2 to the end of exon 3 was inserted into the mouse gene locus, replacing the mouse sequence from the start of exon 2 to the end of exon 4. The sequences of the exon-intron junctions at the start of mouse exon 2 and the end of mouse exon 4 were preserved to ensure proper splicing. [Figure 6](a) Protocol for a T cell transfer assay to evaluate anti-BTLA antibodies in vivo. A mixture of humanized and wild-type OVA-specific CD4 T cells was injected into recipient mice. The following day, the mice were immunized with ovalbumin in alum to activate the transferred cells, and 24 hours later, anti-human BTLA antibody or isotype control was administered. Eight days after the initial cell transfer, the ratio of humanized to wild-type cells in the transfer population in the spleen was assessed by flow cytometry. (b) Clones 11.5.1 and, to a lower degree, 2.8.6 both reduced the proliferation of humanized cells compared to wild-type. The graph shows pooled data from two (11.5.1) or three (2.8.6) replicate experiments. [Figure 7] Effect of anti-BTLA clone 2.8.6 on CD4 T cell proliferation in an in vitro mixed lymphocyte reaction. Humanized C57BL / 6 mouse T cells were stained with CellTraceViolet and added to mitomycin C-treated Balb / c-stimulated cells in the presence of anti-BTLA antibody or isotype control. After 96 hours, the proliferation of humanized CD4 cells was evaluated and normalized to proliferation in the absence of the antibody. Clone 2.8.6 inhibited the proliferation of humanized cells with an IC50 of 0.029 nM and had the greatest effect of inhibiting proliferation by 42%. [Figure 8](a) Effects of clone 2.8.6 in a T-cell colitis model. RAG knockout recipient mice were injected with CD45RBhiCD25-CD4+ T cells derived from humanized BTLA mice and treated with 200 μg of 2.8.6 or isotype control antibody on days 7, 21, and 35. Isotype control treated mice progressively lost weight from 3 weeks onward, while 2.8.6 treated mice were spared. (b) Eight weeks after cell transfer, the colon was processed to extract lamina propria lymphocytes, and the total number of inflammatory cells extracted per colon was calculated. Isotype control treated mice had significantly more infiltrating immune cells than 2.8.6 treated mice. (c) The colon weight-to-length ratio was calculated as a marker of inflammation and thickening. 2.8.6 treatment prevented the increase in weight-to-length ratio seen in isotype control treated mice. [Figure 9] (a) Effects of BTLA antibodies in a parent-to-F1 model of GVHD. C57BL / 6 spleen cells and bone marrow cells derived from humanized BTLA mice were injected into CB6F1 recipient mice and subsequently treated with anti-BTLA antibodies or isotype controls. Untreated mice developed clinical GVHD with progressive weight loss, dermatitis, and diarrhea and were culled upon reaching a pre-defined humane endpoint. Mice treated with 2.8.6 and 11.5.1 antibodies were relatively well-preserved, and their survival was comparable to control mice reconstituted with syngeneic cells. (b) Five weeks after cell transfer, mice were culled, and the colon weight-to-length ratio was calculated as a marker of enteritis. Treatments 2.8.6 and 11.5.1 prevented colonic thickening observed in untreated mice. [Figure 10] (a) Effect of D265A mutant clone 11.5.1 in in vivo T cell transfer assay. This mutant antibody, which does not bind to the Fc receptor, no longer inhibited the proliferation of humanized BTLA cells; instead, it led to increased proliferation due to receptor blockade. (b) The D265A mutant 11.5.1 antibody no longer inhibited T cell proliferation in the mixed lymphocyte reaction. [Figure 11]Anti-BTLA antibodies do not fix complement. Splenocytes from humanized BTLA mice were incubated at 37°C for 1 hour in the presence of 10% rabbit complement, 20 μg / ml BTLA antibody, and an isotype control or positive control (depleting CD20 antibody). Anti-CD20 antibody depleted most B cells and confirmed rabbit complement activity, but BTLA antibody did not deplete either of these populations, even though both B and T cell populations stained positively for BTLA. [Figure 12] Anti-BTLA antibodies do not induce antibody-dependent cell-mediated cytotoxicity. Splenocytes from humanized BTLA mice were incubated at 37°C for 24 hours in the presence of 20 μg / ml BTLA antibody, isotype control, or positive control (depleting CD20 antibody). Anti-CD20 antibody depleted the majority of B cells by inducing ADCC by effector cells in the mixture, while BTLA antibody did not deplete either of these populations, even though both B and T cell populations stained positively for BTLA. [Figure 13] Anti-BTLA antibodies do not deplete B or T cells in vivo. Humanized BTLA mice were injected with 200 μg of 2.8.6 antibody. After 24 hours, the spleen and bone marrow were collected, and cell populations were evaluated by flow cytometry. 2.8.6 did not deplete B or T cells in the spleen, nor did it affect the frequency of different B cell precursor populations in the bone marrow. [Figure 14] BTLA expression levels on humanized mouse B cells or CD4+ T cells after 6 days of in vivo incubation with antibody 2.8.6 or 11.5.1, compared to BTLA expression on mouse-derived cells injected with isotype control antibody. [Modes for carrying out the invention]

[0019] The inventors have identified a particularly potent agonist antibody against BTLA that is more effective than current antibodies in suppressing T cell responses and is therefore expected to be particularly useful in the treatment of immune-mediated disorders.

[0020] In this specification and the accompanying claims, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to "a molecule" may optionally include two or more such molecules.

[0021] In this specification, whenever an aspect is described with the term "contains," it is understood that aspects described in the terms "consisting of" and / or "essentially becoming from," or otherwise similar aspects, are also provided.

[0022] Unless the context clearly indicates otherwise, it should be understood that one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect. Furthermore, the various embodiments may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other aspects of the invention are further described by the following detailed description.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. For example, see Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition. 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000. Oxford University Press provides to those skilled in the art a dictionary of many of the terms used in this disclosure.

[0024] The term “approximately” in this specification refers to the normal range of error for each value, as readily known to those skilled in the art. References to “approximately” values ​​or parameters in this specification include (and are described) in a manner directed toward the value or parameter itself.

[0025] In this specification, amino acids may be referred to by either a commonly known three-letter or one-letter code as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by their commonly recognized one-letter code.

[0026] The numbering of amino acids in the variable domain, CDR, and framework region (FR) of antibodies is as follows, unless otherwise indicated: Kabat et al. Sequences of Proteins. We will follow the Kabat definition as presented in *of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0027] The terms “B and T lymphocyte attenuation factor” and “BTLA” are used interchangeably and refer to either the protein or the gene (or other nucleic acid encoding all or part of BTLA) unless otherwise indicated by context. Human BTLA sequences include all human isotypes and variants. A representative example of full-length human BTLA is disclosed in Genbank under deposit number: AJ717664.1. Another representative polypeptide sequence of human BTLA is disclosed in SEQ ID NO: 23, which differs from that of AJ717664.1 only by two native variant single nucleotide polymorphisms. Despite allelic mutations, human BTLA polypeptide sequences will typically have at least 90% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) to the human BTLA of SEQ ID NO: 23.

[0028] A representative example of a full-length cynomolgus (cyno) BTLA is disclosed in Genbank under deposit number XP_005548224. The reference polypeptide sequence of the cyno BTLA is disclosed in SEQ ID NO: 24. A cyno BTLA polypeptide sequence will typically have at least 90% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) to a cyno BTLA such as the one disclosed in SEQ ID NO: 24.

[0029] The terms and sequence identity are well known in the art. For the purposes of this invention, when determining whether a target sequence satisfies defined constraints (e.g., 90% identity), the BLAST (Basic local alignment search tool) algorithm (Altschul et al. J Mol Biol 215) is used. If identified in this way using either 403-410, 1990 or the Smith-Waterman algorithm (see Smith and Waterman. J Mol. Biol. 147:195-197, 1981), it is considered to satisfy the defined restrictions.

[0030] Antibodies and antigen-binding fragments of antibodies An antibody is an immunoglobulin molecule capable of specifically binding to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen-recognition site located within the variable domain of the immunoglobulin molecule. In particular, as used herein, the term “antibody” includes undamaged polyclonal antibodies, undamaged monoclonal antibodies, multispecific antibodies generated from at least two undamaged antibodies, such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecules containing an antigen-recognition site, insofar as the antibody exhibits the desired biological activity.

[0031] In this specification, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen and contains an FcR binding site which may or may not be functional. In this specification, a BTLA agonist antibody (or antibody fragment) refers to an antibody (or antibody fragment) that binds to BTLA and enhances co-inhibitory signaling to T and / or B cells.

[0032] An antigen-binding site refers to a portion of a molecule that binds to all or part of a target antigen. In an antibody molecule, this site may also be called the antibody-antigen-binding site and includes a portion of the antibody that specifically binds to all or part of the target antigen. If the antigen is large, the antibody may bind to only a specific portion of the antigen; this portion is called an epitope. The antibody-antigen-binding site may be provided by one or more antibody variable domains. Preferably, the antibody-antigen-binding site includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0033] The present invention also includes antibody fragments containing antigen-binding sites. Therefore, when referring to antibodies, the term “the antigen-binding fragment” refers to antibody fragments, such as Fab, Fab', F(ab')2, diabody, Fv fragments, and single-stranded Fv(scFv) mutants, that possess an antigen-recognition site and thus possess the ability to bind to an antigen.

[0034] Antigen-binding immunoglobulin (antibody) fragments are well known in the art. Such fragments do not need to have a functional Fc receptor binding site. In this specification, the terms “antibody fragment molecule of the present invention,” “antibody fragment,” and “antigen-binding fragment thereof” are interchangeable.

[0035] In this specification, the term "BTLA-binding molecule" refers to both an antibody capable of binding to BTLA and its antigen-binding fragment. There are five major classes (i.e., isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are denoted as alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Unless otherwise indicated by contextual constraints, the antibodies of the present invention may be derived from one of these classes or subclasses of antibodies. The heavy chain constant domains corresponding to different classes of antibodies are typically denoted by the corresponding lowercase Greek letters, α, δ, ε, γ, and μ, respectively. The light chains of antibodies derived from vertebrate species can be assigned to one of two distinct types, referred to as kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain.

[0036] "Natural antibodies" are typically heterotetrameric Y-glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide links varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form interfaces between the light and heavy chain variable domains. Each heavy chain contains one variable domain (VH) and a constant domain, and the constant domain is C in the case of IgG, IgA, and IgD antibodies. H 1, C H 2, and C H It includes three domains referred to as 3 (IgM and IgE are the fourth domain, C H (Having 4). In the IgG, IgA, and IgD classes, C H 1 and C HThe two domains are separated by a flexible hinge region, which is a proline and cysteine-rich segment of varying lengths (approximately 10 to 60 amino acids in various IgG subclasses). Both the light and heavy chain variable domains are linked to the constant domain by a "J" region of approximately 12 or more amino acids, and the heavy chain also has a "D" region of approximately 10 additional amino acids. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues. The heavy chain variable region (VH) and light chain variable region (VL) can each be further divided into a hypervariable region called CDR, interspersed with more conserved regions called framework regions (FR). Each VH and VL contains three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, such as diverse cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0037] The antibodies or antigen-binding fragments of the present invention may be derived from any animal species, including mice, rats, humans, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody or antigen-binding antibody fragment is monoclonal, for example, a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or its antigen-binding fragment. Non-human antibodies or antigen-binding fragments may be humanized by recombinant methods to reduce their immunogenicity in humans.

[0038] The antibody or its antigen-binding fragment of the present invention may be identified using known methods. For example, the antigen-binding portion may be selected using phage display or other antigen-binding selection or panning approaches. These antigen-binding portions may then be incorporated into the antibody framework (for example, by fusing them to the constant and hinge regions of an IgG1 or IgG4 molecule).

[0039] The term “monoclonal antibody” (“mAb”), as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting the population are identical except for possible mutations, such as naturally occurring mutations that may exist in small amounts. Therefore, the modifying phrase “monoclonal” indicates the characteristic of an antibody or a fragment of an antibody or antigen-binding fragment that is not a mixture of distinct antibodies or antigen-binding fragments. mAbs are highly specific and target a single antigenic site / epitope. It is directed in response.

[0040] mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art. For example, a monoclonal antibody or its antigen-binding fragment according to the present invention may be produced by the hybridoma method first described by Kohler and Milstein (Nature 256:495, 1975), or by the recombinant DNA method described, for example, U.S. Patents 4,816,567 and 6,331,415. Alternatively, the "monoclonal antibody" may be isolated from a phage antibody library using, for example, the techniques described by Clackson et al., Nature 1991;352:624-628 and Marks et al., J. Mol. Biol. 1991;222:581-597.

[0041] The term monoclonal may also be considered an antigen-binding fragment of the antibody of the present invention. This simply means that the molecule is produced or exists in a single clonal form. A “human” antibody (HumAb) refers to an antibody in which both the framework and CDR region have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo). However, the term “human antibody” as used herein is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence.

[0042] Human antibodies may also be prepared by administering an immunogen / antigen to transgenic animals, such as immunoxenomouses, that are modified to produce undamaged human antibodies or undamaged antibodies containing human variable regions in response to antigen exposure, but whose endogenous loci have been rendered inoperable (see, for example, U.S. Patents 6,075,181 and 6,150,584, for the XENOMOUSE® technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced through human B-cell hybridoma technology. These animals typically contain all or part of the human immunoglobulin locus, which either replaces the endogenous immunoglobulin locus, is located extrachromosomally, or is randomly incorporated into the animal's chromosomes. In these transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also, see, for example, XENOMOUSE. TM U.S. Patents 6,075,181 and 6,150,584 describing the technology; HUMAB TMU.S. Patent No. 5,770,429 describing the technology; K-M MOUSE TM U.S. Patent No. 7,041,870 describing the technology, and VELOCIMOUSE TM See U.S. Patent Application Publication No. US2007 / 0061900 describing the technology. The human variable regions derived from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0043] Also, human antibodies may be produced by methods based on hybridomas. Human myeloma and mouse - human heteromyeloma cell lines for producing human monoclonal antibodies have been described (e.g., Kozbor J. Immunol, 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51 - 63(Marcel Dekker, Inc., New York, 1987 ); and see Boerner et al., J. Immunol., 147:86(1991)). Human antibodies generated through human B - cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557 - 3562(2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26:265 - 268(2006) (describing human - human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20:927 - 937(2005), as well as Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27:185 - 91(2005).

[0044] The terms "human" antibody and "fully human" antibody are used synonymously. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In this specification, “humanized antibody” refers to an antibody in which some, most, or all of the amino acids outside the CDR of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's CDR are replaced with residues derived from a non-human species (donor antibody), e.g., mouse, rat, or rabbit CDR, which have the desired specificity, affinity, and ability. The humanized antibody may also include residues that are not found in either the recipient antibody or the transferred CDR or framework sequence, but are included to further improve and optimize antibody performance. In one embodiment of the humanized form of Ab, some, most, or all of the amino acids outside the CDR are replaced with amino acids derived from human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids may be permissible as long as they do not impede the antibody’s ability to bind to a particular antigen. Humanized antibodies retain antigen specificity similar to that of the original antibody. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are those of the human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulins. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0045] In this specification, “modified antibody” refers to an antibody, which may be a humanized antibody, in which specific residues have been substituted with others in order to reduce undesirable effects or properties. Such substitutions may be within the CD domain. For example, as described herein (see Example 21), the CDRH2 of humanized antibody 3E8 was modified with an N57Q substitution to remove deamidation potential and with a K63S substitution to reduce expected immunogenicity.

[0046] A “chimeric antibody” refers to an antibody in which the variable region originates from one species and the constant region originates from another species, for example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody, or vice versa. The term also includes antibodies that contain a V region from one individual of one species (e.g., the first mouse) and a constant region from another individual of the same species (e.g., the second mouse). The term “antigen (Ag)” refers to a molecular entity used in the immunization of immunocompetent vertebrates to produce an antibody (Ab) that recognizes Ag or to screen an expression library (e.g., phage, yeast, or ribosome display library). In this specification, Ag is used more broadly and is generally intended to include a target molecule that is specifically recognized by Ab, and therefore also includes a molecular part or mimetic used in the immunization process for producing Ab or in library screening for selecting Ab.

[0047] A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. Traditionally, the recombinant production of bispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature). 305:537-539 (1983). Methods for producing bispecific antibodies are within the realm of the art. For example, bispecific antibodies may be produced by a variety of methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai et al., (1990) Clin. Exp. Immunol. 79:315-321 and Kostelny et al., (1992) J Immunol. 148:1547-1553. Furthermore, bispecific antibodies may be formed as "Diabody" (Holliger et al., (1993) PNAS USA 90:6444-6448) or as "Janusin" (Traunecker et al., (1991) EMBO J. 10:3655-3659 and Traunecker et al., (1992) Int. J. Cancer Suppl. 7:51-52).

[0048] Generally, the term “epitope” refers to a region or site of an antigen to which an antibody specifically binds, i.e., a region or site that is in physical contact with the antibody. Therefore, the term “epitope” refers to one or more parts of a molecule in the antigen-binding region of an antibody that are recognized by and can be bound to by the antibody. Typically, an epitope is defined in the context of molecular interactions between an “antibody, or its antigen-binding portion (Ab)” and its corresponding antigen. Epitopes often consist of molecular surfaces such as amino acids or sugar side chains and possess specific three-dimensional structural and charge properties. In some embodiments, epitopes may be protein epitopes. Protein epitopes may be linear or conformational. In linear epitopes, all interaction points between the protein and the interacting molecule (e.g., antibody) occur linearly along the primary amino acid sequence of the protein. A “nonlinear epitope” or “conformation epitope” contains a discontinuous polypeptide (or amino acid) within an antigenic protein to which an antibody specific to the epitope binds. The term “antigenic epitope” is defined herein as a portion of an antigen to which an antibody can specifically bind, as determined by any method known in the art, for example, by conventional immunoassay.

[0049] The term "specifically binding" to an epitope is well understood in the art, and the methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell, protein, or substance more frequently, more quickly, for a longer period of time, and / or with higher affinity than it does with another cell, protein, or substance.

[0050] A variety of assay formats may be used to select antibodies or peptides that specifically bind to molecules of interest. For example, many assays that can be used to identify antibodies that specifically react with an antigen or receptor, or their ligand-binding moiety that specifically binds to a homologous ligand or binding partner, include solid-phase ELISA immunoassays, immunoprecipitation, and Biacore. TM (GE Healthcare, Piscataway, New Jersey), KinExA, Fluorescence-Activated Cell Sorting (FACS), Octet TM (ForteBio, Inc., Menlo Park, California) and Western blot analysis are available. Typically, specific or selective reactions will be at least twice the background signal or noise, more typically more than 10 times the background, more typically more than 50 times the background, more typically more than 100 times the background, more typically more than 500 times the background, more typically more typically more than 1000 times the background, more typically more typically more than 1000 times the background, and more typically more typically more than 10,000 times the background. Also, the antibody has an equilibrium dissociation constant (K, as interchangeably used herein). D If the KD (or KD) is <7nM, it is said to "specifically bind" to the antigen.

[0051] In accordance with a first aspect of the present invention, we provide an isolated antibody or its antigen-binding fragment that specifically binds to BTLA, wherein the antibody has a heavy chain and / or light chain comprising at least one CDR, and the antibody or its antigen-binding fragment is selected from the group consisting of 11.5.1, 2.8.6, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, 6.2, and 831, as disclosed in Table 1 and described herein. In one embodiment, the antibody or its antigen-binding fragment competes with its innate ligand HVEM for binding to BTLA. In another embodiment, the antibody or its antigen-binding fragment does not interfere with the binding of HVEM.

[0052] In a further embodiment, an isolated antibody that binds to human BTLA is selected from the group consisting of 11.5.1 and 2.8.6, and the antibody specifically binds to BTLA and induces signal transduction through the receptor.

[0053] Any antibody or its antigen-binding fragment (whether mouse, humanized, or humanized / manipulated) is defined by an antibody selected from the group consisting of 11.5.1, 2.8.6, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, 6.2, and 831, derived from any of the antibodies disclosed in Table 1, including, for example, VH CDR1, 2, and 3, or VL CDR1, 2, and 3, as well as VL CDR1, 2, and 3.

[0054] In accordance with a modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA is provided, comprising 0 to 3 amino acid modifications, e.g., 0, 1, 2, or 3 amino acid modifications, and comprising at least one VH CDR having an amino acid sequence as shown in any of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In certain embodiments, the amino acid modifications include, but are not limited to, amino acid substitutions, additions, deletions, or chemical modifications, which do not preclude the antibody-binding affinity or T-cell inhibitory effect of the modified amino acid sequence compared to the unmodified amino acid sequence.

[0055] According to a modification of the first aspect of the present invention, the amino acid sequence comprises 0 to 3 amino acid modifications and has an amino acid sequence as shown in any of SEQ ID NO: 193, SEQ ID NO: 194, or SEQ ID NO: 195. The present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA and contains at least one VH CDR.

[0056] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has an amino acid sequence as shown in SEQ ID NO: 7, CDRH2 has an amino acid sequence as shown in SEQ ID NO: 8, and CDRH3 has an amino acid sequence as shown in SEQ ID NO: 9 is provided.

[0057] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has an amino acid sequence as shown in SEQ ID NO: 193, CDRH2 has an amino acid sequence as shown in SEQ ID NO: 194, and CDRH3 has an amino acid sequence as shown in SEQ ID NO: 195 is provided.

[0058] A modification of the first aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising 0 to 3 amino acid modifications and at least one VL CDR having an amino acid sequence such as that shown in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0059] A modification of the first aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising 0 to 3 amino acid modifications and at least one VL CDR having an amino acid sequence such as that shown in SEQ ID NO: 196, SEQ ID NO: 197, or SEQ ID NO: 12.

[0060] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has an amino acid sequence as shown in SEQ ID NO: 10, CDRL2 has an amino acid sequence as shown in SEQ ID NO: 11, and CDRL3 has an amino acid sequence as shown in SEQ ID NO: 12 is provided.

[0061] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has an amino acid sequence as shown in SEQ ID NO: 196, CDRL2 has an amino acid sequence as shown in SEQ ID NO: 197, and CDRL3 has an amino acid sequence as shown in SEQ ID NO: 12 is provided.

[0062] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, with CDRH1 having the amino acid sequence shown in SEQ ID NO: 7, CDRH2 having the amino acid sequence shown in SEQ ID NO: 8, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, with CDRL1 having the amino acid sequence shown in SEQ ID NO: 10, CDRL2 having the amino acid sequence shown in SEQ ID NO: 11, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 12.

[0063] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, with CDRH1 having an amino acid sequence as shown in SEQ ID NO: 193, CDRH2 having an amino acid sequence as shown in SEQ ID NO: 194, and CDRH3 having an amino acid sequence as shown in SEQ ID NO: 195, and the light chain comprises a heavy chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, with CDRL1 having an amino acid sequence as shown in SEQ ID NO: 196, CDRL2 having an amino acid sequence as shown in SEQ ID NO: 197, and CDRL3 having an amino acid sequence as shown in SEQ ID NO: 12.

[0064] According to a modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA is provided, comprising 0 to 3 amino acid modifications and at least one VH CDR having an amino acid sequence such as that shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0065] According to another variation of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA is provided, comprising 0 to 3 amino acid modifications and at least one VH CDR having an amino acid sequence such as that shown in SEQ ID NO: 199, SEQ ID NO: 120, or SEQ ID NO: 201.

[0066] In accordance with a modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has the amino acid sequence shown in SEQ ID NO: 1, CDRH2 has the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 has the amino acid sequence shown in SEQ ID NO: 3 is provided.

[0067] In accordance with a modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has an amino acid sequence as shown in SEQ ID NO: 199, CDRH2 has an amino acid sequence as shown in SEQ ID NO: 200, and CDRH3 has an amino acid sequence as shown in SEQ ID NO: 201 is provided.

[0068] A modification of the first aspect of the present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VL CDR having an amino acid sequence such as that shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0069] A modification of the first aspect of the present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VL CDR having an amino acid sequence such as that shown in SEQ ID NO: 202, SEQ ID NO: 203, or SEQ ID NO: 6.

[0070] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has an amino acid sequence as shown in SEQ ID NO: 4, CDRL2 has an amino acid sequence as shown in SEQ ID NO: 5, and CDRL3 has an amino acid sequence as shown in SEQ ID NO: 6 is provided.

[0071] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has an amino acid sequence as shown in SEQ ID NO: 202, CDRL2 has an amino acid sequence as shown in SEQ ID NO: 203, and CDRL3 has an amino acid sequence as shown in SEQ ID NO: 6 is provided.

[0072] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, with CDRH1 having the amino acid sequence shown in SEQ ID NO: 1, CDRH2 having the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, with CDRL1 having the amino acid sequence shown in SEQ ID NO: 4, CDRL2 having the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 6, is provided.

[0073] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, with CDRH1 having an amino acid sequence as shown in SEQ ID NO: 199, CDRH2 having an amino acid sequence as shown in SEQ ID NO: 200, and CDRH3 having an amino acid sequence as shown in SEQ ID NO: 201, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, with CDRL1 having an amino acid sequence as shown in SEQ ID NO: 202, CDRL2 having an amino acid sequence as shown in SEQ ID NO: 203, and CDRL3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0074] According to another variation of the first aspect of the present invention, (1) SEQ ID NOs: 31, 32, or 33, including 0-3 amino acid modifications; (2) SEQ ID NOs: 37, 38, or 39, including 0-3 amino acid modifications; (3) SEQ ID NOs: 43, 44, or 45, including 0-3 amino acid modifications; (4) SEQ ID NOs: 43, 56, or 57, including 0-3 amino acid modifications; (5) SEQ ID NOs: 61, 62, or 63, including 0-3 amino acid modifications; (6) SEQ ID NOs: 31, 32, or 69, including 0-3 amino acid modifications; (7) SEQ ID NOs: 0-3 amino SEQ ID NOs. 73, 74, or 75, including acid modification; (8) SEQ ID NOs. 79, 80, or 81, including 0-3 amino acid modification; (9) SEQ ID NOs. 85, 86, or 87, including 0-3 amino acid modification; (10) SEQ ID NOs. 61, 92, or 93, including 0-3 amino acid modification; (11) SEQ ID NOs. 97, 98, or 99, including 0-3 amino acid modification; (12) SEQ ID NOs. 103, 104, or 105, including 0-3 amino acid modification; (13) SEQ ID NOs. 109, 110, or 111, including 0-3 amino acid modification. ;(14)Sequence IDs 85, 110, or 117, containing 0-3 amino acid modifications;(15)Sequence IDs 121, 122, or 123, containing 0-3 amino acid modifications;(16)Sequence IDs 127, 128, or 129, containing 0-3 amino acid modifications;(17)Sequence IDs 133, 134, or 135, containing 0-3 amino acid modifications;(18)Sequence IDs 139, 140, or 141, containing 0-3 amino acid modifications;(19)Sequence IDs 145, 146, or 147, containing 0-3 amino acid modifications;(20)0-3 SEQ ID NOs: 31, 32, or 33, including amino acid modifications; (21) SEQ ID NOs: 31, 32, or 159, including amino acid modifications of 0-3; (22) SEQ ID NOs: 169, 170, or 171, including amino acid modifications of 0-3; (23) SEQ ID NOs: 61, 62, or 63, including amino acid modifications of 0-3; (24) SEQ ID NOs: 31, 182, or 183, including amino acid modifications of 0-3; (25) SEQ ID NOs: 187, 188, or 189, including amino acid modifications of 0-3; (26) SEQ ID NOs: 193, including amino acid modifications of 0-3 The present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NOs: 199, 200, or 201, which includes the amino acid modification of 0-3; SEQ ID NOs: 205, 206, or 207, which includes the amino acid modification of 0-3; SEQ ID NOs: 211, 212, or 213, which includes the amino acid modification of 0-3; SEQ ID NOs: 127, 386, or 129, which includes the amino acid modification of 0-3; SEQ ID NOs: 205, 206, or 207, which includes the amino acid modification of 0-3; SEQ ID NOs: 127, 388, or 129, which includes the amino acid modification of 0-3; or (35) SEQ ID NOs: 205, 387, or 207, which includes the amino acid modification of 0-3.

[0075] According to a modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1, CDRH2, and CDRH3 correspond to (1) SEQ ID NOs. 31, 32, and 33, respectively; (2) SEQ ID NOs. 37, 38, and 39, respectively; (3) SEQ ID NOs. 43, 44, and 45, respectively; (4) SEQ ID NOs. 43 and 56, respectively. , and SEQ ID NO: 57; (5) SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively; (6) SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 69, respectively; (7) SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; (8) SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; (9) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively; (10) SEQ ID NO: 61, SEQ ID NO: 92, and SEQ ID NO: 93, respectively; (11) SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (12) Each (13) SEQ ID NOs. 103, 104, and 105; (14) SEQ ID NOs. 109, 110, and 111, respectively; (15) SEQ ID NOs. 85, 110, and 117, respectively; (16) SEQ ID NOs. 127, 128, and 129, respectively; (17) SEQ ID NOs. 133, 134, and 135, respectively; (18) SEQ ID NOs. 139, 140, and 141, respectively. (19) SEQ ID NOs. 145, 146, and 147, respectively; (20) SEQ ID NOs. 31, 32, and 33, respectively; (21) SEQ ID NOs. 31, 32, and 159, respectively; (22) SEQ ID NOs. 169, 170, and 171, respectively; (23) SEQ ID NOs. 61, 62, and 63, respectively; (24) SEQ ID NOs. 31, 182, and 183, respectively; (25) SEQ ID NOs. 187, 188, and 189, respectively;(26) The antibody or its antigen-binding fragment is provided, having an amino acid sequence as shown in SEQ ID NOs. 193, 194, and 195, respectively; (27) SEQ ID NOs. 199, 200, and 201, respectively; (28) SEQ ID NOs. 205, 206, and 207, respectively; (29) SEQ ID NOs. 211, 212, and 213, respectively; (30) SEQ ID NOs. 127, 386, and 129, respectively; (31) SEQ ID NOs. 205, 206, and 207, respectively; (32) SEQ ID NOs. 127, 386, and 129, respectively; or (33) SEQ ID NOs. 205, 387, and 207, respectively, and in any CDR / SEQ ID NO, 0 to 3 amino acid modifications may be present.

[0076] According to a variation of the first aspect of the present invention, (1) Sequence IDs 34, 35, or 36; (2) Sequence IDs 40, 41, or 42; (3) Sequence IDs 46, 47, or 48; (4) Sequence IDs 58, 59, or 60; (5) Sequence IDs 64, 65, or 66; (6) Sequence IDs 34, 35, or 72; (7) Sequence IDs 76, 47, or 78; (8) Sequence IDs 82, 83, or 84; (9) Sequence IDs 88, 89, or 90; (10) Sequence IDs 94, 95, or 96; (11) Sequence IDs 100, 101, or 102; (12) Sequence IDs Numbers 64, 107, or 108; (13) Sequence numbers 88, 89, or 114; (14) Sequence numbers 124, 125, or 126; (15) Sequence numbers 34, 35, or 36; (16) Sequence numbers 136, 137, or 138; (17) Sequence numbers 142, 143, or 144; (18) Sequence numbers 148, 149, or 150; (19) Sequence numbers 136, 137, or 162; (20) Sequence numbers 34, 35, or 36; (21) Sequence numbers 172, 173, or 174; (22) Sequence numbers 64, 65, The present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VL CDR containing an amino acid sequence as shown in (23) SEQ ID NOs: 136, 137, or 186; (24) SEQ ID NOs: 190, 191, or 192; (25) SEQ ID NOs: 196, 197, or 12; (26) SEQ ID NOs: 202, 203, or 6; (27) SEQ ID NOs: 142, 209, or 210; (28) SEQ ID NOs: 214, 35, or 216; (29) SEQ ID NOs: 10, 11, or 12; (30) SEQ ID NOs: 4, 5, or 6; or (31) SEQ ID NOs: 142, 143, or 210, wherein any CDR / SEQ ID NO may contain 0 to 3 amino acid modifications.

[0077] According to another variation of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1, CDRL2, and CDRL3 are (1) SEQ ID NOs. 34, 35, and 36, respectively; (2) SEQ ID NOs. 40, 41, and 42, respectively; (3) SEQ ID NOs. 46, 47, and 48, respectively; (4) SEQ ID NOs. 58 and 5, respectively. 9, and SEQ ID NO: 60; (5) SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; (6) SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 72, respectively; (7) SEQ ID NO: 76, SEQ ID NO: 47, and SEQ ID NO: 78, respectively; (8) SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; (9) SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively; (10) SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; (11) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively; (12 )Sequences 64, 107, and 108 respectively; (13)Sequences 88, 89, and 114 respectively; (14)Sequences 124, 125, and 126 respectively; (15)Sequences 34, 35, and 36 respectively; (16)Sequences 136, 137, and 138 respectively; (17)Sequences 142, 143, and 144 respectively; (18)Sequences 148, 149, and 150 respectively; ( 19) SEQ ID NOs. 136, 137, and 162, respectively; (20) SEQ ID NOs. 34, 35, and 36, respectively; (21) SEQ ID NOs. 172, 173, and 174, respectively; (22) SEQ ID NOs. 64, 65, and 180, respectively; (23) SEQ ID NOs. 136, 137, and 186, respectively; (24) SEQ ID NOs. 190, 191, and 192, respectively; (25) SEQ ID NOs. 196, 197, or 12, respectively;(26) The antibody or its antigen-binding fragment is provided, having an amino acid sequence as shown in SEQ ID NOs. 202, 203, and 6, respectively; (27) SEQ ID NOs. 142, 209, and 210, respectively; (28) SEQ ID NOs. 214, 35, and 216, respectively; (29) SEQ ID NOs. 10, 11, or 12, respectively; (30) SEQ ID NOs. 4, 5, or 6, respectively; or (31) SEQ ID NOs. 142, 143, or 210, respectively, and having 0 to 3 amino acid modifications in any CDR / SEQ ID NO.

[0078] According to another variation of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, and the light chain comprises three CDRs: CDRH1, CDRH2 and CDRH3 DR: Includes a light chain variable region comprising CDRL1, CDRL2, and CDRL3, where (1) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 31, 32, and 33, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 34, 35, and 36, respectively; (2) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 37, 38, and 39, respectively, and CDRL1 (3) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 40, 41, and 42, respectively; (4) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 43, 44, and 45, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 46, 47, and 48, respectively; (5) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 43, 56, and 56, respectively. (7) Having an amino acid sequence as shown in 7, and CDRL1, CDRL2, and CDRL3 having an amino acid sequence as shown in SEQ ID NOs. 58, 59, and 60, respectively; (5) Having an amino acid sequence as shown in SEQ ID NOs. 61, 62, and 63, respectively, and CDRL1, CDRL2, and CDRL3 having an amino acid sequence as shown in SEQ ID NOs. 64, 65, and 66, respectively; (6) Having an amino acid sequence as shown in SEQ ID NOs. 64, 65, and 66, respectively; (7) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 31, 32, and 69, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 34, 35, and 72, respectively; (8) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 73, 74, and 75, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 76, 47, and 78, respectively;(8) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 79, 80, and 81, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 82, 83, and 84, respectively; (9) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 85, 86, and 87, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 88, 87, and 84, respectively. (10) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 61, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; (11) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively, and CDRL1, C (12) Whether DRL2 and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 100, 101, and 102, respectively; (13) Whether CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 103, 104, and 105, respectively, and whether CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 64, 107, and 108, respectively; (14) Whether CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 109, (14) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in sequence number 110 and sequence number 111, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 88, sequence number 89, and sequence number 114, respectively; (14) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 85, sequence number 110, and sequence number 117, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 88, sequence number 89, and sequence number 114, respectively;(15) CDRH1, CDRH2, CDRH3 are; (16) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 121, 122, and 123, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 124, 125, and 126, respectively; (16) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 127, 128, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 34, 35, and 36, respectively (17) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 133, 134, and 135, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 136, 137, and 138, respectively; (18) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 139, 140, and 141, respectively, and CDRL1, CDRL 2. CDRL3 has the amino acid sequence shown in SEQ ID NOs. 142, 143, and 144, respectively; (19) CDRH1, CDRH2, and CDRH3 have the amino acid sequence shown in SEQ ID NOs. 145, 146, and 147, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequence shown in SEQ ID NOs. 148, 149, and 150, respectively; (20) CDRH1, CDRH2, and CDRH3 have the amino acid sequence shown in SEQ ID NOs. 31, 144, and 144, respectively. (21) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NO: 32 and SEQ ID NO: 33, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, respectively; (21) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 159, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 162, respectively;(22) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 169, 170, and 171, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 172, 173, and 174, respectively; (23) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 61, 62, and 63, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 64, (24) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 31, sequence number 182, and sequence number 183, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 136, sequence number 137, and sequence number 186, respectively; (25) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 187, sequence number 188, and sequence number 189, respectively, and (26) CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 190, 191, and 192, respectively; (27) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 193, 194, and 195, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 196, 197, and 12, respectively; (28) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 199, (28) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 200 and 201, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 202, 203, and 6, respectively; (28) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 205, 206, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 142, 209, and 210, respectively;(29) CDRH1, CDRH2, and CDRH3 are those; (30) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 211, 212, and 213, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 214, 35, and 216, respectively; (31) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 127, 386, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 34, 35, and 36, respectively; (32) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 205, 206, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 142, 209, and 210, respectively; (32) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 127, 386, and 129, respectively; (33) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NO: 388 and SEQ ID NO: 129, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or (34) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210, respectively; or (34) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 210, respectively; the antibody or its antigen-binding fragment is provided, wherein any CDR / SEQ ID NO: 388 and SEQ ID NO: 129 have amino acid sequences as shown in SEQ ID NO: 388 and SEQ ID NO: 129, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 210, respectively; any CDR / SEQ ID NO: 142 may contain 0 to 3 amino acid modifications.

[0079] According to another variation of the first aspect of the present invention: (1) at least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; (2) at least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42; (3) at least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48; (4) at least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 43, SEQ ID NO: 56, or SEQ ID NO: 57, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60 CDR; (5) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66; (6) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 69, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 72; (7) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 76, SEQ ID NO: 47, or SEQ ID NO: 78; (8) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 81, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84;(9) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; (10) SEQ ID NO: 61; (11) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 92 or SEQ ID NO: 93, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 94, SEQ ID NO: 95, or SEQ ID NO: 96; (12) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 102; (13) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 64, SEQ ID NO: 107, or SEQ ID NO: 108; (14) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 114 CDR; (14) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 85, SEQ ID NO: 110, or SEQ ID NO: 117, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 114; (15) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 124, SEQ ID NO: 125, or SEQ ID NO: 126; (16) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36 CDR; (17) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138; (18) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144; (19) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 145, SEQ ID NO: 146, or SEQ ID NO: 147, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 148, SEQ ID NO: 149, or SEQ ID NO: 150; (20) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 148, SEQ ID NO: 149, or SEQ ID NO: 150 CDR; (21) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 159, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 162; (22) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 169, SEQ ID NO: 170, or SEQ ID NO: 171, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174; (23) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 180; (24) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 182, or SEQ ID NO: 183, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 186;(25) at least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192; (26) at least one VH containing an amino acid sequence as shown in SEQ ID NO: 193, SEQ ID NO: 194, or SEQ ID NO: 195; CDR, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 196, SEQ ID NO: 197, or SEQ ID NO: 12; (27) SEQ ID NO: 199, SEQ ID NO: 2 (28) At least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 00 or SEQ ID NO: 201, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 202, SEQ ID NO: 203, or SEQ ID NO: 6; (29) At least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 142, SEQ ID NO: 209, or SEQ ID NO: 210; (29) At least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 211, SEQ ID NO: 212, or SEQ ID NO: 213, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 214, SEQ ID NO: 35, or SEQ ID NO: 216; (30) At least one VH CDR containing an amino acid sequence such as that shown in SEQ ID NO: 127, SEQ ID NO: 386, or SEQ ID NO: 129, and at least one VL CDR containing an amino acid sequence such as that shown in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36 CDR; (31) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 209, or SEQ ID NO: 210; (32) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 127, SEQ ID NO: 388, or SEQ ID NO: 129, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; (33) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 205, SEQ ID NO: 387, or SEQ ID NO: 207, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 209, or SEQ ID NO: 210; or (34) At least one VH CDR containing an amino acid sequence as shown in SEQ ID NO: 205, SEQ ID NO: 387, or SEQ ID NO: 207, and at least one VL CDR containing an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 210;This provides an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, and in which any CDR / Sequence ID may contain 0 to 3 amino acid modifications.

[0080] In another embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain, which specifically binds to human BTLA, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13, or a sequence having at least 90% sequence identity to said sequence. In another embodiment, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as that shown in SEQ ID NO: 17 or 21.

[0081] With respect to any aspect disclosed herein that refers to at least 90% sequence identity, it is understood that this includes any sequence identity ranging from 90% to 100%, i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.

[0082] In another embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequences shown in SEQ ID NOs: 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 306, 326, 327, 330, 331, 13, 17, 21, 382, ​​384, 389, 390, and 378.

[0083] In another embodiment, the heavy chain variable region contains up to 10 modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, as in SEQ ID NOs: 301, 302, 303, 305, 306, 307, 308, 309, 310, 311 , containing amino acid sequences such as those shown in 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 306, 326, 327, 330, 331, 13, 17, 21, 382, ​​384, 389, 390 and 378.

[0084] In another embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14, or a sequence having at least 90% sequence identity to said sequence. In another embodiment, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as that shown in SEQ ID NO: 15 or 19.

[0085] In another embodiment, the light chain variable region includes an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as those shown in SEQ ID NOs: 351, 352, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 366, 367, 368, 369, 370, 372, 351, 374, 375, 376, 377, 380, 381, 14, 15, 19, 383, 385, or 378.

[0086] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13, and the light chain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 14.

[0087] In accordance with another modification of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 13, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 14.

[0088] In accordance with another variation of the first aspect of the present invention, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising a heavy chain and a light chain, is provided, wherein: (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 17, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 15; or (2) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 21, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 19.

[0089] An isolated antibody or antigen-binding fragment that specifically binds to human BTLA, comprising a heavy chain and a light chain, wherein: (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 301, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 351; (2) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 302, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 352; (3) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence showing the amino acid sequence shown in SEQ ID NO: 303, and the light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 353; (4) the heavy chain comprises the sequence shown in SEQ ID NO: 305 (5) The heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 355, and the light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 306, and the light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 356, and the light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 307, and the light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 357, and the light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence such as the one shown in SEQ ID NO: 308, and the light chain is sequence number (8) The heavy chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in Sequence ID No. 358; (9) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in Sequence ID No. 309, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in Sequence ID No. 359; (10) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in Sequence ID No. 311, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence as shown in Sequence ID No. 361;(11) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 312, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 362; (12) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 313, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 363; (13) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 314, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 364; (14) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 315, (15) The light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 364; (16) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 316, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 366; (17) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 318, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 368;(18) The heavy chain comprises a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 319, and the light chain comprises a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 369; (19) The heavy chain comprises a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 320, and the light chain comprises at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 370; (20) The heavy chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 321, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 370; (21) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 322, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 372; (22) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 324, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 374; (23) The heavy chain includes an amino acid sequence having at least 90% sequence identity to an amino acid sequence as shown in SEQ ID NO: 306 (24) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 375; (25) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 326; (26) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 377; (27) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17; (28) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 15;(27) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 21, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 19; (28) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 330, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 380; (29) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 331, and the light chain includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 381; (30) The heavy chain includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 382, ​​and (31) The light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 383; (32) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 384, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 385; (33) The heavy chain includes a variable heavy chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 389, and the light chain includes a variable light chain region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 383;Alternatively, (34) the antibody or antigen-binding fragment is provided, wherein the heavy chain comprises a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 390, and the light chain comprises a light chain variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 378.

[0090] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 14.

[0091] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 15.

[0092] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 19.

[0093] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 301, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 351.

[0094] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 302, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 352.

[0095] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 303, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 353.

[0096] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 305, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 355.

[0097] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 306, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 356.

[0098] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 307, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 357.

[0099] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 308, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 358.

[0100] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 309, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 359.

[0101] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 310, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 360.

[0102] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 311, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 361.

[0103] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 312, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 362.

[0104] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 313, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 363.

[0105] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 314, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 364.

[0106] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 315, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 364.

[0107] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 316, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 366.

[0108] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 317, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 367.

[0109] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 318, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 368.

[0110] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 319, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 369.

[0111] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 320, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 370.

[0112] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 321, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 370.

[0113] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 322, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 372.

[0114] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 324, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 374.

[0115] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 306, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 375.

[0116] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 326, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 376.

[0117] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 327, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 377.

[0118] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 15.

[0119] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 19.

[0120] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 330, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 380.

[0121] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 331, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 381.

[0122] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 382, ​​and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 383.

[0123] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 384, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 385.

[0124] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 389, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 383.

[0125] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 390, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 385.

[0126] In one embodiment, the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 390, and the light chain variable region includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 378.

[0127] In another embodiment, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 13.

[0128] In another embodiment, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 17.

[0129] In another embodiment, the heavy chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 21.

[0130] In another embodiment, the heavy chain variable region polypeptides are SEQ ID NOs: 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, The sequences disclosed in 315, 316, 317, 318, 319, 320, 321, 322, 324, 306, 326, 327, 330, 331, 382, ​​384, 389, or 390 have at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity.

[0131] In another embodiment, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 14.

[0132] In another embodiment, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 15.

[0133] In another embodiment, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence disclosed in SEQ ID NO: 19.

[0134] In other embodiments, the light chain variable region polypeptide has at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequences disclosed in SEQ ID NOs: 351, 352, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 364, 366, 367, 368, 369, 370, 372, 374, 375, 376, 377, 380, 381, 383, 385, or 378.

[0135] In accordance with another modification of the first aspect of the present invention, an isolated antibody or its antigen-binding fragment is provided, having a primary VH domain, primary VL domain, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any antibody clone as shown in Table 1. In a particular embodiment, what is provided herein is an isolated antibody selected from the antibody clones shown in Table 1.

[0136] Table 1. Exemplary BTLA agonist antibodies

[0137] [Table 1]

[0138] In certain embodiments, the heavy or light chain also includes a constant region. If the molecule is a full-length IgG antibody molecule, the heavy chain may include three constant domains. In a particular embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA may be up to approximately 10 x 10 -9 K regarding the binding of M to human BTLA D This shows that, in a particular embodiment, an isolated antibody or its antigen-binding fragment that specifically binds to human BTLA is up to approximately 4 x 10⁶. -9 K regarding the binding of M to human BTLA D This shows that, in a particular embodiment, the isolated antibody or its antigen-binding fragment that specifically binds to human BTLA is up to approximately 1x1 0 -9 K regarding the binding of M to human BTLA D This indicates.

[0139] In certain embodiments, the isolated antibody of the present invention (e.g., a humanized antibody) is approximately 10 nM (1 x 10⁻¹⁶) at 37°C. -8 M) not exceeding; appropriately, K not exceeding approximately 1 nM D It binds to human BTLA; more appropriately, the antibody is at approximately 500 pM (5x10) at 37°C. -10 M), 200 pM, 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or K not exceeding 2 pM. D This refers to a mode that has a value. In this context, the term "approximately" means + / - 10%.

[0140] In certain embodiments, the isolated antibody of the present invention (e.g., a humanized antibody) is subjected to at least 1.0 x 10⁻¹⁶ temperatures at 37°C. 5 It binds to human BTLA at an on-rate of (1 / Ms). In certain embodiments, the isolated antibody of the present invention (e.g., a humanized antibody) is converted to at least 2.0 x 10⁻¹⁶ at 37°C. 5 (1 / Ms), 3.0x10 5 (1 / Ms), 4.0x10 5 (1 / Ms), 5.0x10 5 (1 / Ms), 6.0x10 5 (1 / Ms), or 7.0x10 5It binds to human BTLA at an on-rate of (1 / Ms).

[0141] In a particular embodiment, the isolated antibody of the present invention (e.g., a humanized antibody) is converted to 1.0 x 10⁻¹⁶ at 37°C. -3 It binds to human BTLA at an off-rate not exceeding or less than (1 / s). In certain embodiments, the isolated antibody of the present invention (e.g., a humanized antibody) binds at 37°C, 3.0 x 10⁻¹⁶ -4 It binds to human BTLA at an off-rate not exceeding or less than (1 / s). In certain embodiments, the isolated antibody of the present invention (e.g., a humanized antibody) binds at 37°C to 2.0 x 10⁻¹⁶ -4 (1 / s) or 1.0 x 10 -4 It binds to human BTLA at an off-rate not exceeding (1 / s) or less.

[0142] In a particular aspect of the first facet of the present invention, the present invention provides an agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, wherein the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2; does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM), as determined by, for example, surface plasmon resonance (SPR), as described in Example 4; and inhibits T cell proliferation in vitro, as determined by, for example, a mixed lymphocyte reaction assay, as described in Example 9. In some embodiments, the antibody or its antigen-binding fragment, when determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, has a value of at least 5.0 x 10⁻¹⁶. 5It binds to human B and T lymphocyte attenuation factor (BTLA) at an on-rate of (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment is determined at 3.0 x 10⁻¹⁶ when measured by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 The antibody binds to human B and T lymphocyte attenuation factor (BTLA) with an off-rate of less than 1 / s. In some embodiments, the antibody or its antigen-binding fragment is determined at 3.0 x 10⁻¹⁶ at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2. -4 (1 / s) ~ 1.0 x 10 -3 The antibody or its antigen-binding fragment binds to human B and T lymphocyte attenuation factor (BTLA) at an off rate of (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:D52, P53, E55, E57, E83, Q86, E103, L106 and E92, as determined by X-ray crystallography of the mutant receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or The antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from N65 and A64.

[0143] Methods for characterizing the properties of the antibody or antigen-binding fragment of the present invention are well known in the art. A suitable method for determining binding specificity at 37°C using surface plasmon resonance (SPR) is described in Example 2. A suitable method for determining whether the test antibody / fragment inhibits the binding of BTLA to herpesvirus entry mediator (HVEM) is described in Example 4; this also uses surface plasmon resonance (SPR). A suitable method for determining whether the test antibody / fragment inhibits T cell proliferation in vitro is a mixed lymphocyte reaction assay, such as that described in Example 9. A suitable method for determining the binding site of the antibody / fragment to BTLA may utilize X-ray crystallography or flow cytometry of the mutagenic receptor, such as the method described in Example 5.

[0144] In a particular aspect of the first aspect of the present invention, a method such as that described in Example 2 is used, and when determined at 37°C by surface plasmon resonance (SPR), at least 5.0 x 10⁻¹⁰ 5 This specification provides an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on rate of (1 / Ms), wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM) when determined, for example, by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro when determined, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or its antigen-binding fragment, when determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, exhibits a rate of 3.0 x 10⁻¹⁶ -4The antibody or its antigen-binding fragment binds to human B and T lymphocyte-decaying factor (BTLA) with an off-rate of less than (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to human B and T lymphocyte-decaying factor (BTLA) with a KD of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey BTLA with a KD of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography or flow cytometry of the mutant receptor, as described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from:N65 and A64.

[0145] In a particular aspect of the first aspect of the present invention, a method such as that described in Example 2 is used, and when determined at 37°C by surface plasmon resonance (SPR), the result is 3.0 x 10⁻¹⁰. -4 (1 / s) ~ 1.0 x 10 -3 An isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an off-speed of (1 / s), wherein the antibody is subjected to surface plasmon resonance, for example, using a method such as that described in Example 4. The present specification provides an antibody or antigen-binding fragment that, when determined by surface plasmon resonance (SPR), does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro when determined by a mixed lymphocyte reaction assay, for example, using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM when determined by surface plasmon resonance (SPR) at 37°C, using a method such as that described in Example 2. In some embodiments, the antibody or antigen-binding fragment binds to cynomolgus monkey BTLA with a KD of less than 20 nM when determined by surface plasmon resonance (SPR) at 37°C, using a method such as that described in Example 2. In some embodiments, the antibody or antigen-binding fragment binds to at least 5.0 x 10 when determined by surface plasmon resonance (SPR) at 37°C, using a method such as that described in Example 2. 5 The antibody or its antigen-binding fragment binds to human B and T lymphocyte attenuation factor (BTLA) at an on rate of (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using methods such as those described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to BTLA residues selected from N65 and A64.

[0146] In a specific embodiment of the first aspect of the present invention, when measured at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, the result was 1.0 x 10⁻¹⁰. -3 Off speed less than (1 / s) and at least 5.0x10 5This specification provides an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on-rate of (1 / Ms), wherein the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM) when determined, for example, by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro when determined, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or its antigen-binding fragment binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM when determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey BTLA with a KD of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0147] In a particular aspect of the first facet of the present invention, the present invention provides an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 2 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, wherein the antibody inhibits the binding of BTLA to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay, as determined by a method such as that described in Example 9. In some aspects, the antibody or its antigen-binding fragment has a KD of 1.0 x 10⁻¹⁰ when determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 6 The antibody binds to human B and T lymphocyte attenuation factor (BTLA) at an on-rate of less than (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment is determined at 1.0 x 10⁻¹⁶ when measured by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -3The antibody or its antigen-binding fragment binds to human B and T lymphocyte-decaying factor (BTLA) with an off-rate of less than (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey B and T lymphocyte-decaying factor (BTLA) with a KD of less than 10 nM, as determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutant receptor or by flow cytometry, as described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0148] In a particular aspect of the first aspect of the present invention, when determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, 1 x 10⁻¹⁰ -3This specification provides an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an off-rate of less than (1 / s), wherein the antibody inhibits the binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay, for example, using a method such as that described in Example 9. In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 2 nM, as determined by surface plasmon resonance (SPR) at 37°C, using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D52, P53, E55, E57, E83, Q86, E103, L106 and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA residues selected from D35, T78, K81, S121 and L123. The antibody binds to a selected human BTLA residue. In some embodiments, the antibody or its antigen-binding fragment binds to residue H68 of human BTLA. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0149] In a specific aspect of the first facet of the present invention, an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA), wherein the antibody binds to cynomolgus monkey BTLA with a KD of at least 5 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2; inhibits the binding of BTLA to herpesvirus entry mediator (HVEM), as determined by surface plasmon resonance (SPR), as determined by a method such as that described in Example 4; and inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay, as determined by a method such as that described in Example 9, is provided herein. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0150] In a specific aspect of the first facet of the present invention, an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA), wherein the antibody binds to cynomolgus monkey BTLA with a KD of at least 50 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2; and does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro as determined by, for example, a mixed lymphocyte reaction assay using a method such as that described in Example 9, is provided herein. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0151] In a particular aspect of the first aspect of the present invention, an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of 1400 nM to 3500 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2; and the antibody, as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4, The present specification provides an antibody or antigen-binding fragment that does not inhibit the binding of BTLA to lupesvirus entry mediator (HVEM) and inhibits T cell proliferation in vitro, as determined by, for example, a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment exhibits a growth of at least 2.0 x 10⁻¹⁶ when determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 It binds to human BTLA at an on-rate of (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment is determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, with a value of 10.0 x 10⁻¹⁶. -1 The antibody or its antigen-binding fragment binds to human BTLA at an off-rate of less than (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:D52, P53, E55, E57, E83, Q86, E103, L106 and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:D35, T78, K81, S121 and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from:N65 and A64.

[0152] In a particular aspect of the first aspect of the present invention, when determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, the result was 1.7 x 10⁻¹⁰. 5 (1 / Ms) ~ 2.5 x 10 5The following is provided herein: an isolated agonist antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on rate of (1 / Ms); and the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM) when determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro when determined by, for example, a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or its antigen-binding fragment, when determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2, has an on rate of 3.0 x 10 -1 It binds to human BTLA at an off-rate of less than (1 / s). In some embodiments, the antibody or its antigen-binding fragment is determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, with a value of 3.0 x 10⁻¹⁶. -1 (1 / s) ~ 5.0 x 10 -1The antibody or its antigen-binding fragment binds to human BTLA at an off-rate of (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA with a KD of at least 150 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to human BTLA with a KD of 150 nM to 1500 nM, as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. In some embodiments, the antibody or its antigen-binding fragment binds to an epitope that blocks the binding of the 286 antibody. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D52, P53, E55, E57, E83, Q86, E103, L106 and E92, as determined by X-ray crystallography or flow cytometry of the mutagenic receptor using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from:D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In this configuration, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0153] In a particular aspect of the first facet of the present invention, an isolated agonist antibody or its antigen-binding fragment is provided herein, which specifically binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of 40 nM to 1200 nM as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2; and which does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and which inhibits T cell proliferation in vitro as determined by, for example, a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some aspects, the antibody or its antigen-binding fragment has a KD of at least 1.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 It binds to human BTLA at an on-rate of (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment is determined at 37°C by surface plasmon resonance (SPR) using a method such as that described in Example 2, with a value of 1.0 x 10⁻¹⁶. 5 (1 / Ms) ~ 10x10 5 It binds to human BTLA at an on-rate of (1 / Ms). In some embodiments, the antibody or its antigen-binding fragment is determined at 6.0 x 10⁻¹⁶ when measured by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 It binds to human BTLA at an off-rate of less than (1 / s). In some embodiments, the antibody or its antigen-binding fragment is determined at 6.0 x 10⁻¹⁶ when measured by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s) ~ 10.0 x 10 -2The antibody or its antigen-binding fragment binds to human BTLA at an off-rate of (1 / s). In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D52, P53, E55, E57, E83, Q86, E103, L106, and E92, as determined by X-ray crystallography of the mutagenic receptor or by flow cytometry, using a method such as that described in Example 5. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or its antigen-binding fragment binds to a human BTLA residue selected from D35, T78, K81, S121, and L123. In some embodiments, the antibody or its antigen-binding fragment binds to the human BTLA residue H68. In some embodiments, the antibody or its antigen-binding fragment binds to a BTLA residue selected from N65 and A64.

[0154] In a particular embodiment, an isolated antibody or antigen-binding fragment of the present invention that specifically binds to human BTLA increases BTLA activity and / or signaling through the receptor. In certain embodiments, the antibody of the present invention is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0155] In a particular embodiment, the antigen-binding fragment of the present invention is: scFv, sc(Fv) 2 Selected from the group consisting of dsFv, Fab, Fab', (Fab')2, and deabody. In certain embodiments, heavy and light chain molecules forming an antigen-binding fragment are linked by a flexible linker. Many commonly used flexible linkers exist, and the selection of a linker can be made by those skilled in the art.

[0156] The scFv peptide linker, which links the VH and VL domains, ligates the carboxyl terminus of one variable domain to the amino terminus of another variable domain without significantly impairing VH-VL pair formation and antigen-binding site fidelity. The peptide linker can be diverse, ranging in length from 10 to 25 amino acids, and is typically, though not always, glycine. It is composed of hydrophilic amino acids such as (G) and serine (S). The linker is found in natural multidomain proteins (e.g., Argos P. J Mol Biol. 211:943-958, 1990; and Heringa G. Protein See Eng. 15:871-879, 2002 (or an adaptation thereof).

[0157] The most commonly used flexible linkers have sequences consisting mainly of stretches of Gly and Ser residues ("GS" linkers). An example of the most widely used flexible linker is (Gly-Gly-Gly-Gly-Ser). n It has the following sequence. By adjusting the copy number "n", the length of this GS linker may be modified to achieve proper separation of functional domains or to maintain the necessary interdomain interactions. Generally, the (GGGGS)3 peptide is used as the scFv peptide linker (Leith et al., Int. J. Oncol. 24:765-771, 2004; Holiger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). This 15-amino acid linker sequence [referred to as the (GGGGS)3 linker] is used in the recombinant phage antibody system (RPAS kit) commercially available from Amersham. Several other linkers have also been used to generate scFv molecules (e.g., KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST; Bird et al., Science 242:432-426, 1988).

[0158] The inventors mapped epitopes on BTLA to which potent 2.8.6 and 11.5.1 agonists and other antibodies disclosed herein bind. In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to a human BTLA residue selected from: D52, P53, E55, E57, E83, Q86, E103, L106, E92, Y39, K41, R42, Q43, E45, S47, D35, T78, K81, S121, L123, H68, N65, A64.

[0159] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to a human BTLA residue selected from:D52,P53,E55,E57,E83,Q86,E103,L106, andE92.

[0160] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to at least two residues of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0161] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to at least three residues of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0162] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to at least five residues of human BTLA selected from:D52, P53, E55, E57, E83, Q86, E103, L106, and E92.

[0163] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to all human BTLA residues selected from:D52,P53,E55,E57,E83,Q86,E103,L106, andE92.

[0164] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to a human BTLA residue selected from Y39, K41, R42, Q43, E45, and S47. In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to at least two residues of human BTLA selected from Y39, K41, R42, Q43, E45, and S47.

[0165] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to all human BTLA residues selected from Y39, K41, R42, Q43, E45, and S47. In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to a human BTLA residue selected from D35, T78, K81, S121, and L123.

[0166] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to at least two residues of human BTLA selected from D35, T78, K81, S121, and L123.

[0167] In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to residue H68 of human BTLA. In a particular embodiment, the antibody or antigen-binding fragment of the present invention binds to a human BTLA residue selected from N65 and A64.

[0168] In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to both the N65 and A64 residues of human BTLA. Residue numbering, for example K41, refers to the amino acid (K; lysine) at position 41; here, the numbering refers to the position in the human BTLA polypeptide as disclosed in Sequence ID No. 23.

[0169] In certain embodiments, the antibody of the present invention is an IgG1, IgG2, or IgG4 antibody. In certain embodiments, the antibody is a mouse or human antibody. In certain embodiments, the antibody or antigen-binding fragment of the present invention is a humanized antibody.

[0170] In certain embodiments, the antibody or antigen-binding fragment of the present invention is a fully human antibody. In certain embodiments, the antibody or antigen-binding fragment of the present invention acts as an agonist that induces signal transduction through the BTLA receptor.

[0171] The antibody (or its antigen-binding fragment) of the present invention is a particularly potent agonist. In a particular embodiment, the antibody or antigen-binding fragment of the present invention has an EC50 not exceeding 1 nM.

[0172] The agonist antibody (or its antigen-binding fragment) of the present invention exhibits particularly high efficacy. In certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits T cell proliferation by at least 20%, preferably at least 30%, and more preferably at least 40%.

[0173] In certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits T-cell IFN-gamma production by at least 50%, preferably at least 75%, and more preferably at least 95%, in an in vitro mixed lymphocyte reaction, as measured, for example, by ELISA of the supernatant.

[0174] In certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits T cell IL-2 production by at least 50%, preferably at least 75%, and more preferably at least 95%, in an in vitro mixed lymphocyte reaction, as measured, for example, by ELISA of the supernatant.

[0175] In certain embodiments, the antibody or antigen-binding fragment of the present invention is mixed in vitro. In lymphocyte reactions, for example, when measured by ELISA of the supernatant, it inhibits T cell IL-17 production by at least 50%, preferably at least 75%, and more preferably at least 95%.

[0176] In certain embodiments, the antibody or antigen-binding fragment of the present invention reduces mortality in a mouse GVHD model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 12.

[0177] In certain embodiments, the antibody or antigen-binding fragment of the present invention reduces weight loss in a mouse T-cell colitis model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 11.

[0178] In certain embodiments, the antibody or antigen-binding fragment of the present invention reduces colitis in a mouse T-cell colitis model by at least 50%, preferably at least 75%, and more preferably at least 95%, using methods such as those described in Example 11.

[0179] In certain aspects, the present invention also relates to isolated polypeptides comprising a VL domain or a VH domain of any of the antibodies or antigen-binding fragments described herein. In certain embodiments, the isolated polypeptide comprises an amino acid sequence such as that shown in SEQ ID NO: 13 or 14, or a sequence having at least 90% identity to said sequence.

[0180] nucleic acid molecule The antibody or its antigen-binding fragment of the present invention will be encoded by nucleic acids. The antibody or its antigen-binding fragment may be encoded by a single nucleic acid molecule, or by two or more nucleic acid molecules. For example, since the antigen-binding site is typically formed by the combination of a heavy-chain variable polypeptide region and a light-chain variable polypeptide region, the two variable (heavy-chain and light-chain) polypeptide regions may be encoded by separate nucleic acid molecules. Alternatively, for example in the case of scFv, these may be encoded by the same nucleic acid molecule.

[0181] In accordance with a second aspect of the present invention, one or more nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof, according to a first aspect of the present invention, are provided. From the primary amino acid sequence of the polypeptide(s) encoding the antibody or antigen-binding fragment of the present invention, a person skilled in the art can determine a suitable nucleotide sequence(s) encoding the polypeptide(s), and preferably one that is codon-optimized (see, for example, Mauro and Chappell. Trends Mol Med. 20(11):604-613, 2014).

[0182] Where the preceding aspects of the Invention are referred to herein, for example, "according to a first (or second, etc.) aspect of the Invention," it is understood that this includes any variation of the aforementioned aspect referred to (for example, a variation of the first (or second, etc.) aspect).

[0183] In accordance with a modification of a second aspect of the present invention, an isolated nucleic acid is provided comprising a nucleotide sequence encoding a heavy-chain variable-region polypeptide or a light-chain variable-region polypeptide of the present invention. The heavy-chain variable-region polypeptide or light-chain variable-region polypeptide of the present invention refers to an individual polypeptide chain comprising amino acids that constitute part of an antigen-binding site. Of course, the polypeptide may also include other domains such as a constant domain, a hinge region, and an Fc region, for example, one or more Fc receptor-binding sites.

[0184] According to another variation of the second aspect of the present invention, an isolated nucleic acid is provided comprising one or more nucleotide sequences encoding a polypeptide capable of forming the antibody or antigen-binding fragment of the present invention. In certain embodiments, the polypeptide may also comprise a constant domain, a hinge region, and other domains, such as an Fc region, or one or more Fc receptor-binding sites.

[0185] One nucleic acid molecule may encode only a polypeptide sequence containing the VL domain of an antibody or a fragment thereof. Another nucleic acid molecule may encode only a polypeptide sequence containing the VH domain of an antibody or a fragment thereof. However, the nucleic acid molecule may also encode both the VH and VL domains containing polypeptide sequences capable of forming the antibody or an antibody fragment of the present invention.

[0186] For example, a nucleic acid molecule (one or more) encoding an antibody or antigen-binding fragment of the present invention, in accordance with the first aspect of the present invention, may be a vector (e.g., a plasmid vector, cosmid vector or viral vector, or artificial chromosome), or part of such a vector, which may also contain other functional regions (elements), such as one or more promoters, one or more origins of replication, one or more selectable markers (one or more), and one or more other elements typically found in expression vectors. Cloning and expression of nucleic acids encoding proteins, including antibodies, are well-established and well within the scope of the art.

[0187] In accordance with a third aspect of the present invention, a vector comprising nucleic acids of the second aspect of the present invention is provided. In a particular embodiment, the vector is a plasmid vector, a cosmid vector, a viral vector, or an artificial chromosome.

[0188] The nucleic acid of the present invention, which includes a vector nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of forming the antibody of the present invention or its antigen-binding fragment, may be in a purified / isolated form. The isolated / purified nucleic acids encoding the antibodies or antigen-binding fragments of the present invention are substances that naturally associate with each other, for example, in their natural environment, or such preparations are made in vitro or in Recombinant DNA technology performed in vivo will not contain, or substantially contain, other proteins or nucleic acids found in the environment in which it is prepared (e.g., cell culture).

[0189] In certain embodiments, the nucleic acids of the present invention are pure to a degree greater than 80%, for example, greater than 90%, greater than 95%, greater than 97%, and greater than 99%. Accordingly, according to another variation of the third aspect of the present invention, a vector comprising a nucleic acid or nucleotide sequence encoding a heavy-chain variable polypeptide or a light-chain variable polypeptide of the present invention is provided. In a particular embodiment, the vector comprises a nucleic acid encoding both a heavy-chain and a light-chain variable region. In a particular embodiment, the polypeptide may also also comprise other domains, such as a constant domain, a hinge region, and an Fc region, such as one or more Fc receptor binding sites.

[0190] The nucleic acids and / or vectors of the present invention may be introduced into host cells. The introduction may be carried out using any available technique. With respect to eukaryotic cells, suitable techniques may include transduction using calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and retroviruses or other viruses, such as vaccinia, or, in the case of insect cells, baculoviruses. The introduction of nucleic acids into host cells, particularly eukaryotic cells, may be carried out using virus or plasmid-based systems. Plasmid systems may be maintained in episomes or incorporated into host cells or artificial chromosomes. Incorporation may be carried out at one or more gene loci. This may be by random or targeted incorporation of one or more copies. With respect to bacterial cells, appropriate techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages.

[0191] In one embodiment, the nucleic acid of the present invention is incorporated into the genome (e.g., chromosomes) of a host cell. Incorporation may be facilitated by the inclusion of sequences that promote recombination with the genome, according to standard techniques.

[0192] host cell A further aspect of the present invention provides host cells containing nucleic acids as disclosed herein. Such host cells may be in vitro or in culture.

[0193] The host cell may be derived from any species, such as bacteria or yeast, but preferably the host cell is a mammalian cell, such as a human cell, or a rodent cell, such as a HEK293T cell or a CHO-K1 cell.

[0194] Accordingly, in accordance with the fourth aspect of the present invention, a host cell is provided comprising a nucleic acid according to the second aspect of the present invention, or a vector according to the third aspect of the present invention. For example, host cells may be treated to induce or enable the expression of the protein of the present invention from nucleic acids by culturing the host cells under conditions for the expression of the encoding nucleic acid. Purification of the expressed product may be achieved by methods known to those skilled in the art.

[0195] Accordingly, the nucleic acids of the present invention, comprising a vector nucleic acid containing a nucleotide sequence encoding a polypeptide capable of forming the antibody or antigen-binding fragment of the present invention, may be present in isolated host cells. Host cells are typically part of a clonal population of host cells. In this specification, reference to host cells also includes the clonal population of said cells. The clonal population is one that has grown from a single parent host cell. Host cells may be of any suitable origin. Suitable host cells include bacterial, fungal, or mammalian cells.

[0196] The host cell may act to assist in the amplification of the vector nucleic acid (e.g., using a plasmid) or may act as a biological factory expressing the polypeptide(s) of the present invention that form the BTLA antibody or its antigen-binding fragment. Suitable hosts for amplifying the vector nucleic acid may be bacterial or fungal cells, such as Escherichia coli cells or Saccharomyces cerevisiae cells. Suitable hosts for expressing the protein of the present invention (i.e., the polypeptide constituting the human BTLA-binding antibody or its antigen-binding fragment) may be mammalian cells, such as HEK293T or CHO-K1 cells. In certain embodiments, the host cell is a mammalian cell, such as HEK293T or CHO-K1 cell.

[0197] A variety of host-expression vector systems may be used to express BTLA-binding molecules as described herein (see, for example, U.S. Patent No. 5,807,715). For example, mammalian cells, such as Chinese hamster ovary cells (CHO), combined with a vector, such as a major intermediate early gene promoter element derived from human cytomegalovirus, are an effective expression system for the CEA protein (Foecking et al., Gene, 45:101 (1986); and Cockett et al., Bio / Technology, 8:2 (1990)). Different host cells are used for the post-translational processing of proteins and gene products. They possess characteristic and specific mechanisms for syncing and modification. Appropriate cell lines or host systems may be selected to ensure correct modification and processing of the proteins of this disclosure. For this purpose, eukaryotic host cells possessing cellular mechanisms for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, HEK, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O and HsS78Bst cells.

[0198] Antibody production A method for producing an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, according to the fifth aspect of the present invention, comprising the steps of culturing a host cell according to the fourth aspect of the present invention under conditions for the production of the antibody or antigen-binding fragment, and optionally isolating and / or purifying the antibody or antigen-binding fragment.

[0199] A method for producing an antibody or antigen-binding fragment that binds to human BTLA, according to a fifth aspect of the present invention, comprising the steps of culturing host cells containing nucleic acids encoding polypeptides (one or more) that form an antibody or antigen-binding fragment that binds to human BTLA, under conditions for the production of the antibody or antigen-binding fragment, and optionally further comprising the steps of isolating / purifying the antibody or antigen-binding fragment.

[0200] Through isolation and purification, the inventors mean that the antibodies of the present invention or their antigen-binding fragments, or the polypeptides constituting these molecules, will not contain or substantially contain other proteins or nucleic acids found together with naturally associated substances, such as in their natural environment, or in the environment in which such preparations are made (e.g., cell culture) if such preparations are carried out by recombinant DNA technology in vitro or in vivo.

[0201] A method for preparing an antibody or antigen-binding fragment that specifically binds to human BTLA, according to a fifth aspect of the present invention: a) Provide a host cell containing one or more nucleic acid molecules that, when expressed, can be combined to produce a human BTLA-binding molecule, encoding amino acid sequences of heavy-chain variable domains and light-chain variable domains; b) Culture host cells expressing the encoded amino acid sequence; and c) Isolate the antibody or antigen-binding fragment molecule. The present invention provides the method, including the steps involved.

[0202] One or more nucleic acid molecules are those described above, which encode polypeptides capable of forming the antibody of the present invention or its antigen-binding fragments that specifically bind to human BTLA. In a particular embodiment, the antibody or its antigen-binding fragment is: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO: 1, CDRH2 has the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 has the amino acid sequence shown in SEQ ID NO: 3; and ii) A light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO: 4, CDRL2 has the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 has the amino acid sequence shown in SEQ ID NO: 6. Includes.

[0203] In a particular embodiment, the antibody or its antigen-binding fragment is: i) three CDRs: CDRH1 , a heavy chain variable region comprising CDRH2 and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO: 7, CDRH2 has the amino acid sequence shown in SEQ ID NO: 8, and CDRH3 has the amino acid sequence shown in SEQ ID NO: 9; and ii) A light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO: 10, CDRL2 has the amino acid sequence shown in SEQ ID NO: 11, and CDRL3 has the amino acid sequence shown in SEQ ID NO: 12. Includes.

[0204] In a particular embodiment, the antibody or its antigen-binding fragment is: i) an amino acid sequence disclosed in Sequence ID No. 13, or a heavy chain variable region containing a sequence having at least 90% sequence identity with said sequence; and ii) A light chain variable region containing the amino acid sequence disclosed in Sequence ID No. 14, or a sequence having at least 90% sequence identity with said sequence. Includes.

[0205] In various embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, where the heavy chain comprises a heavy chain variable region containing three CDRs: CDRH1, CDRH2, and CDH3, and the light chain comprises a light chain variable region containing three CDRs: CDRL1, CDRL2, and CDRL3, where (1) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 31, 32, and 33, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 34 and 35, respectively. (2) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; (3) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively, and CDRL 1. CDRL2 and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 46, 47, and 48, respectively; (4) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 43, 56, and 57, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 58, 59, and 60, respectively; (5) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 61, sequence (6) CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 62 and sequence number 63, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 64, sequence number 65, and sequence number 66, respectively; (6) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 31, sequence number 32, and sequence number 69, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 34, sequence number 35, and sequence number 72, respectively;(7) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 73, 74, and 75, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 76, 47, and 78, respectively; (8) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 79, 80, and 81, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 82, 83, and 84, respectively; (9) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 85, 86, and 87, respectively, and CDRL1, CDRL2, and CDRL3 are respectively; (10) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 88, 89, and 90, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 94, 95, and 96, respectively; (11) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 97, 98, and 99, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 100, 101, and 102, respectively; (12) CDRH1, CDRH2, CDRH3 have amino acid sequences as shown in SEQ ID NOs. 103, 104, and 105, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 64, 107, and 10 (13) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 109, 110, and 111, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 88, 89, and 114, respectively? (14) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 85, 110, and 117, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 88, 89, and 114, respectively? (15) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 121, 122, and 123, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 124, 125, and 126, respectively?(16) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 127, 128, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively; (17) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 133, 134, and 135, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 136, (18) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 139, sequence number 140, and sequence number 141, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 142, sequence number 143, and sequence number 144, respectively; (19) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 145, sequence number 146, and sequence number 147, respectively, and (20) CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 148, 149, and 150, respectively; (21) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 31, 32, and 33, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in SEQ ID NOs. 148, 149, and 150, respectively; (22) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in SEQ ID NOs. 31, 149, and 150, respectively; (22) CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 32 and sequence number 159, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 136, sequence number 137, and sequence number 162, respectively; (22) CDRH1, CDRH2, and CDRH3 have amino acid sequences as shown in sequence number 169, sequence number 170, and sequence number 171, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as shown in sequence number 172, sequence number 173, and sequence number 174, respectively;(23) CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NO: 64 and SEQ ID NO: 6, respectively; (24) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 182, and SEQ ID NO: 183, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 186, and (25) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, and (26) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 12 (27) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 199, 200, and 201, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 202, 203, and 6, respectively? (28) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 205, 206, and 207, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 142, 209, and 210, respectively? (29) Do CDRH1, CDRH2, and CDRH3 have the amino acid sequences shown in SEQ ID NOs. 211, 212, and 213, respectively, and do CDRL1, CDRL2, and CDRL3 have the amino acid sequences shown in SEQ ID NOs. 214, 35, and 216, respectively?(30) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 127, SEQ ID NO: 386, and SEQ ID NO: 129, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; (31) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210; (32) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NO: 127, SEQ ID NO: 388, and SEQ ID NO: 129, and CDRL1, CDRL2, and C (33) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36; (34) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NOs. 205, 387, and 207, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NOs. 142, 209, and 210; or (34) CDRH1, CDRH2, and CDRH3 each have the amino acid sequences shown in SEQ ID NOs. 205, 387, and 207, and CDRL1, CDRL2, and CDRL3 each have the amino acid sequences shown in SEQ ID NOs. 142, 143, and 210; 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO.

[0206] The conditions for producing the antibody or antigen-binding fragment of the present invention and for purifying the said molecule are well known in the art. One method for handling this is to prepare a clonal population of cells capable of expressing the antibody or fragment of the present invention, and to culture these in a suitable growth medium for a period and temperature that promotes the proliferation / growth of the cell population and the expression of the protein(s) of interest. If the protein(s) of interest (e.g., the antibody(s) of the present invention) is expressed in the host cells, the cells are lysed (e.g., using a mild surfactant or sonication) to separate the cell contents (and therefore the protein(s) of interest) into the surrounding tissue. The cells may be released into a surrounding medium (which may be a culture medium or another medium in which the cells are reconstituted), and this medium is then subjected to the purification process. If the protein(s) of interest (e.g., the antibody(s) of the present invention) are secreted into the growth medium, the medium is subjected to the purification process. Antibody purification involves the isolation of antibodies from hybridoma cell lines, for example from the culture medium or from the culture supernatant, using well-established methods, typically involving chromatography (e.g., affinity chromatography, anion and / or cation exchange chromatography, size exclusion chromatography or other separation techniques) to separate the protein of interest from undesirable host-derived proteins and other cellular contaminants (e.g., nucleic acids, carbohydrates, etc.).

[0207] The purified protein may also be subjected to a virus inactivation process. Finally, the purified protein of interest may be freeze-dried or compounded, for example, in preparation for storage, transport, and subsequent use. Preferably, the protein of interest (e.g., the antibody of the present invention or its antigen-binding fragment) will be substantially free of any contaminating proteins originally present in the culture medium after expression or cell lysis.

[0208] In certain embodiments, the antibody or antigen-binding fragment of the present invention will be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0209] The protein of the present invention (the antibody or antigen-binding fragment thereof) may be incorporated into a suitable composition. composition BTLA-binding molecules may be administered alone, but in certain embodiments, the administration is of a pharmaceutical composition in which the BTLA-binding molecules are combined with at least one pharmaceutically acceptable excipient. The excipient may be a suitable pharmaceutically acceptable carrier solute. Such carriers are well known in the art and include phosphate-buffered saline, water, liposomes, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers may be formulated according to well known conventional methods. These pharmaceutical compositions may be administered to the subject in an appropriate dose. The administration measure will be determined by the attending physician and clinical factors.

[0210] In accordance with the sixth aspect of the present invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or one produced by the fifth aspect. In a particular embodiment, the composition comprises phosphate-buffered saline.

[0211] A "pharmaceutical composition" refers to a preparation that is in a form that enables the effective biological activity of the active ingredient and does not contain any further components that would be unacceptably toxic to the subject to which the formulation will be administered. A pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. In this context, the term excipient refers to any additive, such as fillers, solubilizers, carriers, vehicles, or other additives.

[0212] The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients, such as water, ion exchangers, proteins, buffers, and salts. Preservatives and other additives may also be present. The excipients may be solvents or dispersion media. Suitable formulations for use in the therapies disclosed herein are described in Remington's Pharmaceutical Sciences, 16th edition, edited by A. Osol (1980).

[0213] A "pharmaceutically acceptable" excipient is one that can be reasonably administered to the target mammal in order to provide an effective dose of the active ingredient used. The pharmaceutical compositions of the present invention are prepared for storage by mixing the composition with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980)) in the form of a lyophilized formulation or an aqueous solution. Acceptable excipients are non-toxic to the recipient at the dosage and concentration used, and include, for example, buffers of phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g. These include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN(TM), PLURONICS(TM), or polyethylene glycol (PEG). A lyophilized HER2 antibody formulation is described in WO97 / 04801.

[0214] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane. The route of administration of a BTLA-binding molecule, such as an antibody or an antigen-binding fragment thereof, may be, for example, oral, parenteral, inhalation or topical. As used herein, the term parenteral includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0215] Pharmaceutical compositions for parenteral administration include sterile aqueous or non-aqueous solutions, and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, aqueous solutions, or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. Furthermore, the composition may, in certain embodiments of human origin, include a proteinaceous carrier such as serum albumin or immunoglobulin. For intravenous injection, or injection at the site of affliction, the active ingredient is in the form of an aqueous solution acceptable for parenteral use, which is pyrogen-free and has the appropriate pH, isotonicity and stability. A suitable person skilled in the art can adequately prepare a suitable solution using, for example, an isotonic vehicle, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary. As described above, all of these are referred to as excipients herein.

[0216] The injectable composition may be administered with a medical device known in the art. For example, a hypodermic needle. Needleless injection devices, such as those disclosed in U.S. Pat. Nos. 6,620,135 and 5,312,335, may also be utilized.

[0217] Pharmaceutical compositions for oral administration are in the form of tablets, capsules, powders, liquids or semi-solids This may be so. Tablets may contain a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other sugar solutions or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, may be included as required.

[0218] The antibody of the present invention or its antigen-binding fragment may be formulated in liquid, semi-solid or solid form according to the physicochemical properties of the molecule and the delivery route. The formulation may contain excipients, or a combination of excipients, such as: sugars, amino acids and surfactants. The liquid formulation may contain a wide range of antibody concentrations and pH values. Solid formulations may be produced, for example, by freeze-drying, spray-drying or drying by supercritical fluid technology.

[0219] The pharmaceutical composition may be administered as a single dose, multiple doses or by infusion over a defined period. The dosing regimen may also be adjusted to provide an optimal desired response (e.g., a therapeutic or prophylactic response). In particular, parenteral formulations may be a single bolus dose, an infusion or a loading bolus dose followed by one or more maintenance doses. These compositions may be administered at specific fixed or variable intervals, such as once a day or "as needed".

[0220] Dosage The therapeutically effective amount of BTLA-binding molecules, or pharmaceutical formulations containing such molecules, may be determined by standard clinical techniques, for example, through dose-ranging clinical trials. Furthermore, in vitro assays may be optionally used to assist in identifying the optimal dosage range. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the physician's judgment and the individual patient's circumstances. The effective dose may be extrapolated from dose-response curves obtained from in vitro or animal model test systems. The dosage of the composition to be administered may be determined by those skilled in the art without excessive experimentation, in combination with standard dose-response studies. Relevant circumstances to be considered when making these decisions include the single or multiple conditions to be treated, the selection of the composition to be administered, the individual patient's age, weight, and response, and the severity of the patient's symptoms. For example, the dose of the formulation in milliliters (mL) to be administered may be calculated using the actual patient weight. Downward adjustment to an "ideal" weight may not be possible. In such circumstances, the appropriate dose may be calculated by the following formula: Dosage (ml) = [Patient weight (kg) x Dose level (mg / kg) / Drug concentration (mg / mL)] The therapeutically effective dose of a pharmaceutical composition for the treatment of BTLA-related diseases or disorders, as discussed herein, will vary depending on many different factors, including the means of administration, the target site, the patient's physiological state, the patient's weight, the patient's sex, the patient's age, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The therapeutically effective dose is likely to be determined from clinical trials and is determinable by the attending physician using treatment guidelines. Typically, the patient is human, but non-human mammals may also be treated. To optimize safety and efficacy, the therapeutic dosage may be potentiated using routine methods known to those skilled in the art.

[0221] BTLA-binding molecules are administered at concentrations of approximately 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg, according to various methods.

[0222] The pharmaceutical compositions of the present invention may be administered alone, simultaneously, or sequentially, in combination with other treatments, depending on the condition to be treated. Such combinations may include other immunosuppressants, such as those selected from corticosteroids, cyclosporine, azathioprine, sulfasalazine, methotrexate, mycophenolate, tacrolimus, and fingolimod, or other biological agents, such as infliximab, adalimumab, ustekinumab, tocilizumab, and rituximab.

[0223] A method for preparing a pharmaceutical composition according to a seventh aspect of the present invention is provided, comprising the step of incorporating an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, or one produced according to a fifth aspect of the present invention, into a composition comprising at least one further component. In a particular embodiment, the at least one further component is a pharmaceutically acceptable excipient.

[0224] kit Furthermore, the product (e.g., a BTLA-binding molecule or its pharmaceutical composition) may be packaged and sold in kit form. Such manufactured articles may have labels or package inserts indicating the product and instructions for the proper use of the product for the treatment of subjects suffering from or prone to a disease or disorder.

[0225] Accordingly, according to one aspect of the present invention, a kit is provided comprising an antibody or its antigen-binding fragment according to the first aspect of the present invention, or a pharmaceutical composition according to the sixth aspect of the present invention. Preferably, such a kit includes a package insert containing instructions for use.

[0226] therapy / medical use The antibody or its antigen-binding fragment of the present invention, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, may be used as a typical drug in therapy.

[0227] In certain embodiments, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, may be used to treat or prevent any disease or condition in a subject for which such treatment is necessary.

[0228] BTLA is involved in downregulating the immune response and is therapeutic for many diseases or conditions by suppressing host T cells and / or B cells (see, for example, Crawford & Wherry. Editorial: Therapeutic potential of targeting BTLA. J Leukocyte Biol. 86:5-8, 2009). Diseases or conditions that may benefit from treatment with anti-BTLA agonists are referred to herein as “BTLA-related diseases.” BTLA-related diseases include inflammatory or autoimmune diseases and disorders of excessive immune cell proliferation.

[0229] Specific BTLA-related diseases that can be treated with the BTLA-binding molecule of the present invention include: Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular endocrine syndrome, Behçet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, and polycystic sclerosis. This includes rheumatoid arthritis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0230] In accordance with the eighth aspect of the present invention, an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or a pharmaceutical composition according to the sixth aspect of the present invention, is provided for use in therapy.

[0231] In certain embodiments, therapy is the treatment or prevention of BTLA-related disorders. In certain embodiments, BTLA-related diseases are caused by decreased BTLA expression and / or activity in the subject. In particular, any disease or disorder characterized by the presence or activity of T or B cells may be treated with the BTLA agonist antibody or antigen-binding fragment of the present invention.

[0232] In one embodiment, BTLA-related diseases are inflammatory diseases (e.g., rheumatoid arthritis), autoimmune diseases or disorders (e.g., graft-versus-host), or proliferative diseases or disorders (e.g., cancer).

[0233] In certain embodiments, the therapy is for the treatment or prevention of inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation. According to a variation of the eighth aspect of the present invention, there is provided a method of treating a patient in need thereof, the method comprising administering to the patient an antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the sixth aspect of the present invention. In certain embodiments, the patient in need of treatment or to be treated has (or suffers from) a BTLA-related disease. In certain embodiments, the patient in need of treatment or to be treated has (or suffers from) an inflammatory disease, an autoimmune disease, or a disorder of excessive immune cell proliferation.

[0234] In certain embodiments, an antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the sixth aspect of the present invention, is administered to a patient in need thereof in a pharmaceutically acceptable amount.

[0235] According to a variation of this aspect, there is provided an antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the sixth aspect of the present invention, for use in a method of treating a patient in need thereof. In certain embodiments, the method is for treating or preventing a BTLA-related disease. In certain embodiments, the method is for treating or preventing an inflammatory or autoimmune disease, and a disorder of excessive immune cell proliferation.

[0236] According to a further variation of this aspect, there is provided the use of an antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the sixth aspect of the present invention, in the manufacture of a medicament for the treatment of a patient in need thereof.

[0237] In one embodiment, the therapy is for the treatment of BTLA-related diseases. Appropriately, BTLA-related diseases are inflammatory diseases (e.g., asthma), autoimmune diseases or disorders (e.g., rheumatoid arthritis), or immunoproliferative diseases or disorders (e.g., lymphoma).

[0238] In a particular embodiment, the antibody of the present invention or its antigen-binding fragment, or the antibody or Pharmaceutical compositions containing the antigen-binding fragment are used to suppress T cells and / or B cells.

[0239] In a particular embodiment, the antibody or antigen-binding fragment of the present invention, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is used for: Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyglandular endocrine syndrome, Behçet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease (GVHD), Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, It is used to treat or prevent diseases or conditions in subjects in need, selected from the group consisting of hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis (MS), myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0240] In certain embodiments, the antibody or its antigen-binding fragment of the present invention, or a pharmaceutical composition comprising the antibody or its antigen-binding fragment, is used to treat or prevent a disease or condition in a subject in need, selected from the group consisting of GVHD, colitis, rheumatoid arthritis, psoriasis, and MS. In one embodiment, the immunoproliferative disorder is cancer. Preferably, the cancer is leukemia or lymphoma.

[0241] In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in preventing or treating graft rejection.

[0242] In another embodiment, the present invention relates to the prevention or treatment of graft-versus-host disease. In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in the treatment of rheumatoid arthritis.

[0243] In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in the treatment of diabetes, such as type 1 diabetes.

[0244] In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in the treatment of psoriasis. In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in the treatment of multiple sclerosis.

[0245] In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the antibody or its antigen-binding fragment, is intended for use in the treatment of colitis. The term “effective dose” or “therapeutic effective dose” refers to the amount of medication or drug that is sufficient to alleviate symptoms in a patient or to achieve a desired biological outcome, such as increased tumor cell death, reduced tumor size, increased progression-free survival, or increased overall survival, for example in cancer. As disclosed elsewhere herein, the effective dose will typically be evaluated through thorough human clinical studies.

[0246] Throughout this description and claims, the terms “comprise” and “contain” and their variations mean “to include, but not limited to,” and they are not intended (and do not exclude) any other parts, appendices, components, integers or processes. Throughout this description and claims, singular forms include plural forms unless the context requires otherwise. In particular, where the indefinite article is used, this specification is understood to expect both singular and plural forms unless the context requires otherwise.

[0247] Features, integers, properties, compounds, chemical parts or groups described in combination with specific aspects, embodiments or examples of the present invention shall be understood to be applicable to any other aspects, embodiments or examples described herein, unless incompatible therewith. All features disclosed herein (including any accompanying claims, abstracts and figures) and / or all steps of any method or process thus disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to any of the details of any of the embodiments described herein. The present invention extends to any novel one or any novel combination of features disclosed herein (including any accompanying claims, abstracts and figures), or any novel one or any novel combination of steps of any method or process thus disclosed.

[0248] The reader's attention is directed to all articles and documents submitted in connection with this Specified, either concurrently with or prior to this Specified, and which are open to public access in this Specified, and the contents of all such articles and documents are incorporated herein by reference.

[0249] The present invention will be further described herein with reference to the following non-limiting embodiments and accompanying figures. [Examples]

[0250] In subsequent examples, antibodies 11.5.1 and 2.8.6 are shown to bind to human BTLA with high affinity. Using transgenic mice expressing the human receptor, these antibodies are shown to inhibit T cell responses in vitro and in vivo following binding to BTLA, and to induce disease remission in mouse models of inflammatory bowel disease and graft-versus-host disease. While these agonist effects are dependent on Fc receptor binding, the antibodies do not cause depletion of BTLA-expressing cells through cytotoxicity and do not induce receptor downregulation.

[0251] Example 1. Generation and sequencing of anti-BTLA antibodies Human BTLA extracellular region (BTLA K31~R151 By immunizing mice with ), BioGenes GmbH generated antibodies that recognize the human immune cell receptor BTLA. Splenocytes derived from immunized mice were fused with Sp2 / 0-Ag14 myeloma cells, and the resulting hybridomas were selected for their reactivity with human BTLA by ELISA of the supernatant, in combination with dilution cloning. Antibodies were isotyped from the hybridoma supernatant using a rapid mouse isotyping kit (RayBiotech). Antibodies produced by clones 2.8.6 and 11.5.1 were both found to be IgG1k.

[0252] To sequence the immunoglobulin variable domain, RNA was extracted from hybridomas using TRIzol reagent (ThermoFisher) according to the manufacturer's instructions. Primers specific to the first constant domain of the heavy chain or the constant domain of the light chain were used, and Super Script II reverse transcriptase (Invitr) was used according to the manufacturer's instructions. RNA was reverse transcribed using ogen to produce cDNA.

[0253] Next, using primers that target conserved regions of immunoglobulin gene loci, as previously described (Tille et al., J Immunol Methods). (350:183-193, 2009), PCR was performed, and the PCR products were sequenced. In some cases, the identification of functional light chains was complicated by the abundance of non-functional kappa light chain cDNA from fusion myeloma cell lines. To address this, a previously described technique was used to force partial excision of abnormal chain products by over-adding primers specific to the non-functional CDR3 (Yuan et al. J Immunol Methods. 294:39553-61, 2005).

[0254] The variable domain sequence was evaluated using the NCBI IgBlast tool to determine the location of the CDR. Example 2. Binding to soluble human and cynomolgus monkey BTLA. The binding affinity and dynamics of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to human or cynomolgus monkey BTLA were determined by surface plasmon resonance using Biacore T200 (GE Healthcare). Series S CM5 sensor chips (GE Healthcare) were coated with polyclonal anti-mouse IgG using a mouse antibody capture kit (GE Healthcare). The anti-BTLA antibody was then captured on the biosensor surface, and a negative control antibody (clone Mopc21; Biolegend) was captured in the reference channel. The binding affinity and dynamics of monomer-soluble human BTLA extracellular domains (BTLA) at various concentrations were then determined in single-cycle dynamics analysis. K31~R151 ) (derived from Sequence ID No. 23) or soluble cynomolgus monkey BTLA extracellular domain (BTLA K31~R151 (derived from Sequence ID No. 24) in 10mM buffer. Clone 2.8.6 bound to human BTLA with a KD of 0.65 nM and 0.005% v / v surfactant P20, pH 7.4 (HBS-P), at 37°C (Figure 1a). After reference and blank subtraction, the association and dissociation rates were fitted and the dissociation constants were calculated using BiaEvaluation software (GE Healthcare) (Figure 1b). Clone 2.8.6 bound to human BTLA with a KD of 0.65 nM and to cynomolgus monkey BTLA with a KD of 7.89 nM. Clone 11.5.1 bound to human BTLA with a KD of 0.75 nM and to cynomolgus monkey BTLA with a KD of 0.99 nM. In separate experiments with human BTLA only, clone 2.8.6 bound to human BTLA with a KD of 0.37 nM, and clone 11.5.1 bound to human BTLA with a KD of 0.53 nM.

[0255] Example 3. Binding to BTLA on cells The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to bind to human or cynomolgus monkey BTLA expressed on the cell surface was evaluated by flow cytometry. Full-length human or cynomolgus monkey BTLA was expressed in Jurkat T cell lines using a lentiviral transfection system. 1 x 10⁶ wells 5 Cells were plated in 96-well U-bottom plates. BTLA antibody binding to mIgG1 isotype control (clone MOPC-21, Biolegend #400165) was evaluated at 12 concentrations in FACS buffer (PBS, 2% FCS, 0.05% sodium azide) using 1:3 serial dilutions, starting at 90 μg / ml. Nonspecific antibody binding was prevented by the addition of Fc block (Biolegend #101319). Antibodies were incubated on ice for 30 minutes, then cells were washed twice with FACS buffer and stained with AF647-conjugated anti-mIgG1 secondary antibody (Biolegend #406618). Secondary antibodies were incubated on ice for 30 minutes, then cells were washed and resuspended in FACS buffer for analysis on a flow cytometer. The geometric mean fluorescence intensity of the secondary antibodies was plotted for each concentration, and Gr Using aphPad Prism software, the receptor binding EC50 was calculated by nonlinear curve fitting. Clone 11.5.1 binds to human BTLA-expressing cells with an EC50 of 0.016 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.0057 nM. Clone 2.8.6 binds to human BTLA-expressing cells with an EC50 of 0.085 nM and to cynomolgus monkey BTLA-expressing cells with an EC50 of 0.16 nM (Figure 1c-d).

[0256] Example 4. Competition with the natural ligand HVEM for binding to BTLA. The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to block native ligand binding to BTLA was evaluated by surface plasmon resonance using Biacore T200 (GE Healthcare). Amine coupling was used to evaluate the ability of the human BTLA extracellular domain (BTLA) to block ligand binding. 31K~151R The BTLA was co-coupled to a CM5 sensor chip. Subsequently, human HVEM extracellular domains fused to mouse IgG1 Fc were injected onto the immobilized hBTLA in HBS-P buffer at 37°C, allowing for complete dissociation. A saturated amount of anti-BTLA antibody (2.8.6 or 11.5.1) was then injected, followed immediately by a second injection of human HVEM-mFc at the same concentration as the first injection (Figure 2a). After saturation of BTLA with antibody, equilibrium HVEM binding (resonance units) was expressed as a percentage of binding before antibody injection (Figure 2b). If HVEM binding after saturation with antibody was >90% of binding before antibody injection, the antibody was considered non-blocking.

[0257] Example 5. Binding epitope of antibody 11.5.1 on human BTLA The functional epitopes of antibody 11.5.1 on human BTLA were determined by flow cytometry evaluation of binding to a single-residue mutant panel of receptors expressed on the cell surface. Constructs encoding the human extracellular domain of BTLA and the transmembrane and intracellular domains of mouse CD28 were cloned into the nicistronic mammalian expression vector pGFP2-n2 (BioSignal Packard Ltd), which also encodes GFP. Mutant constructs differing by one amino acid were prepared using a "dramatic" mutagenesis approach (Davis et al. Proc Natl Acad Sci USA. 95, 5490-4 (1998)). Plasmids (2 μg / well) were transfected into HEK-293T cells in 6-well plates using Genejuice transfection reagent (Novagen; 6 μl / well). Spurious and untransfected controls were included in each experiment. Cells were harvested after 48 hours and stained with 10 μg / ml of fluorescent dye-conjugated anti-BTLA antibody in PBS, 0.05% azide, and 2% FCS (FACS buffer) along with survival / death markers at 4°C for 1 hour. Cells were washed, pelletized, and resuspended in 200 μl FACS buffer, then analyzed by BD FACScan on flow cytometry. GFP-positive (transfection) viable cells were gated and analyzed for anti-BTLA antibody binding (an example of binding analysis for clone 11.5.1 is shown in Figure 3a). For each mutant, the geometric mean of anti-BTLA antibody binding to transfection cells was expressed as a percentage of binding to the wild-type receptor (Figure 3b). A panel of anti-BTLA antibodies was evaluated, and any mutations that eliminated binding of all antibodies were excluded from the analysis, as it was assumed that such mutations led to dramatic changes in protein folding or expression rather than exhibiting antibody epitopes. Mutations Y39R and K41E completely invalidated the binding of antibody 11.5.1, but the binding of 2.8.6 remained unaffected.These mutations were mapped onto the human BTLA crystal structure (Compaan et al., J Biol Chem. 280:39553-61, 2005), showing the binding epitope of 11.5.1 (black residue) in Figure 3c. The residues required for HVEM binding (Gln37, Arg42, Pro59, His127; from Japanese Patent Publication No. WO2017004213) are also mapped onto the structure in gray, indicating that 11.5.1 binds to an epitope very close to the HVEM binding site.

[0258] Example 6. Crystal structure of Fab' fragment of 2.8.6 in complex with human BTLA The structural epitope of antibody 2.8.6 on human BTLA was determined by solving the crystal structure of antibody Fab in complex with the extracellular domain of human BTLA. The heavy and light chain variable domains of antibody 2.8.6 were cloned into pOPINVH and pOPINVL expression vectors (Addgene) encoding the first constant domain of mouse IgG1 heavy chain (including 6x histidine tag) and the constant domain of mouse Ig kappa chain, respectively. These vectors were transiently and simultaneously transfected into HEK293T cells to produce anti-BTLA 2.8.6 Fab' fragments, which were purified by Ni-NTA purification. S33~D135The BTLA was cloned into a pGMT7 vector and expressed in BL21(DE3)pLysS E. coli cells (Novagen) to produce inclusion bodies. The inclusion bodies were isolated from the cell pellet by sonication and repeatedly washed with a washing solution containing 0.5% Triton X-100. The purified BTLA inclusion bodies were solubilized in a denaturation solution containing 6M guanidine hydrochloride. The solubilized protein solution was slowly diluted to a final protein concentration of 1–2 μM in refolding buffer [0.1M Tris-HCl (pH 8.0), 0.6M L-arginine, 2 mM ethylenediaminetetraacetic acid, 3.73 mM cystamine, and 6.73 mM cysteamine], and then stirred at 4°C for 48 hours. The BTLA refolded mixture was then concentrated on a VIVA FLOW50 system (Sartorius). BTLA was purified by gel filtration on a Superdex 75 column (GE Healthcare).

[0259] Purified BTLA and Fab' were mixed and purified as a complex by size exclusion chromatography. Crystals suitable for data acquisition were obtained by suspension vapor diffusion in 0.2 M calcium acetate, 0.1 M imidazole, pH 8.0, and 10% (w / v) PEG8000 at 293°K. The final dataset was collected using Photon Factory, and the structures were determined by molecular substitution using the structures of BTLA (PDB ID: 2AW2 chain A) and anti-PD1-Fab (PDB ID: 5GGS chain C, D) as search probes.

[0260] The residues on the BTLA at the interface with antibody 2.8.6 are A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, and D135.

[0261] Example 7. Development of humanized BTLA mice To provide a platform for evaluating anti-human BTLA antibodies in a mouse model, we developed a knock-in line of C57Bl / 6 mice expressing a chimeric form of BTLA including the human extracellular region and the mouse transmembrane and signaling regions. A section of human genomic DNA from the start of exon 2 to the end of exon 3 was inserted into the mouse locus, replacing the mouse sequence from the start of exon 2 to the end of exon 4. The sequences of the exon-intron junctions at the start of mouse exon 2 and the end of mouse exon 4 were preserved to ensure proper splicing (Figure 5).

[0262] Example 8. Inhibition of antigen-specific T cell proliferation in vivo. The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit antigen-specific T cell proliferation in vivo was evaluated using a susceptible T cell transfer assay (Figure 6a). In this assay, purified OTII (TCR transgenic) CD4 cells, specific to ovalbumin (OVA), were obtained from mice expressing homozygous human BTLA (hBTLA) and from OT-II mice (The Jackson Laboratory) expressing wild-type mouse BTLA receptors. + Contains a mixture of T cells 5x10 5T cells were transferred to non-transgenic C57BL / 6 recipients. Transferred cells were distinguished from host cells using the CD45.2 (against CD45.1) allotyping marker. Wild-type donor cells also expressed green fluorescent protein under the control of the human ubiquitin C promoter, allowing them to be distinguished from humanized donor cells by flow cytometry. The day after T cell transfer, recipient mice were immunized with 100 μg of ovalbumin (Sigma-Aldrich) in 100 μl PBS mixed with 100 μl Imject Alum (ThermoFisher) to induce T cell proliferation. On day 2, mice were intraperitoneally administered 200 μg of antibody. Eight days after the initial T cell transfer, the ratio of humanized BTLA expression to wild-type OVA-specific T cells in the spleen was determined by flow cytometry. This method allowed us to track the proliferation or decrease of humanized cells that bind to anti-human BTLA antibodies compared to wild-type controls that do not bind to anti-human BTLA antibodies. Both antibodies 2.8.6 and 11.5.1 led to decreased proliferation of humanized BTLA cells compared to wild-type controls, indicating that these antibodies induce signaling through inhibitory BTLA receptors, which leads to decreased T cell proliferation (Figure 6b).

[0263] Example 9. Inhibition of T cell proliferation in mixed lymphocyte reaction. The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit the proliferation of primary T cells derived from humanized mice in vitro was evaluated using a mixed lymphocyte reaction (MLR). Splenocytes derived from Balb / c mice were treated with mitomycin C at 37°C for 30 minutes, then washed and used as stimulating cells. T cells were purified from the spleens of humanized BTLA mice by negative selection using magnetically activated cell sorting (Mojosort mouse CD3 T cell isolation kit, Biolegend #480023), stained with the CellTrace Violet cell proliferation kit (ThermoFisher), and used as reaction cells. 4 x 10⁶ cells per well. 5 Stimulated cells and 2x105 Reactive cells were mixed in 96-well U-bottom plates with various concentrations of anti-BTLA or isotype control antibody (clone MOPC-21, Biolegend #400165). For a total of 10 concentrations, sequential 1:3 dilutions of the antibody were evaluated, starting at a concentration of 1 μg / ml. Polyclonal anti-mHVEM antibody (R&D Systems #AF2516) was also added to all wells at 1 μg / ml to block any baseline signaling through the BTLA pathway and highlight the effect of the agonist antibody. After 96 hours, the dilution of CellTrace Violet in reactive cells was evaluated by flow cytometry as a growth marker. Growth in the presence of anti-BTLA antibody or isotype control was compared to growth in the absence of the antibody. CD4 + and CD8 + The populations were gated and analyzed separately. Both antibodies 2.8.6 and 11.5.1 reduced the proliferation of human BTLA-expressing T cells, indicating that these antibodies induce inhibitory signaling through the human BTLA receptor. Clone 2.8.6 inhibited CD4 T cells with an IC50 of 0.029 nM and had a maximum effect of 42% inhibition of proliferation (Figure 7). Clone 11.5.1 inhibited CD4 T cells with an IC50 of 0.016 nM and had a maximum effect of 33% inhibition of proliferation.

[0264] Example 10. Inhibition of NFκB signaling in human BTLA or cynomolgus monkey BTLA-transfected Jurkat T cell lines. The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit NFκB signaling was evaluated using a BTLA transfection reporter T cell line. Jurkat T cell lines, stably transfected with an expression cassette containing an NFκB-reactive transcription element upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences #TR850A-1), were used as the reporter cell line for NFκB signaling. Full-length human or cynomolgus monkey BTLA was expressed in this reporter cell line using a lentiviral transfection system. These cells were mixed with a stimulating cell line consisting of bw5147 cells expressing an anti-CD3 scFv construct on their surface, as described by Leitner et al. J Immunol Methods. 2010 Oct 31;362(1-2):131-41. The stimulating cell line was also transfected with mouse FcγRIIB to provide Fc receptors for presentation of the agonist BTLA antibody. 5 x 10⁶ cells per well. 4 Reporter cells were subjected to a 5x10⁶ experiment in the presence of various concentrations of BTLA antibody or isotype control (clone MOPC-21, Biolegend #400165). 4Stimulated cells were mixed in a 96-well U-bottom plate. After incubation at 37°C for 24 hours, the cells were pelleted and stained for flow cytometry using a viability dye (Zombie Aqua, Biolegend #423101) and a mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830) to separate stimulated (mouse) cells from reacted (human) cells. The geometric mean of GFP expression was evaluated for each antibody concentration and normalized to GFP expression in the absence of the antibody. Clone 2.8.6 inhibited human BTLA-transfected cells with an IC50 of 0.06 nM and cynomolgus monkey BTLA-transfected cells with an IC50 of 0.22 nM. Clone 11.5.1 inhibited human BTLA-transfected cells with an IC50 of 0.033 nM and cynomolgus monkey BTLA-transfected cells with an IC50 of 0.14 nM.

[0265] Example 11. Treatment of a T-cell-driven mouse model of colitis with antibody 2.8.6 The ability of the BTLA agonist antibody 2.8.6 to induce remission in a T-cell-driven model of colitis was evaluated using humanized mice. This T-cell transfer model has been previously described as a mouse model of inflammatory bowel disease (Ostanin et al., Am J Physiol Gastrointest Liver Physiol. 296:G135-46, 2009). CD45RB cells sorted from the spleen and lymph nodes of humanized BTLA mice were also evaluated. hi CD25-CD4+ T cells were introduced at 5x10⁶ per mouse. 5 Cell dose, Rag1 KO recipient (Rag1 tm1Mom The Jackson The T cells were intraperitoneally injected into mice (in the laboratory). The transferred T cells induced inflammatory colitis, which developed approximately 3 weeks later, leading to diarrhea and weight loss. Rag1 KO same-cage mice that had not received T cell transfer served as non-disease controls. Seven, 21, and 35 days after T cell transfer, recipient mice were intraperitoneally injected with 200 μg of 2.8.6 or isotype control antibody. All mice were regularly weighed, and at 8 weeks, colon weight and dimensions were measured, and inflammatory infiltration was assessed by histology, as well as by cell counting and flow cytometry of extracted lamina propria leukocytes. Antibody 2.8.6 prevented weight loss (Figure 8a) and significantly reduced inflammatory infiltration of the colon (Figure 8b). Colon inflammation in diseased mice led to an increase in colon weight:length ratio, which was not observed in 2.8.6-treated mice (Figure 8c).

[0266] Example 12. Treatment of a mouse model of graft-versus-host disease (GVHD) The effect of anti-BTLA agonist antibodies was evaluated in a GVHD model from non-lethal parent to F1 generation. Bone marrow cells (BMC) and spleen cells were used in humanized BTLA donor mice (C57BL / 6 background; H2 b ) was collected from 2x10 7 BMC and 107 spleen cells were irradiated lethally with 9 Gy total irradiation to CB6F1(H2 b / d The cells were intravenously infused into the recipients. Irradiated CB6F1 mice reconstituted with syngeneic BMC and spleen cells served as non-disease controls. On the day of immune cell transfer, mice were intraperitoneally infused with 200 μg of anti-BTLA antibody or isotype control. GVHD was monitored by periodic weight measurement of the mice, calculation of relative body weight loss, and clinical observation. Five weeks after immune cell transplantation, or upon reaching humane endpoint (including >20% body weight loss compared to starting body weight in the first 14 days, or >15% body weight loss at any other point in time), the mice were disposed of. At death, the weight and dimensions of the colon were measured, and the colonic weight was calculated. The volume-to-length ratio was calculated as a marker for colitis, a major clinical feature of GVHD. Both antibodies 2.8.6 and 11.5.1 significantly reduced weight loss, led to increased survival (Figure 9a), and prevented colitis (Figure 9b).

[0267] Example 13. The agonist activity of antibody 11.5.1 is dependent on Fc receptor binding. Antibody 11.5.1 was recombinantly expressed as mIgG1k containing the D265A mutation, which was previously described as significantly reducing Fc receptor binding (Clynes et al., Nat Med. 6:443-446, 2000). This mutant antibody was evaluated using the T cell transfer assay described in Example 8. The parental antibody 11.5.1 inhibited the proliferation of humanized T cells, as its pure effect is BTLA receptor agonism. However, the FcR-null D265A mutation led to increased proliferation of humanized T cells, suggesting that the FcR-null mutation eliminates the antibody's agonist effect, leaving only the receptor blockade effect (Figure 10a).

[0268] Furthermore, the D265A mutant 11.5.1 antibody was used in vitro as described in Example 9. The assay was also evaluated using the MLR assay. Again, the parental 11.5.1 antibody inhibited the proliferation of humanized T cells, as its pure effect is BTLA receptor agonism. The FcR-null D265A mutation removed the antibody's agonist effect, and therefore this antibody showed no effect in this assay (Figure 10b). The FcR-null 11.5.1 antibody did not enhance the proliferation of humanized cells in this assay because HVEM was blocked (by the addition of a polyclonal anti-HVEM antibody), and therefore there was no baseline signaling through the pathway that should be blocked by the BTLA-blocking antibody.

[0269] Example 14. Antibodies 2.8.6 and 11.5.1 were not composed of complement-fixed antibodies in vitro. Splenocytes derived from humanized mice were incubated at 37°C for 15 minutes with 10% baby rabbit complement (BioRad) and 20 μg / ml of anti-BTLA antibody (or isotype control or positive control depleted anti-CD20 antibody; clone SA271G2 of Biolegend). The anti-CD20 antibody was B220. + While it depletes the majority of B cells, B220 + or CD4 + Both cell populations were stained positive for BTLA, but the anti-BTLA antibody did not deplete either population (Figure 11).

[0270] Example 15. Antibodies 2.8.6 and 11.5.1 do not induce ADCC in vitro. Humanized mouse-derived whole spleen cells (including bone marrow effector cells) were incubated with 20 μg / ml anti-BTLA antibody (or isotype control or depleted anti-CD20 antibody SA271G2) at 37°C for 24 hours. The anti-CD20 antibody was B220. + While it depletes most of the cells, B220 + or CD4 + Both cell populations were stained positive for BTLA, but the anti-BTLA antibody did not deplete either population (Figure 12).

[0271] Example 16. Antibodies 2.8.6 and 11.5.1 do not deplete BTLA-expressing cells in vivo. Humanized BTLA mice were intraperitoneally injected with 200 μg of anti-BTLA antibody or an isotype control. After 24 hours, the spleen was harvested, and the frequencies of different cell populations were identified by flow cytometry. The anti-BTLA antibody did not affect the frequency or absolute number of B or T cells in the spleen, nor the number of B cell precursors in the bone marrow (Figure 13).

[0272] Example 17. Antibodies 2.8.6 and 11.5.1 stabilize BTLA expression on immune cells in vivo. Humanized mice were intraperitoneally injected with 10 mg / kg of antibody 2.8.6 or 11.5.1. Six days after injection, the mice were humanely sacrificed, and the spleens were harvested and processed into single-cell suspensions for evaluation by flow cytometry. The cells were stained with an antibody cocktail that identifies immune cell subsets and a fluorescent-conjugated anti-BTLA antibody that has an epitope non-competitive with the injected antibody. After incubation with the anti-BTLA antibody in vivo, the geometric mean of BTLA staining was normalized to the geometric mean of BTLA staining after incubation with an isotype control (using the same staining antibody). BTLA expression was significantly higher on B cells and CD4 T cells from mice injected with either clone 2.8.6 or 11.5.1 compared to mice injected with an isotype control (Figure 14). This suggests that clones 2.8.6 and 11.5.1 stabilize the expression of BTLA on the cell surface in vivo rather than inducing receptor downregulation, as has been observed with other BTLA antibodies in the prior art (M.-L. del Rio et al. / Immunobiology 215 (2010) 570-578). For the purpose of immunosuppression, agonist antibodies that stabilize receptor expression present the advantage of allowing for an extended high level of inhibitory signaling through the pathway compared to downregulating antibodies.

[0273] Example 18. Tolerability and Side Effects in Animal Models No tolerability issues or side effects were observed in any animal studies using antibody 2.8.6 or 11.5.1.

[0274] Example 19. Humanization of Antibody 2.8.6 Antibody 2.8.6 was humanized by CDR grafting onto a homologous human germline framework region (see SEQ ID NOs: 13-14). IGHV2-5 * 08 was used for the heavy chain, and IGKV3-11 * 01 was used for the light chain. After humanization, binding to BTLA was evaluated by SPR. Humanized 2.8.6 had a K of 0.73 nMD Then, it bound to the monomer BTLA.

[0275] Example 20. Characterization of an exemplary BTLA antibody This example describes the characterization of exemplary mIgG1 BTLA antibodies provided herein, in addition to sections 2.8.6 and 11.5.1. A variety of clones, listed in Table 1, were evaluated for their binding affinity to BTLA and their lymphocyte inhibitory efficiency (Table 2). For each antibody, the association rate ("on rate") and dissociation rate ("off rate") for binding to human BTLA, as well as the KD for binding to human or cynomolgus monkey BTLA, were measured according to the method described in Example 2, and curves for injection of BTLA extracellular domains at single concentrations were fitted.

[0276] The inhibitory efficiency of individual antibodies against T cells was also evaluated at a single concentration of 10 μg / ml. For each individual antibody, an MLR assay was performed according to the method described in Example 9 (two biological replicates as shown in Table 3); an anti-CD3 assay was performed according to the method described below (two biological replicates, Table 3); and the inhibition of NFκB signaling in human BTLA-transfected Jurkat T cell lines by each antibody was determined according to the method described in Example 10 (Table 3). In various in vitro stimulation assays for each exemplary antibody, the mean inhibition of T cells compared to the isotype control was calculated as the average of the inhibition percentages of all assay results (Tables 2 and 3).

[0277] The ability of BTLA agonist antibodies to inhibit anti-CD3 and anti-CD28-induced T cell activation was evaluated as follows: Splenocytes derived from humanized BTLA mice were processed into single-cell suspensions and treated with ACK buffer to lyse red blood cells. Staining with E (Biolegend catalog number 423801) allowed for tracking of cell proliferation. 2 x 10⁶ wells 5Cells were plated in 96-well U-bottom plates with 50 ng / ml of soluble anti-CD3 antibody (clone 145.2C11; Biolegend #100339) and anti-CD28 antibody (clone 37.51; Biolegend #102115), respectively, and with 10 μg / ml of soluble anti-BTLA antibody or isotype control. After 72 hours, cells were analyzed by flow cytometry to assess proliferation ("anti-CD3 / CD28 (CD4 T cell proliferation)") and T cell activation by staining for a surface expression activation marker ("anti-CD3 / CD28 (CD69+ CD4 T cell)"). For each BTLA antibody, the inhibition percentage compared to the isotype control antibody was calculated.

[0278] Furthermore, for each BTLA antibody, its ligand-blocking ability, such as competition with HVEM for binding to BTLA, was evaluated according to the method described in Example 4, and the results were indicated as "yes" for inhibition of HVEM-BTLA binding by more than 90% and as "no" for inhibition of HVEM-BTLA binding by less than 10%. The functional epitopes of each BTLA antibody were also determined according to the method described in Example 5. The "Epitope" column in Table 2 summarizes the epitope groups to which each individual BTLA antibody binds. Antibodies 2.8.6, 6.2, 831, 16H2, 7A1, 16F10, 6G8, 3E8, 4E8, 15C6, 12F11, 10B1, 15B6, 4D3, 16E1, 4D5, and 3A9 all bind to a primary epitope (referred to as "Epitope 1" in the table) containing at least one definitive residue selected from the list: D52, P53, E55, E57, E83, Q86, E103, L106, and E92. Antibodies that bind to Epitope 1 do not compete with ligand HVEM for binding to BTLA. Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, and 26F3 all bind to a different second epitope ("Epitope 2") containing at least one definitive residue selected from the list: Y39, K41, R42, Q43, E45, and S47. Antibodies that bind to Epitope 2 compete with ligand HVEM for binding to BTLA. Antibody 26B1 binds to a third epitope ("Epitope 3") containing at least one definitive residue selected from the list: D35, T78, K81, S121, and L123. Antibodies that bind to Epitope 3 compete with ligand HVEM for binding to BTLA. Antibodies 24H7, 4B1, 8B4, and 4H4 all bind to a different fourth epitope ("Epitope 4") containing the definitive residue H68. The antibody that binds to epitope 4 does not compete with ligand HVEM for binding to BTLA. Antibody 21C7 binds to a different fifth epitope ("epitope 5") containing at least one definitive residue selected from list:N65 and A64. The antibody that binds to epitope 5 does not compete with ligand HVEM for binding to BTLA.

[0279] Example 21. Humanization and CDR operation of BTLA antibodies 3E8 and 6.2 The variable domains of 3E8 and 6.2 were humanized by germlining them into homologous human germline framework regions (sequence numbers 382-385). For 3E8, the selected acceptor frameworks were VH1-1-08 and JH6 for the heavy chain, and VK3-L6 and JK2 for the light chain. For 6.2, the selected acceptor frameworks were VH3-3-21 and JH6 for the heavy chain, and VK2-A19 and JK4 for the light chain.

[0280] In some cases, it is possible to remove undesirable properties without significantly affecting target binding by substituting specific residues in the CDR of an antibody. The CDRH2 of humanized antibody 6.2 was modified with D54E and N56Q substitutions (SEQ ID NO: 387) to remove deamidation potential (the modified humanized VH sequence of 6.2 is provided in SEQ ID NO: 390). Similarly, the CDRH of humanized antibody 3E8 was modified as determined by Lonza's Epibase analysis. Sequence 2 was modified with an N57Q substitution (SEQ ID NO: 386) to remove its deamidation potential, and then modified with a K63S substitution to reduce its expected immunogenicity (providing the modified humanized VH sequence of 3E8 as SEQ ID NO: 389).

[0281] Table 2. Characterization of binding affinity and inhibitory effects of exemplary antibodies.

[0282] [Table 2]

[0283] Table 3. Results of assays showing the inhibitory effect of exemplary antibodies

[0284] [Table 3]

[0285] Sequence List

[0286] [ka]

[0287] [ka]

[0288] [ka]

[0289] [ka]

[0290] Table 4. Exemplary CDR sequences

[0291] [Table 4-1]

[0292] [Table 4-2]

[0293] Table 5. Exemplary primary VH and VL sequences

[0294] [Table 5-1]

[0295] [Table 5-2]

[0296] [Table 5-3]

[0297] Table 5-4

[0298] Table 5-5

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VH CDR having an amino acid sequence such as that shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and containing 0 to 3 amino acid modifications.

2. Contains 0-3 amino acid modifications, including sequence numbers 31, 32, 33, 37, 38, 39, 43, 44, 45, 56, 57, 61, 62, 63, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 117, 121, 122, 123, 127, 128, 129, 133 An isolated antibody or antigen-binding fragment thereof that binds to human BTLA, comprising at least one VH CDR having an amino acid sequence as shown in any of 134, 135, 139, 140, 141, 145, 146, 147, 159, 169, 170, 171, 182, 183, 187, 188, 189, 193, 194, 195, 199, 200, 201, 205, 206, 207, 211, 212, 213, 386, 387, and 388.

3. An isolated antibody or its antigen-binding fragment that specifically binds to human BTLA, comprising at least one VL CDR having an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

4. At least one VL having an amino acid sequence containing 0 to 3 amino acid modifications, such as those shown in SEQ ID NOs: 34, 35, 36, 40, 41, 42, 46, 47, 48, 58, 59, 60, 64, 65, 66, 72, 76, 78, 82, 83, 84, 88, 89, 90, 94, 95, 96, 100, 101, 102, 107, 108, 114, 124, 125, 126, 136, 137, 138, 142, 143, 144, 148, 149, 150, 162, 172, 173, 174, 180, 186, 190, 191, 192, 196, 197, 202, 203, 209, 210, 214, or 216. An isolated antibody containing CDR that specifically binds to human BTLA, or an antigen-binding fragment thereof.

5. An isolated human antibody or its antigen-binding fragment that specifically binds to B and T lymphocyte attenuation factors (BTLAs), comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, wherein (i) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, comprising 0 to 3 amino acid modifications; or (ii) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, comprising 0 to 3 amino acid modifications.

6. An isolated human antibody or its antigen-binding fragment that specifically binds to B and T lymphocyte attenuation factors (BTLAs), comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1, CDRH2, and CDRH3 are (1) comprising 0 to 3 amino acid modifications, as in SEQ ID NOs. 31, 32, or 33; (2) comprising 0 to 3 amino acid modifications, as in SEQ ID NOs. 37, 38, or 39; (3) comprising 0 to 3 amino acid modifications, as in SEQ ID NOs. 43, 44, and or 45; (4) SEQ ID NOs. 43, 56, or 57, including 0-3 amino acid modifications; (5) SEQ ID NOs. 61, 62, or 63, including 0-3 amino acid modifications; (6) SEQ ID NOs. 31, 32, or 69, including 0-3 amino acid modifications; (7) SEQ ID NOs. 73, 74, or 75, including 0-3 amino acid modifications; (8) SEQ ID NOs. 79, 80, or 81, including 0-3 amino acid modifications; (9) SEQ ID NOs. 85, 86, or 87, including 0-3 amino acid modifications; (10) SEQ ID NOs. 0-3 amino acid modifications , SEQ ID NOs. 61, 92, or 93; (11) SEQ ID NOs. 97, 98, or 99, including 0-3 amino acid modifications; (12) SEQ ID NOs. 103, 104, or 105, including 0-3 amino acid modifications; (13) SEQ ID NOs. 109, 110, or 111, including 0-3 amino acid modifications; (14) SEQ ID NOs. 85, 110, or 117, including 0-3 amino acid modifications; (15) SEQ ID NOs. 121, 122, or 123, including 0-3 amino acid modifications; (16) SEQ ID NOs. 12, including 0-3 amino acid modifications 7, 128, or 129; (17) SEQ ID NOs: 133, 134, or 135, including 0-3 amino acid modifications; (18) SEQ ID NOs: 139, 140, or 141, including 0-3 amino acid modifications; (19) SEQ ID NOs: 145, 146, or 147, including 0-3 amino acid modifications; (20) SEQ ID NOs: 31, 32, or 33, including 0-3 amino acid modifications; (21) SEQ ID NOs: 31, 32, or 159, including 0-3 amino acid modifications; (22) SEQ ID NOs: 169, 170, including 0-3 amino acid modifications. or 171; (23) SEQ ID NOs. 61, 62, or 63, including 0-3 amino acid modifications; (24) SEQ ID NOs. 31, 182, or 183, including 0-3 amino acid modifications; (25) SEQ ID NOs. 187, 188, or 189, including 0-3 amino acid modifications; (26) SEQ ID NOs. 193, 194, or 195, including 0-3 amino acid modifications; (27) SEQ ID NOs. 199, 200, or 201, including 0-3 amino acid modifications; (28) SEQ ID NOs. 205, 206, or 20 7; (29) SEQ ID NO: 211, 212, or 213, including 0-3 amino acid modifications; (30) SEQ ID NO: 127, 386, or 129, including 0-3 amino acid modifications; (33) SEQ ID NO: 205, 206, or 207, including 0-3 amino acid modifications; (34) SEQ ID NO: 127, 388, or 129, including 0-3 amino acid modifications; or (35) The antibody or its antigen-binding fragment having an amino acid sequence as shown in SEQ ID NO: 205, 387, or 207, including 0-3 amino acid modifications.

7. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (i) CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs: 4, 5, and 6, comprising 0 to 3 amino acid modifications; or (ii) CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs: 10, 11, and 12, comprising 0 to 3 amino acid modifications.

8. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1, CDRL2, and CDRL3 each contain: (1) 0 to 3 amino acid modifications, respectively, as in SEQ ID NOs: 34, 35, and 36; (2) 0 to 3 amino acid modifications, respectively, as in SEQ ID NOs: 40, 41, and 42; (3) 0 to 3 amino acid modifications, respectively, as in SEQ ID NOs: 46, 47, and 48; (4) 0 to 3 amino acid modifications, respectively, as in SEQ ID NOs: 58, 59, and 60; (5) 0 to 3 amino acid modifications, respectively, as in SEQ ID NOs: 64, 65, and 66; (6) 0 to 3 amino acid modifications, respectively (7) SEQ ID NOs: 76, 47, and 78, each containing 0 to 3 amino acid modifications; (8) SEQ ID NOs: 82, 83, and 84, each containing 0 to 3 amino acid modifications; (9) SEQ ID NOs: 88, 89, and 90, each containing 0 to 3 amino acid modifications; (10) SEQ ID NOs: 94, 95, and 96, each containing 0 to 3 amino acid modifications; (11) SEQ ID NOs: 100, 101, and 102, each containing 0 to 3 amino acid modifications; (12) SEQ ID NOs: 64, 107, and 108, each containing 0 to 3 amino acid modifications; (13) SEQ ID NOs: 88, 89, and 114, each containing 0 to 3 amino acid modifications; ( 14) Sequence IDs 124, 125, and 126, each containing 0 to 3 amino acid modifications; (15) Sequence IDs 34, 35, and 36, each containing 0 to 3 amino acid modifications; (16) Sequence IDs 136, 137, and 138, each containing 0 to 3 amino acid modifications; (17) Sequence IDs 142, 143, and 144, each containing 0 to 3 amino acid modifications; (18) Sequence IDs 144, 143, and 144, each containing 0 to 3 amino acid modifications. (19) SEQ ID NO. 148, SEQ ID NO. 149, and SEQ ID NO. 150; (20) SEQ ID NO. 136, SEQ ID NO. 137, and SEQ ID NO. 162, each containing 0 to 3 amino acid modifications; (21) SEQ ID NO. 172, SEQ ID NO. 173, and SEQ ID NO. 174, each containing 0 to 3 amino acid modifications; (22) SEQ ID NO. 64, SEQ ID NO. 65, and SEQ ID NO. 180, each containing 0 to 3 amino acid modifications; (23) SEQ ID NOs: 136, 137, and 186, each containing 0 to 3 amino acid modifications; (24) SEQ ID NOs: 190, 191, and 192, each containing 0 to 3 amino acid modifications; (25) SEQ ID NOs: 196, 197, and 12, each containing 0 to 3 amino acid modifications; (26) SEQ ID NOs: 202, 203, and 6, each containing 0 to 3 amino acid modifications; (27) SEQ ID NOs: 14, each containing 0 to 3 amino acid modifications 2. The antibody or its antigen-binding fragment having an amino acid sequence as shown in SEQ ID NO: 209 and SEQ ID NO: 210; (28) SEQ ID NO: 214, SEQ ID NO: 35 and SEQ ID NO: 216, each containing 0 to 3 amino acid modifications; (29) SEQ ID NO: 10, 11, or 12, each containing 0 to 3 amino acid modifications; (30) SEQ ID NO: 4, 5, or 6, each containing 0 to 3 amino acid modifications; or (31) SEQ ID NO: 142, 143, or 210, each containing 0 to 3 amino acid modifications.

9. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has the amino acid sequence shown in SEQ ID NO: 1, CDRH2 has the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 has the amino acid sequence shown in SEQ ID NO: 3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has the amino acid sequence shown in SEQ ID NO: 4, CDRL2 has the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 has the amino acid sequence shown in SEQ ID NO:

6.

10. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDRH3, where CDRH1 has the amino acid sequence shown in SEQ ID NO: 7, CDRH2 has the amino acid sequence shown in SEQ ID NO: 8, and CDRH3 has the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, where CDRL1 has the amino acid sequence shown in SEQ ID NO: 10, CDRL2 has the amino acid sequence shown in SEQ ID NO: 11, and CDRL3 has the amino acid sequence shown in SEQ ID NO:

12.

11. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDH3, where (1) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NOs: 31, 32, and 33, each comprising 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs: 34, 35, and 36, each comprising 0 to 3 amino acid modifications. (2) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NOs: 37, 38, and 39, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs: 40, 41, and 42, each containing 0 to 3 amino acid modifications; (3) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NOs: 43, 44, and 45, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs: 6, 47, and 48, each containing 0 to 3 amino acid modifications; (4) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NOs: 43, 56, and 57, each containing 0 to 3 amino acid modifications (5) Having a sequence, and CDRL1, CDRL2, and CDRL3 each having an amino acid sequence as shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, each containing 0 to 3 amino acid modifications; (6) Having an amino acid sequence as shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each having an amino acid sequence as shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, each containing 0 to 3 amino acid modifications; (7) Having an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 69, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each having an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 72, each containing 0 to 3 amino acid modifications;(7) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 73, 74, and 75, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs. 76, 47, and 78, each containing 0 to 3 amino acid modifications; (8) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 79, 80, and 81, each containing 0 to 3 amino acid modifications, and CD (9) Whether RL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs. 82, 83, and 84, each containing 0 to 3 amino acid modifications; (10) C (11) DRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 61, 92, and 93, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs. 94, 95, and 96, each containing 0 to 3 amino acid modifications; (12) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 97, 98, and 99, each containing 0 to 3 amino acid modifications, and CDRL1 (12) CDRL2 and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 100, 101, and 102, each containing 0 to 3 amino acid modifications; (2) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs: 103, 104, and 105, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 64, 107, and 108, each containing 0 to 3 amino acid modifications;(13) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 114, each containing 0 to 3 amino acid modifications; (14) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NOs. 85, 110, and 117, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NOs. 88, 89, and 114, each containing 0 to 3 amino acid modifications; (15) CDRH1, CDRH2, and CDRH3 each contain 0 to 3 amino acid (16) Having an amino acid sequence as shown in SEQ ID NOs: 121, 122, and 123, including modifications, and CDRL1, CDRL2, and CDRL3 having an amino acid sequence as shown in SEQ ID NOs: 124, 125, and 126, each including 0 to 3 amino acid modifications; (16) Having an amino acid sequence as shown in SEQ ID NOs: 127, 128, and 129, each including 0 to 3 amino acid modifications (17) CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, each containing 0 to 3 amino acid modifications; (17) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135, each containing 0 to 3 amino acid modifications; (18) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs: 139, 140, and 141, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 142, 143, and 144, each containing 0 to 3 amino acid modifications;(19) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, each containing 0 to 3 amino acid modifications; (20) CDRH1, CDRH2, and CDRH3 each contain 0 to 3 amino acid modifications (1) Having an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and CDRL1, CDRL2, and CDRL3 each having an amino acid sequence as shown in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, and having an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 15933, and CDRL1, CDRL2, and CDRL3 each having an amino acid modification of 0 to 3 amino acids; (21) Having an amino acid sequence as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 159, and (22) CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 162, each containing 0 to 3 amino acid modifications; (22) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, each containing 0 to 3 amino acid modifications (1) (2) (3) (4) (5) (6) (1(24) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NO: 31, SEQ ID NO: 182, and SEQ ID NO: 183, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 186, each containing 0 to 3 amino acid modifications; (25) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have; (26) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs: 190, 191, and 192, which include 0 to 3 amino acid modifications; and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 193, 194, and 195, which include 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 196, 197, and 12, which include 0 to 3 amino acid modifications. (27) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 6, each containing 0 to 3 amino acid modifications; (28) CDRH1, CDRH2, and CDRH3 each contain 0 to 3 amino acid modifications (29) CDRH1, CDRH2, CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 211, SEQ ID NOs. 212, and SEQ ID NOs. 210, and CDRL1, CDRL2, and CDRL3 each have amino acid modifications of 0 to 3; (29) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs. 211, SEQ ID NOs. 212, and SEQ ID NOs. 213, and CD (30) RL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 214, SEQ ID NO: 35, and SEQ ID NO: 216, each containing 0 to 3 amino acid modifications; (30) CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 127, SEQ ID NO: 386, and SEQ ID NO: 129, and CDRL1, CDRL2, and CDRL3 each contain 0 to 3 amino acid modifications, SEQ ID NO: 34, SEQ ID NO: 216 (31) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs: 205, 206, and 207, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 142, 209, and 210, each containing 0 to 3 amino acid modifications; (32) CDRH1, CDRH2, and CDRH3 each have amino acid sequences as shown in SEQ ID NOs: 127, 388, and 129, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have amino acid sequences as shown in SEQ ID NOs: 34, 35, and 36, each containing 0 to 3 amino acid modifications. (33) The antibody or its antigen-binding fragment, wherein CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210, each containing 0 to 3 amino acid modifications; or (34) The antibody or its antigen-binding fragment, wherein CDRH1, CDRH2, and CDRH3 each have an amino acid sequence as shown in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207, each containing 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each have an amino acid sequence as shown in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 210, each containing 0 to 3 amino acid modifications.

12. An isolated human antibody or its antigen-binding fragment, comprising a heavy chain and a light chain, which specifically binds to BTLA, wherein the heavy chain comprises a variable region having an amino acid sequence as shown in SEQ ID NO: 13, or a sequence having at least 90% identity to said sequence.

13. Isolated human antibodies, including heavy and light chains, that specifically bind to BTLA, or their antigen binding. An antibody or antigen-binding fragment comprising a light chain variable region having an amino acid sequence as shown in Sequence ID No. 14, or a sequence having at least 90% identity to said sequence.

14. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 14, and the antibody binds to BTLA.

15. An isolated human antibody or its antigen-binding fragment, comprising a heavy chain and a light chain, which specifically binds to BTLA, wherein the heavy chain comprises a variable heavy chain region having an amino acid sequence as shown in SEQ ID NO: 17 or 21, or a sequence having at least 90% identity to said sequence.

16. An isolated human antibody or its antigen-binding fragment, comprising a heavy chain and a light chain, which specifically binds to BTLA, wherein the light chain comprises a variable region having an amino acid sequence as shown in SEQ ID NOs. 15 and 19, or a sequence having at least 90% identity to said sequence.

17. (1) The heavy chain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 15; or (2) The heavy chain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 21, and the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, and the antibody specifically binds to BTLA, the antibody or its antigen-binding fragment according to any of the preceding claims.

18. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable heavy chain region containing an amino acid sequence as shown in SEQ ID NOs: 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 326, 327, 330, 331, 382, ​​384, 389, or 390, or a sequence having at least 90% identity to said sequence.

19. An isolated human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a variable region of the light chain containing an amino acid sequence as shown in SEQ ID NOs: 351, 352, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 366, 367, 368, 369, 370, 372, 374, 375, 376, 377, 378, 380, 381, 383, or 385, or a sequence having at least 90% identity to said sequence.

20. (1) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 301, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 351; (2) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 302, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 352; (3) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 303, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 353; (4) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 305, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 355; (5) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 306, and the light chain includes an amino acid sequence as shown in SEQ ID NO: 356 (6) The heavy chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 307, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 357; (7) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 308, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 358; (8) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 309, and the light chain includes an amino acid sequence as shown in SEQ ID NO: 359 (9) The heavy chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO 310, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO 360; (10) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO 311, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO 361; (11) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO 312, and the light chain includes an amino acid sequence as shown in SEQ ID NO 362 (12) The heavy chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 313, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 363; (13) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 314, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 364; (14) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 315, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 364 (15) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 316, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 366; (16) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 317, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 367; (17) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 318, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 368;(18) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 319, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 369; (19) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 320, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 370; (20) Whether the heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 321, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 370; (21) (22) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 322, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 372; (23) The heavy chain includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 306, and the light chain includes a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 375; (24) The heavy chain is (25) The heavy chain contains a variable heavy chain region containing an amino acid sequence as shown in sequence number 326, and the light chain contains a variable light chain region containing an amino acid sequence as shown in sequence number 376; (26) The heavy chain contains a variable heavy chain region containing an amino acid sequence as shown in sequence number 327, and the light chain contains a variable light chain region containing an amino acid sequence as shown in sequence number 377; (27) The heavy chain contains a variable heavy chain region containing an amino acid sequence as shown in sequence number 17, and the light chain contains a variable light chain region containing an amino acid sequence as shown in sequence number 15; (28) The heavy chain contains an amino acid sequence as shown in sequence number 21 (29) The heavy chain includes a variable heavy chain region containing an amino acid sequence as shown, and the light chain includes a variable light chain region containing an amino acid sequence as shown in SEQ ID NO: 19; (30) The heavy chain includes a variable heavy chain region containing an amino acid sequence as shown in SEQ ID NO: 330, and the light chain includes a variable light chain region containing an amino acid sequence as shown in SEQ ID NO: 380; and the antibody specifically binds to BTLA;(31) The heavy chain includes a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 382, ​​and the light chain includes a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 383; (32) The heavy chain is SEQ ID NO: 3; (33) an antibody or antigen-binding fragment of any of the preceding claims, comprising a heavy chain variable region having an amino acid sequence as shown in 84, and a light chain comprising a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 385; (34) a heavy chain comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 390, and a light chain comprising a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 385; or (35) an antibody or antigen-binding fragment of any of the preceding claims, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 390, and a light chain comprising a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 378, and the antibody binds to BTLA.

21. An antibody or antigen-binding fragment of any of the preceding claims, wherein the heavy chain or light chain also includes a constant region.

22. An antibody or antigen-binding fragment according to any of the preceding claims, wherein a heavy chain and a light chain are linked by a flexible linker to form a single-chain antibody.

23. An antibody or antigen-binding fragment of any of the preceding claims, which binds to a residue of BTLA selected from A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, D135.

24. The antibody or antigen-binding fragment of claim 23, which binds to a residue of BTLA selected from Y39 and / or K41.

25. An antibody or antigen-binding fragment of claim 23 or 24, which binds to a BTLA residue selected from Y39 and K41.

26. An antibody or antigen-binding fragment thereof according to any of the preceding claims, which is an IgG1, IgG2, or IgG4 antibody.

27. An antibody according to any of the preceding claims, selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., bispecific antibodies).

28. An antigen-binding fragment according to any one of claims 1 to 26, selected from the group consisting of scFv, sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and a deabody.

29. A monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims.

30. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment agonizes human BTLA expressed on the surface of an immune cell, and the immune cell may be a T cell.

31. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the binding of the antibody or antigen-binding fragment to human BTLA expressed on the surface of an immune cell reduces the proliferation of the immune cell compared to a comparable immune cell to which the antibody or antigen-binding fragment is not bound, and the cell may be a T cell.

32. The antibody or antigen-binding fragment of claim 31, wherein the reduction in cell proliferation is measured by the assay described in Example 8 or Example 9.

33. The antibody or antigen-binding fragment of claim 31 or 32, wherein the reduction in cell proliferation is measured in vitro or in vivo.

34. The antibody or antigen-binding fragment of any one of claims 31 to 33, wherein the reduction in cell proliferation is at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%.

35. The antibody or antigen-binding fragment of any one of claims 31 to 33, wherein the reduction in cell proliferation is approximately 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 50%, 20% to 40%, or 20% to 30%.

36. An antibody or antigen-binding fragment of any of the preceding claims, comprising a domain that binds to an Fc receptor.

37. The antibody or antigen-binding fragment of claim 36, wherein the Fc receptor is expressed on the surface of immune cells.

38. The antibody or antigen-binding fragment of claim 37, wherein the immune cells are antigen-presenting cells.

39. The antibody or antigen-binding fragment according to claim 38, wherein the antigen-presenting cell is a dendritic cell, macrophage, monocyte, or neutrophil.

40. An antibody or antigen-binding fragment according to any one of claims 36 to 39, which binds to human BTLA expressed on the surface of T cells.

41. The antibody or antigen-binding fragment of claim 40, wherein, as a result of the binding of the antibody or antigen-binding fragment to the Fc receptor expressed on the surface of the immune cells and the binding of the antibody or antigen-binding fragment to human BTLA on the surface of the T cells, the cell surfaces of the immune cells and the T cells are within 250 Å, 200 Å, 150 Å, or 100 Å.

42. The antibody or antigen-binding fragment according to claim 40, wherein, as a result of the binding of the antibody or antigen-binding fragment to the Fc receptor expressed on the surface of the immune cells and the binding of the antibody or antigen-binding fragment to human BTLA on the surface of the T cells, the cell surfaces of the immune cells and the T cells are within 250 Å to 100 Å, 200 Å to 100 Å, 150 Å to 100 Å, 200 Å to 150 Å, or 250 Å to 150 Å.

43. The antibody or antigen-binding fragment according to any one of claims 36 to 42, wherein the Fc receptor is FcγRIIB.

44. The antibody or antigen-binding fragment according to any of the preceding claims, wherein the binding of the antibody or antigen-binding fragment to human BTLA expressed on the surface of an immune cell reduces NFκB signaling of the immune cell compared to a comparable immune cell to which the antibody or antigen-binding fragment is not bound, and the immune cell may be a T cell.

45. The antibody or antigen-binding fragment of claim 44, wherein the reduction in NFκB signaling of the immune cells is measured by the assay described in Example 10.

46. The antibody or antigen-binding fragment of claim 44 or 45, wherein the reduction in NFκB signaling of the immune cells is at least about 10%, 15%, 20%, 25%, 30%, or 40%.

47. The antibody or antigen-binding fragment of claim 44 or 45, wherein the reduction in NFκB signaling of the immune cells is about 10% to 40%, 10% to 30%, 10% to 20%, 20% to 40%, or 20% to 30%.

48. The antibody or antigen-binding fragment according to any of the preceding claims, wherein the binding of the antibody or antigen-binding fragment to human BTLA expressed on the surface of immune cells reduces the dephosphorylation of the cytoplasmic domain of the human BTLA.

49. The antibody or antigen-binding fragment of claim 48, wherein the dephosphorylation is mediated by CD45 expressed on the surface of the immune cell.

50. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte-decaying factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C, and the antibody binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C; does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

51. At 37°C, at least 5.0 x 10 ５ The antibody or antigen-binding fragment of claim 50, which binds to human B and T lymphocyte-decaying factor (BTLA) at an on-rate of (1 / Ms).

52. 37℃, 3.0 x 10 －４ An antibody or antigen-binding fragment of claim 50 or 51 that binds to human B and T lymphocyte-decaying factor (BTLA) at an off-rate of less than (1 / s).

53. 3.0 x 10 －４ (1 / s) ~1.0x10 －３ An antibody or antigen-binding fragment according to any one of claims 50 to 52, which binds to human B and T lymphocyte-decaying factor (BTLA) at an off-rate of (1 / s).

54. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment, when determined by surface plasmon resonance (SPR) at 37°C, has a density of at least 5.0 x 10⁻¹⁶ ５ An antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on rate of (1 / Ms), the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM), and the antibody inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay.

55. 3.0 x 10 －４ The antibody or antigen-binding fragment of claim 54, which binds to human B and T lymphocyte-decaying factor (BTLA) at an off-rate of less than (1 / s).

56. The antibody or antigen-binding fragment of claim 54 or 55, which binds to human B and T lymphocyte-decaying factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C.

57. An antibody or antigen-binding fragment according to any one of claims 54 to 56, which binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C.

58. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment, when measured by surface plasmon resonance (SPR) at 37°C, has a coefficient of 3.0 x 10⁻¹⁶. －４ (1 / Ms) ~1.0x10 －３ The antibody or its antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an off rate of (1 / Ms), the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM), and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

59. The antibody or antigen-binding fragment of claim 58, which binds to human B and T lymphocyte-decaying factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C.

60. The antibody or antigen-binding fragment of claim 58 or 59, which binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C.

61. At least 5.0 x 10 ５ An antibody or antigen-binding fragment thereof according to any one of claims 58 to 60, which binds to human B and T lymphocyte attenuator (BTLA) at an on-rate of (1 / Ms).

62. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment, when measured at 37°C by surface plasmon resonance (SPR), has a coefficient of 1.0 x 10⁻¹⁶. －３ Off speed less than (1 / Ms) and at least 5.0 x 10 ５ An antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on rate of (1 / Ms), the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM), and the antibody inhibits T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay.

63. The antibody or antigen-binding fragment of claim 62, which binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C.

64. The antibody or antigen-binding fragment of claim 62 or 63, which binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C.

65. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA) with a KD of less than 2 nM as determined by surface plasmon resonance (SPR) at 37°C, the antibody inhibits the binding of BTLA to herpesvirus entry mediator (HVEM), and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

66. When determined by surface plasmon resonance (SPR) at 37°C, it was 1.0 x 10⁻⁶. ６ The antibody or antigen-binding fragment of claim 65, which binds to human B and T lymphocyte-decaying factor (BTLA) at an on-rate of less than 1 / Ms.

67. When determined by surface plasmon resonance (SPR) at 37°C, it was 1.0 x 10⁻⁶. －３ An antibody or antigen-binding fragment of claim 65 or 66 that binds to human B and T lymphocyte-decaying factor (BTLA) at an off-rate of less than (1 / s).

68. An antibody or antigen-binding fragment according to any one of claims 65 to 67, which binds to cynomolgus monkey B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C.

69. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment comprises 1 x 10 －３ The antibody or its antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an off-rate of less than (1 / s), thereby inhibiting the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibiting T cell proliferation in vitro, as determined by a mixed lymphocyte reaction assay.

70. The antibody or antigen-binding fragment of claim 69, which binds to cynomolgus monkey B and T lymphocyte attenuation factor (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37°C.

71. An antibody or antigen-binding fragment of claim 69 or 70, which binds to human B and T lymphocyte-decaying factor (BTLA) with a KD of less than 2 nM as determined by surface plasmon resonance (SPR) at 37°C.

72. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA), the antibody binds to cynomolgus monkey BTLA with a KD of at least 5 nM as determined by surface plasmon resonance (SPR) at 37°C, the antibody inhibits the binding of BTLA to herpesvirus entry mediator (HVEM), and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

73. An antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA), the antibody binds to cynomolgus monkey BTLA with a KD of at least 50 nM as determined by surface plasmon resonance (SPR) at 37°C, and the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM), and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

74. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA) at a KD of 1400 nM to 3500 nM as measured by surface plasmon resonance (SPR) at 37°C; the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

75. When measured at 37°C by surface plasmon resonance (SPR), at least 2.0 x 10⁻¹⁰ units were found. ５ The antibody or antigen-binding fragment of claim 74, which binds to human BTLA at an on-rate of (1 / Ms).

76. When measured at 37°C using surface plasmon resonance (SPR), the result was 10.0 x 10⁻⁶. －１ An antibody or antigen-binding fragment of claim 74 or 75 that binds to human BTLA at an off-rate of less than (1 / s).

77. An antibody or antigen-binding fragment according to any one of claims 74 to 76, which binds to an epitope that blocks the binding of antibody 4H4.

78. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment, when measured by surface plasmon resonance (SPR) at 37°C, has a reading of 1.7 x 10⁻¹⁴. ５ (1 / Ms) ~2.5x10 ５ An antibody or its antigen-binding fragment that specifically binds to human B and T lymphocyte attenuation factor (BTLA) at an on-rate of (1 / Ms); and the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

79. When measured at 37°C using surface plasmon resonance (SPR), the result was 3.0 x 10⁻⁶. －１ The antibody or antigen-binding fragment of claim 78, which binds to human BTLA at an off-speed of less than (1 / s).

80. When measured at 37°C using surface plasmon resonance (SPR), the result was 3.0 x 10⁻⁶. －１ (1 / s) ~5.0x10 －１ The antibody or antigen-binding fragment of claim 78 or 79, which binds to human BTLA at an off-speed of (1 / s).

81. An antibody or antigen-binding fragment according to any one of claims 78 to 80, which binds to human BTLA with a KD of at least 150 nM when measured at 37°C by surface plasmon resonance (SPR).

82. An antibody or antigen-binding fragment according to any one of claims 78 to 81, which binds to human BTLA with a KD of 150 nM to 1500 nM when measured at 37°C by surface plasmon resonance (SPR).

83. An antibody or antigen-binding fragment according to any one of claims 78 to 82, which binds to an epitope that blocks the binding of the 286 antibody.

84. An antibody or antigen-binding fragment according to any of the preceding claims, wherein the antibody or antigen-binding fragment specifically binds to human B and T lymphocyte attenuation factor (BTLA) at a KD of 40 nM to 1200 nM as measured by surface plasmon resonance (SPR) at 37°C; the antibody does not inhibit the binding of BTLA to herpesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

85. When measured at 37°C by surface plasmon resonance (SPR), at least 1.0 x 10⁻¹⁰ ５ The antibody or antigen-binding fragment of claim 84, which binds to human BTLA at an on-rate of (1 / Ms).

86. When measured at 37°C using surface plasmon resonance (SPR), the result was 1.0 x 10⁻⁶. ５ (1 / Ms) ~10x10 ５ The antibody or antigen-binding fragment of claim 84 or 85, which binds to human BTLA at an on-rate of (1 / Ms).

87. When measured at 37°C using surface plasmon resonance (SPR), the result was 6.0 x 10⁻¹⁰. －１ An antibody or antigen-binding fragment according to any one of claims 84 to 86, which binds to human BTLA at an off-rate of less than (1 / s).

88. When measured at 37°C using surface plasmon resonance (SPR), the result was 6.0 x 10⁻¹⁰. －１ (1 / s) ~10.0x10 －２ An antibody or antigen-binding fragment according to any one of claims 84 to 87, which binds to human BTLA at an off-speed of (1 / s).

89. An isolated nucleic acid comprising one or more nucleotide sequences encoding a polypeptide capable of forming an antibody or antigen-binding fragment as claimed in any of claims 1 to 88.

90. A host cell comprising the nucleic acid sequence described in claim 89.

91. A method for producing an antibody or antigen-binding fragment thereof that binds to BTLA, comprising the step of culturing the host cells of claim 90 under conditions for the production of the antibody or antigen-binding fragment thereof, and further comprising the step of isolating and / or purifying the antibody or antigen-binding fragment thereof.

92. A method for preparing a human antibody or its antigen-binding fragment that specifically binds to BTLA, comprising: 1) Provide a host cell containing one or more nucleic acid molecules that, when expressed, can be combined to produce an antibody or an antigen-binding molecule, encoding heavy-chain and light-chain amino acid sequences; 2) Culture host cells that express the encoded amino acid sequence; and 3) Isolating an antibody or antigen-binding molecule according to any of claims 1 to 88. The method, including the steps.

93. A pharmaceutical composition comprising a therapeutically effective amount of any antibody or antigen-binding fragment according to claims 1 to 88, and at least one pharmaceutically acceptable excipient.

94. An antibody or its antigen-binding fragment according to any one of claims 1 to 88, or a pharmaceutical composition according to claim 93, for use in therapy.

95. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 88, or a pharmaceutical composition according to claim 93, for use in the treatment or prevention of inflammatory or autoimmune diseases and disorders of excessive immune cell proliferation.

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