BTLA antibodies

Abstract

To provide new and useful agents, such as antibodies or antigen-binding antibody fragments, capable of modulating the BTLA.SOLUTION: The invention provides: agonistic antibodies that bind the human B and T lymphocyte attenuator (BTLA)and modulate its activity; and their use in treating inflammatory, autoimmune and proliferative diseases and disorders.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] FIELD OF THEINVENTION The present invention relates generally to antibodies or antigen-binding fragments that bind to human B and T lymphocyte attenuator (BTLA) and uses thereof. More specifically, the present invention relates to agonistic antibodies that bind to and modulate the activity of human BTLA and their use in treating inflammatory, autoimmune and proliferative diseases and disorders. [Background technology]

[0003] The immune system must achieve a balance between the destruction of pathogens or dangerously mutated cells and the tolerance of healthy self-tissues and harmless symbionts. To facilitate this balance, the activity of immune cells is influenced by the integration of signals from a multitude of stimulatory and inhibitory receptors that adapt the cell to its environment. These surface-expressed receptors present attractive targets for therapeutic modulation of the immune response. Many human diseases result from aberrant or unwanted activation of the immune system, including autoimmune diseases, graft rejection and graft-versus-host disease. Agonistic agents capable of inducing signaling through inhibitory receptors could attenuate these unwanted immune responses.

[0004] B and T lymphocyte attenuation factor (BTLA; also called CD272) is an inhibitory member of the CD28 family of receptors, 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 myeloid and lymphocytes (Han et al., J Immunol. 172:5931-9, 2004). After binding of its ligand, herpesvirus entry mediator (HVEM), BTLA recruits the phosphatases SHP-1 and SHP-2 to the cytoplasmic domain of BTLA (Sedy et al., Nat Immunol. 6:90-8, 2005), which then inhibits the signaling cascade of activated receptors. Mice lacking an intact BTLA gene show hyperproliferative B and T cell responses in vitro, higher titers following DNP-KLH immunization, and increased susceptibility to EAE (Watanabe et al., Nat. Immunol, 4:670-679, 2003). When aged BTLA knockout mice were observed to spontaneously develop autoantibodies, autoimmune hepatitis-like disease occurred, and inflammatory cells infiltrated multiple organs (Oya et al., Arthritis Rheum 58:2498-2510, 2008). This evidence indicates that BTLA inhibitory receptors play a crucial role in maintaining immune homeostasis and inhibiting autoimmunity. Furthermore, HVEM-BTLA signaling is involved in the control of mucosal inflammation and infection immunity (Shui et al., J Leukoc Biol. 89:517-523, 2011).

[0005] Therapeutic agents capable of modulating BTLA function to inhibit autoreactive lymphocytes in the context of autoimmune disorders would be highly desirable. It has been previously shown that monoclonal antibodies that bind to mouse BTLA can act as agonists, inducing signaling through the receptor and inhibiting immune cell responses. In the presence of agonistic anti-BTLA antibodies (mAbs), anti-CD3 and anti-CD28 activated T cells were Cells exhibit reduced IL-2 production and proliferation (Kreig et al., J. Immunol., 175, 6420-6472, 2005).

[0006] Additionally, anti-mouse BTLA agonistic antibodies have been shown to ameliorate disease in murine models of graft-versus-host disease (Sakoda et al., Blood. 117:2506-2514; Albring et al., J Exp Med. 207:2551-9, 2010). Agonistic antibodies targeting the human BTLA receptor have been shown to inhibit T cell responses ex vivo (Otsuki et al., Biochem Biophys Res Commun 344:1121-7, 2006; and see WO2011 / 014438), but have not been translated to the clinic. [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 Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need in the art to discover new 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 anti-human BTLA agonist antibodies or antibody fragments thereof that have one or more desirable properties, including high binding affinity, high agonist potency, high agonist efficacy, excellent pharmacokinetics, and low antigenicity in human subjects. The present invention also relates to the use of the antibodies or antibody fragments of the present invention in the treatment of disease.

[0011] According to a first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to BTLA, wherein the antibody 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, The present invention provides an antibody or antigen-binding fragment thereof having a heavy chain and / or a light chain comprising at least one complementarity determining region (CDR) as presented in an antibody selected from the group consisting of 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, 6.2 and 831.

[0012] According to a second aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding a heavy or light chain polypeptide of an isolated antibody, or antigen-binding fragment thereof, of the first aspect of the invention.

[0013] According to a third aspect of the invention there is provided a vector comprising a nucleic acid of the second aspect of the invention. According to a fourth aspect of the invention there is provided a host cell comprising a nucleic acid sequence according to the second aspect of the invention or a vector according to the third aspect of the invention.

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

[0015] According to a sixth aspect of the invention there is provided a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or produced according to the fifth aspect of the invention.

[0016] According to a seventh aspect of the invention there is provided a method of preparing a pharmaceutical composition comprising the step of formulating an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or one produced according to the fifth aspect of the invention, into a composition comprising at least one further component, in a particular embodiment the at least one further component is a pharma- ceutically acceptable excipient.

[0017] According to an eighth aspect of the invention there is provided a method of treating a BTLA-associated disease in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the first aspect of the invention, or a pharmaceutical composition of the sixth aspect of the invention. [Brief description of the drawings]

[0018] [Figure 1] Binding of antibodies to soluble and cell-expressed human and cynomolgus BTLA. (a) Surface plasmon resonance (SPR) binding curves for soluble monomeric human BTLA extracellular domain injected at increasing concentrations onto immobilized anti-BTLA antibody; graph shows SPR signal after reference and blank subtraction. (b) Association and dissociation rates for binding to human or cynomolgus BTLA calculated by curve fitting using BiaEvaluation software. (c) Binding of antibody 2.8.6 compared to an isotype control antibody to human BTLA or cynomolgus BTLA expressing Jurkat cell lines. (d) EC50 for antibody binding to transfected cell lines as calculated by nonlinear curve fitting using GraphPad Prism software. [Diagram 2] (a) Blocking of ligand binding by anti-BTLA antibodies was assessed by SPR. Human BTLA extracellular domain was immobilized on a sensor chip. Human HVEM was injected to confirm binding and then allowed to dissociate completely. A saturating concentration of anti-BTLA antibody was then injected immediately followed 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. Saturation of BTLA with clone 11.5.1, but not clone 2.8.6, blocked subsequent binding of ligand. [Diagram 3]Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfected with BTLA constructs in a bicistronic vector that also expresses 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 carrying the Y39R mutation (bottom). (b) Binding to each BTLA mutant construct was expressed as a percentage of binding to wild-type BTLA for clones 2.8.6 and 11.5.1. (c) Mutations Y39R and K41E that selectively eliminate binding of clone 11.5.1 were mapped onto the crystal structure of human BTLA (black residues). Residues critical for binding of the ligand HVEM are highlighted in grey. [Figure 4] (a) Crystal structure of the human BTLA extracellular domain in complex with the Fab' fragment of clone 2.8.6. Residues on BTLA that are buried at the interface are highlighted in black. (b) The epitope of antibody 2.8.6 is shown (black residues) in relation to the HVEM binding site (grey residues). [Diagram 5] (a) Strategy for the generation of a chimeric BTLA gene in a humanized BTLA mouse. A section of human genomic DNA from the start of exon 2 to the end of exon 3 was inserted into the mouse locus to replace the mouse sequence from the start of exon 2 to the end of exon 4. The exon-intron junction sequences at the start of mouse exon 2 and the end of mouse exon 4 were left intact to ensure proper splicing. [Figure 6](a) Protocol for 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 next day, mice were immunized with ovalbumin in alum to activate the transferred cells, and 24 hours later, anti-human BTLA antibodies or isotype control were administered. Eight days after the initial cell transfer, the ratio of humanized to wild-type cells in the transferred population in the spleen was assessed by flow cytometry. (b) Both clones 11.5.1 and, to a lesser extent, 2.8.6, reduced the proliferation of humanized cells compared to wild-type. Graphs show 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. T cells from humanized C57BL / 6 mice were stained with CellTraceViolet and added to mitomycin C-treated Balb / c stimulator cells in the presence of anti-BTLA antibody or isotype control. After 96 hours, proliferation of humanized CD4 cells was assessed and normalized to proliferation in the absence of antibody. Clone 2.8.6 inhibited proliferation of humanized cells with an IC50 of 0.029 nM and had a maximal effect of 42% inhibition of proliferation. [Figure 8](a) Effect of clone 2.8.6 in a T-cell colitis model. RAG knockout recipient mice were injected with CD45RBhiCD25-CD4+ T cells from humanized BTLA mice and treated with 200 μg 2.8.6 or isotype control antibody on days 7, 21, and 35. Isotype control-treated mice progressively lost weight from 3 weeks onwards, while 2.8.6-treated mice were spared. (b) Eight weeks after cell transfer, colons were 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) Colon weight-to-length ratio was calculated as a marker of inflammation and hypertrophy. 2.8.6 treatment prevented the increase in weight-to-length ratio seen in isotype control-treated mice. [Figure 9] (a) Effect of BTLA antibodies in a parent-to-F1 model of GVHD. C57BL / 6 splenocytes and bone marrow cells from humanized BTLA mice were injected into CB6F1 recipient mice, which were then treated with anti-BTLA antibodies or isotype control. Untreated mice developed clinical GVHD with progressive weight loss, dermatitis and diarrhea, and were culled upon reaching a pre-specified humane endpoint. 2.8.6 and 11.5.1 antibody-treated mice were relatively spared, with survival comparable to syngeneic cell-reconstituted control mice. (b) Five weeks after cell transfer, mice were culled and colon weight-to-length ratios were calculated as a marker of intestinal inflammation. 2.8.6 and 11.5.1 treatment prevented the colon thickening seen in untreated mice. [Figure 10] (a) Effect of the D265A mutant clone 11.5.1 in an in vivo T cell transfer assay. This mutant antibody, which does not bind to Fc receptors, no longer inhibited proliferation of humanized BTLA cells, but instead led to enhanced proliferation due to receptor blockade. (b) The D265A mutant 11.5.1 antibody no longer inhibited T cell proliferation in a mixed lymphocyte reaction. [Figure 11]Anti-BTLA antibodies do not fix complement. Spleen cells from humanized BTLA mice were incubated with 10% rabbit complement in the presence of 20 μg / ml BTLA antibody, isotype control or positive control (depleting CD20 antibody) for 1 hour at 37° C. The anti-CD20 antibody depleted the majority of B cells, confirming rabbit complement activity, whereas the BTLA antibody did not deplete either the B or T cell populations, even though both populations stained positive for BTLA. [Figure 12] Anti-BTLA antibodies do not cause antibody-dependent cell-mediated cytotoxicity. Spleen cells from humanized BTLA mice were incubated for 24 hours at 37° C. in the presence of 20 μg / ml BTLA antibody, isotype control or positive control (depleting CD20 antibody). The anti-CD20 antibody depleted the majority of B cells by inducing ADCC by effector cells in the mixture, whereas the BTLA antibody did not deplete either the B or T cell populations, even though both populations stained positive 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. 24 hours later, spleens and bone marrow were harvested and cell populations were assessed by flow cytometry. 2.8.6 did not deplete B or T cells in the spleen or affect the frequency of different B cell precursor populations in the bone marrow. [Figure 14] BTLA expression levels on B cells or CD4+ T cells from humanized mice following 6 days of in vivo incubation with antibodies 2.8.6 or 11.5.1 compared to BTLA expression on cells from mice injected with an isotype control antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

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

[0022] It is to be understood that one, some or all of the features of the various embodiments described herein may be applied to any aspect, unless the context clearly indicates otherwise. Moreover, the various embodiments may be combined to form other aspects 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 detailed description that follows.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised ed., 2000, Oxford University Press provides one of ordinary skill in the art with a common dictionary definition of many of the terms used in this disclosure.

[0024] The term "about" as used herein refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference to "about" a value or parameter herein includes (and describes) aspects directed to the value or parameter itself.

[0025] Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0026] The numbering of amino acids in the variable domains, CDRs and framework regions (FRs) of antibodies is according to Kabat et al., Sequences of Proteins, 1999, 1999, unless otherwise indicated. The definition follows the Kabat definition as set forth in The Immunological Interest, 5th ed. 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, unless the context indicates otherwise, refer to either the protein or the gene (or other nucleic acid encoding all or a portion of BTLA). Human BTLA sequences include all human isotypes and variant forms. 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 naturally occurring variant single nucleotide polymorphisms. Despite allelic variation, 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 monkey (cyno) BTLA is disclosed in Genbank under accession number XP_005548224. A reference polypeptide sequence for 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 cyno BTLA as disclosed in SEQ ID NO: 24.

[0029] The term sequence identity is well known in the art. For the purposes of the present invention, in determining whether a target sequence meets a defined restriction (e.g., 90% identity), the BLAST (Basic local alignment search tool) algorithm (Altschul et al. J Mol Biol 215: A sequence is considered to satisfy the defined restrictions if it is so identified using the Smith-Waterman algorithm (see Smith and Waterman. J. Mol. Biol. 147:195-197, 1981).

[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 of the above, through at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. In particular, as used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies generated from at least two intact antibodies, such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule that contains an antigen recognition site, so long as the antibody exhibits the desired biological activity.

[0031] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen and contains an FcR binding site, which may or may not be functional. As used herein, a BTLA agonist antibody (or antibody fragment) refers to an antibody (or antibody fragment) that binds to BTLA and enhances a coinhibitory signal to T and / or B cells.

[0032] An antigen-binding site refers to a portion of a molecule that binds to all or a portion of a target antigen. In an antibody molecule, the site may be referred to as an antibody antigen-binding site and includes the portion of the antibody that specifically binds to all or a portion of a target antigen. If the antigen is large, the antibody may only bind to a specific portion of the antigen, which portion is referred to as an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. Preferably, an 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 that contain an antigen-binding site. Thus, the term "antigen-binding fragment thereof" when referring to an antibody refers to antibody fragments, such as Fab, Fab', F(ab')2, diabodies, Fv fragments and single chain Fv (scFv) mutants, that possess the antigen recognition site and thus possess the ability to bind to the antigen.

[0034] Antigen-binding immunoglobulin (antibody) fragments are well known in the art. Such fragments do not necessarily have a functional Fc receptor binding site. As used herein, the terms "antibody fragment molecule of the invention", "antibody fragment" and "antigen-binding fragment thereof" are used interchangeably herein.

[0035] The term "BTLA-binding molecule" as used herein refers to both antibodies and antigen-binding fragments thereof that are capable of binding to BTLA. There are five major classes (i.e. isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are designated alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the 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 the different classes of antibodies are typically designated by the corresponding lower case Greek letters, α, δ, ε, γ, and μ, respectively. Optional The light chains of antibodies from most vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0036] A "native antibody" is usually a heterotetrameric Y-shaped glycoprotein of about 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 linkages varies among the 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 end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains. Each heavy chain contains one variable domain (VH) and a constant domain, which in the case of IgG, IgA, and IgD antibodies is C H 1. C H 2, and C H IgM and IgE contain a fourth domain, C H 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 variable length (about 10 to about 60 amino acids in the various IgG subclasses). The variable domains of both the light and heavy chains are linked to the constant domain by a "J" region of about 12 or more amino acids, and the heavy chain also has a "D" region of about 10 additional amino acids. Each class of antibody further contains inter- and intrachain disulfide bonds formed by paired cysteine ​​residues. The heavy chain variable region (VH) and the light chain variable region (VL) can each be further divided into hypervariable regions, termed CDRs, interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL contains three CDRs and four FRs, which are arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various 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 thereof of the present invention may be from any animal species, including murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibodies or antigen-binding antibody fragments are monoclonal, e.g., monoclonal antibodies. In some embodiments, the antibodies or antigen-binding fragments thereof are human or humanized antibodies or antigen-binding fragments thereof. Non-human antibodies or antigen-binding fragments thereof may be humanized by recombinant methods to reduce immunogenicity in humans.

[0038] The antibody or antigen-binding fragment of the present invention may be identified using well-known methods. For example, antigen-binding portions may be selected using phage display or other antigen-binding selection or panning approaches. These antigen-binding portions may then be incorporated into antibody frameworks (e.g., fused to the constant and hinge regions of IgG1 or IgG4 molecules).

[0039] The term "monoclonal antibody" ("mAb"), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of an antibody or fragment thereof as not being a mixture of distinct antibodies or antigen-binding fragments. mAbs are highly specific, binding to a single antigenic site / epitope. It is directed towards.

[0040] mAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art. For example, monoclonal antibodies or antigen-binding fragments thereof according to the invention may be made by the hybridoma method first described by Kohler and Milstein (Nature 256:495, 1975), or by recombinant DNA methods, e.g., as described in U.S. Patent Nos. 4,816,567 and 6,331,415. "Monoclonal antibodies" may also be isolated from phage antibody libraries, e.g., using the techniques described in 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 referred to as an antigen-binding fragment of an antibody of the present invention, which simply means that the molecule is produced or exists in a single clonal type. "Human" antibody (HumAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" is not intended herein to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0042] Human antibodies may be prepared by administering immunogen / antigen to transgenic animals, e.g., immunized xeno-mouse, that have been modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584, for XENOMOUSE® technology). Also see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies generated through human B-cell hybridoma technology. Such animals typically contain all or part of the human immunoglobulin loci, which either replace the endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, e.g., XENOMOUSE TM US Patent Nos. 6,075,181 and 6,150,584, which describe the HUMAB technology; TMUS Patent No. 5,770,429 describes the technology; KM MOUSE TM No. 7,041,870, which describes the technology, and VELOCIMOUSE TM See US Patent Application Publication No. US2007 / 0061900 which describes the technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example by combining with a different human constant region.

[0043] Human antibodies may also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of 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 Boerner et al., J. Immunol., 147:86(1991). Human antibodies produced through human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, 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), and 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 interchangeably. This definition of a human antibody specifically excludes humanized antibodies which comprise non-human antigen-binding residues. As used herein, "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDRs of a non-human antibody have been replaced with the corresponding amino acids from a human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDRs have been replaced by residues from the CDRs of a non-human species (donor antibody), e.g., mouse, rat or rabbit, with the desired specificity, affinity and capacity. A humanized antibody may contain residues that are not found in either the recipient antibody or in the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. In one embodiment of a humanized version of an Ab, some, most or all of the amino acids outside the CDRs have been replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids may be tolerated as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., 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, e.g., 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. Pat. Nos. 6,982,321 and 7,087,409.

[0045] As used herein, "engineered antibody" refers to an antibody, which may be a humanized antibody, in which certain residues have been substituted for others to reduce adverse 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 eliminate deamidation potential and a K63S substitution to reduce expected immunogenicity.

[0046] "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody, or vice versa. The term also includes antibodies that contain a V region from one individual (e.g., a first mouse) from one species and a constant region from another individual (e.g., a second mouse) of the same species. The term "antigen (Ag)" refers to a molecular entity that is used to immunize an immunocompetent vertebrate to produce an antibody (Ab) that recognizes the Ag or to screen an expression library (e.g., phage, yeast, or ribosome display library, among others). In this specification, Ag is used more broadly and is generally intended to include the target molecule that is specifically recognized by Ab, and thus also includes portions or mimetics of molecules used in the immunization process to generate Abs or in library screening to select Abs.

[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 co-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 making bispecific antibodies are within the skill of the art. Bispecific antibodies may be produced by a variety of methods, including, for example, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai et al., (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J Immunol. 148:1547-1553. Additionally, bispecific antibodies may be formed as "diabodies" (Holliger et al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (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 the region or site of an antigen to which an antibody specifically binds, i.e., the region or site that physically contacts the antibody. Thus, the term "epitope" refers to the portion of a molecule capable of being recognized and bound by an antibody at one or more of the antigen-binding regions of the antibody. Typically, an epitope is defined in the context of molecular interactions between an "antibody, or antigen-binding portion thereof (Ab)" and its corresponding antigen. Epitopes often consist of surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, an epitope may be a protein epitope. Protein epitopes may be linear or conformational. In a linear epitope, all of the interaction points between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) within an antigenic protein to which an epitope-specific antibody 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 immunoassays.

[0049] An antibody that "specifically binds" to an epitope is a term well understood in the art, and 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 rapidly, for a longer period, 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 a molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore immunoassays, and the like are among the many assays that may be used to identify antibodies that specifically react with an antigen or receptor, or ligand-binding portions thereof that specifically bind to a cognate ligand or binding partner. TM (GE Healthcare, Piscataway, NJ), KinExA, Fluorescence Activated Cell Sorting (FACS), Octet TM (ForteBio, Inc., Menlo Park, Calif.) and Western blot analysis. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically greater than 10 times background, even more typically greater than 50 times background, even more typically greater than 100 times background, even more typically greater than 500 times background, even more typically greater than 1000 times background, and even more typically greater than 10,000 times background. Antibodies also have an equilibrium dissociation constant (K D or KD) < 7 nM.

[0051] According to a first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to BTLA, wherein the antibody has a heavy and / or light chain comprising at least one CDR, and the antibody 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 antigen-binding fragment thereof competes with its natural ligand, HVEM, for binding to BTLA. In another embodiment, the antibody or antigen-binding fragment thereof does not interfere with binding of HVEM.

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

[0053] 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 as disclosed in Table 1 and described herein, it is meant any antibody or antigen-binding fragment thereof (whether murine, humanized or humanized / engineered) that comprises one or more of, for example, VH CDR1, 2 and 3, or VL CDR1, 2 and 3, or VH CDR1, 2 and 3, and VL CDR1, 2 and 3 from any of the antibodies disclosed in Table 1.

[0054] According to a variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VH CDR that contains 0-3 amino acid modifications, for example 0, 1, 2, or 3 amino acid modifications, and has an amino acid sequence as set forth 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 that do not eliminate the antibody binding affinity or T cell inhibitory effect of the modified amino acid sequence when compared to the unmodified amino acid sequence.

[0055] According to a variation of the first aspect of the invention, a polypeptide comprising 0-3 amino acid modifications and having an amino acid sequence as set forth in any of SEQ ID NO:193, SEQ ID NO:194, or SEQ ID NO:195 is provided. The present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, the antibody comprising at least one VH CDR comprising:

[0056] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:7, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:8, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:9.

[0057] According to another variant of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:193, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:194, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:195.

[0058] According to a variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VL CDR having an amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and comprising 0-3 amino acid modifications.

[0059] According to a variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, the antibody or antigen-binding fragment comprising at least one VL CDR having an amino acid sequence as set forth in SEQ ID NO:196, SEQ ID NO:197, or SEQ ID NO:12, and comprising 0-3 amino acid modifications.

[0060] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:10, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:11, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:12.

[0061] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:196, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:197, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:12.

[0062] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:7, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:8, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:9, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:10, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:11, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:12.

[0063] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:193, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:194, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:195, and the light chain comprises a heavy chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:196, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:197, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:12.

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

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

[0066] According to a variant of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:1, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:2, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:3.

[0067] According to a variant of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:199, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:200, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:201.

[0068] According to a variant of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VL CDR comprising an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0069] According to a variant of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VL CDR comprising an amino acid sequence as set forth in SEQ ID NO:202, SEQ ID NO:203, or SEQ ID NO:6.

[0070] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:6.

[0071] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:202, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:203, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:6.

[0072] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:1, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:2, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:6.

[0073] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:199, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:200, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:201, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:202, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:203, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:6.

[0074] According to another variation of the first aspect of the invention, there is provided a method for the preparation of a medicament for use in a medicament for which the medicament for the invention is selected from the group consisting of: (1) SEQ ID NO: 31, 32, or 33, which comprises from 0 to 3 amino acid modifications; (2) SEQ ID NO: 37, 38, or 39, which comprises from 0 to 3 amino acid modifications; (3) SEQ ID NO: 43, 44, or 45, which comprises from 0 to 3 amino acid modifications; (4) SEQ ID NO: 43, 56, or 57, which comprises from 0 to 3 amino acid modifications; (5) SEQ ID NO: 61, 62, or 63, which comprises from 0 to 3 amino acid modifications; (6) SEQ ID NO: 31, 32, or 69, which comprises from 0 to 3 amino acid modifications; (7) SEQ ID NO: 43, 44, or 45, which comprises from 0 to 3 amino acid modifications; (8) SEQ ID NO: 79, 80, or 81, including 0 to 3 amino acid modifications; (9) SEQ ID NO: 85, 86, or 87, including 0 to 3 amino acid modifications; (10) SEQ ID NO: 61, 92, or 93, including 0 to 3 amino acid modifications; (11) SEQ ID NO: 97, 98, or 99, including 0 to 3 amino acid modifications; (12) SEQ ID NO: 103, 104, or 105, including 0 to 3 amino acid modifications; (13) SEQ ID NO: 109, 110, or 111, including 0 to 3 amino acid modifications. (14) SEQ ID NO: 85, 110, or 117, containing 0 to 3 amino acid modifications; (15) SEQ ID NO: 121, 122, or 123, containing 0 to 3 amino acid modifications; (16) SEQ ID NO: 127, 128, or 129, containing 0 to 3 amino acid modifications; (17) SEQ ID NO: 133, 134, or 135, containing 0 to 3 amino acid modifications; (18) SEQ ID NO: 139, 140, or 141, containing 0 to 3 amino acid modifications; (19) SEQ ID NO: 145, 146, or 147, containing 0 to 3 amino acid modifications; (20) SEQ ID NO: (21) SEQ ID NO: 31, 32, or 33, including 0 to 3 amino acid modifications; (22) SEQ ID NO: 169, 170, or 171, including 0 to 3 amino acid modifications; (23) SEQ ID NO: 61, 62, or 63, including 0 to 3 amino acid modifications; (24) SEQ ID NO: 31, 182, or 183, including 0 to 3 amino acid modifications; (25) SEQ ID NO: 187, 188, or 189, including 0 to 3 amino acid modifications; (26) SEQ ID NO: 193, including 0 to 3 amino acid modifications. (27) SEQ ID NO: 199, 200, or 201, including 0 to 3 amino acid modifications; (28) SEQ ID NO: 205, 206, or 207, including 0 to 3 amino acid modifications; (29) SEQ ID NO: 211, 212, or 213, including 0 to 3 amino acid modifications; (30) SEQ ID NO: 127, 386, or 129, including 0 to 3 amino acid modifications; (33) SEQ ID NO: 205, 206, or 207, including 0 to 3 amino acid modifications; (34) SEQ ID NO: 127, 388, or 129, including 0 to 3 amino acid modifications; or (35) SEQ ID NO: 205, 387, or 207, including 0 to 3 amino acid modifications.

[0075] According to a variant of the first aspect of the invention, there is provided 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 wherein CDRH1, CDRH2, CDRH3 are selected from the group consisting of: (1) SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; (2) SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; (3) SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively; and (4) SEQ ID NO:43, SEQ ID NO:56, respectively. (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) SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively; (13) SEQ ID NO:109, SEQ ID NO:110, and SEQ ID NO:111, respectively; (14) SEQ ID NO:85, SEQ ID NO:110, and SEQ ID NO:117, respectively; (15) SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:123, respectively; (16) SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively; (17) SEQ ID NO:133, SEQ ID NO:134, and SEQ ID NO:135, respectively; (18) SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141, respectively. (19) SEQ ID NO:145, SEQ ID NO:146, and SEQ ID NO:147, respectively; (20) SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; (21) SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:159, respectively; (22) SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171, respectively; (23) SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, respectively; (24) SEQ ID NO:31, SEQ ID NO:182, and SEQ ID NO:183, respectively; (25) SEQ ID NO:187, SEQ ID NO:188, and SEQ ID NO:189, respectively;(26) SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195, respectively; (27) SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201, respectively; (28) SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, respectively; (29) SEQ ID NO: 211, 212, and 213, respectively; (30) SEQ ID NO: 127, 386, and 129, respectively; (31) SEQ ID NO: 205, 206, and 207, respectively; (32) SEQ ID NO: 127, 386, and 129, respectively; or (33) SEQ ID NO: 205, 387, and 207, respectively, and optionally 0 to 3 amino acid modifications are present in any CDR / SEQ ID NO.

[0076] According to a variation of the first aspect of the invention, the present invention provides a method for the preparation of a nucleic acid sequence comprising the steps of: (1) SEQ ID NO: 34, 35, or 36; (2) SEQ ID NO: 40, 41, or 42; (3) SEQ ID NO: 46, 47, or 48; (4) SEQ ID NO: 58, 59, or 60; (5) SEQ ID NO: 64, 65, or 66; (6) SEQ ID NO: 34, 35, or 72; (7) SEQ ID NO: 76, 47, or 78; (8) SEQ ID NO: 82, 83, or 84; (9) SEQ ID NO: 88, 89, or 90; (10) SEQ ID NO: 94, 95, or 96; (11) SEQ ID NO: 100, 101, or 102; (12) the sequence (13) SEQ ID NO: 88, 89, or 114; (14) SEQ ID NO: 124, 125, or 126; (15) SEQ ID NO: 34, 35, or 36; (16) SEQ ID NO: 136, 137, or 138; (17) SEQ ID NO: 142, 143, or 144; (18) SEQ ID NO: 148, 149, or 150; (19) SEQ ID NO: 136, 137, or 162; (20) SEQ ID NO: 34, 35, or 36; (21) SEQ ID NO: 172, 173, or 174; (22) SEQ ID NO: 64, 65, or 180; (23) SEQ ID NO: 136, 137, or 186; (24) SEQ ID NO: 190, 191, or 192; (25) SEQ ID NO: 196, 197, or 12; (26) SEQ ID NO: 202, 203, or 6; (27) SEQ ID NO: 142, 209, or 210; (28) SEQ ID NO: 214, 35, or 216; (29) SEQ ID NO: 10, 11, or 12; (30) SEQ ID NO: 4, 5, or 6; or (31) SEQ ID NO: 142, 143, or 210, and wherein 0-3 amino acid modifications may be present in any CDR / SEQ ID NO.

[0077] According to another variation of the first aspect of the invention, there is provided 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, wherein CDRL1, CDRL2, CDRL3 are selected from the group consisting of: (1) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; (2) SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively; (3) SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; (4) SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 (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 ) SEQ ID NO:64, SEQ ID NO:107, and SEQ ID NO:108, respectively; (13) SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:114, respectively; (14) SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, respectively; (15) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; (16) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:138, respectively; (17) SEQ ID NO:142, SEQ ID NO:143, and SEQ ID NO:144, respectively; (18) SEQ ID NO:148, SEQ ID NO:149, and SEQ ID NO:150, respectively; (19) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:162, respectively; (20) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; (21) SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, respectively; (22) SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:180, respectively; (23) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:186, respectively; (24) SEQ ID NO:190, SEQ ID NO:191, and SEQ ID NO:192, respectively; (25) SEQ ID NO:196, SEQ ID NO:197, or SEQ ID NO:12, respectively;(26) the antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:6, respectively; (27) SEQ ID NO:142, SEQ ID NO:209, and SEQ ID NO:210, respectively; (28) SEQ ID NO:214, SEQ ID NO:35, and SEQ ID NO:216, respectively; (29) SEQ ID NO:10, 11, or 12, respectively; (30) SEQ ID NO:4, 5, or 6, respectively; or (31) SEQ ID NO:142, 143, or 210, respectively, and optionally has 0 to 3 amino acid modifications in any CDR / SEQ ID NO.

[0078] According to another variation of the first aspect of the invention, there is provided 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: CDRH4, CDRH5 and CDRH6. DR: A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein (1) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or (2) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and CDRL1 (3) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; (4) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:56, and SEQ ID NO:57, respectively. (5) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth 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 as set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, respectively; (6) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, respectively; (6) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:69, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:72, respectively; (7) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:76, SEQ ID NO:47, and SEQ ID NO:78, respectively;(8) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84, respectively; (9) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, respectively; (10) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:61, 92, and 93, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:94, 95, and 96, respectively; (11) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:97, 98, and 99, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:98, 99, and 99, respectively. (12) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:103, SEQ ID NO:104, and SEQ ID NO:105, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:64, SEQ ID NO:107, and SEQ ID NO:108, respectively; (13) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:109, SEQ ID NO:110, and SEQ ID NO:111, respectively. (14) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:85, 110, and 117, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:88, 89, and 114, respectively; (15) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:85, 110, and 117, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:88, 89, and 114, respectively;(15) CDRH1, CDRH2, and CDRH3; (15) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:127, 128, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:34, 35, and 36, respectively; or (16) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:127, 128, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:34, 35, and 36, respectively. (17) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 133, 134, and 135, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 136, 137, and 138, respectively; (18) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 139, 140, and 141, respectively, and CDRL1, CDRL (19) CDRH1, CDRH2, CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 145, 146, and 147, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NOs: 148, 149, and 150, respectively; (20) CDRH1, CDRH2, CDRH3 have the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively. (21) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:31, 32, and 159, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:136, 137, and 162, respectively;(22) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, respectively; (23) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth 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 as set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, respectively; (24) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:182, and SEQ ID NO:183, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:186, respectively; (25) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NO:187, SEQ ID NO:188, and SEQ ID NO:189, respectively, and (26) CDRH1, CDRH2, CDRH3 have the amino acid sequences set forth in SEQ ID NO:193, SEQ ID NO:194, and SEQ ID NO:195, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences set forth in SEQ ID NO:196, SEQ ID NO:197, and SEQ ID NO:12, respectively; (27) CDRH1, CDRH2, CDRH3 have the amino acid sequences set forth in SEQ ID NO:199, (27) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:205, 206, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:142, 209, and 210, respectively;(29) CDRH1, CDRH2, and CDRH3; (30) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 386, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 34, 35, and 36, respectively; (31) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 205, 206, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 142, 209, and 210, respectively; (32) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 386, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: (32) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs: 34, 35, and 36, respectively; (33) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs: 205, 387, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs: 142, 209, and 210, respectively; or (34) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs: 205, 387, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs: 142, 143, and 210, respectively; and optionally there are 0 to 3 amino acid modifications in any CDR / SEQ ID NO.

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

[0080] According to another variation of the first aspect of the invention, there is provided 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 set forth in SEQ ID NO: 13, or a sequence having at least 90% sequence identity thereto. In other embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 17 or 21.

[0081] With respect to any embodiment disclosed herein referring to at least 90% sequence identity, this is understood to include any sequence identity between 90% and 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 other embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence as set forth 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 other embodiments, the heavy chain variable region comprises up to 10 modifications therein, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, such as SEQ ID NOs: 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390 , 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] According to another variation of the first aspect of the invention, there is provided 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 an amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 90% sequence identity thereto. In other embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 15 or 19.

[0085] In other embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 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] According to another variation of the first aspect of the invention, there is provided 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 set forth in SEQ ID NO:13, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:14.

[0087] According to another variation of the first aspect of the invention, there is provided 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 set forth 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 set forth in SEQ ID NO:14.

[0088] According to another variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof 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 an amino acid sequence as set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO:19.

[0089] According to another variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof 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 an amino acid sequence as set forth 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 an amino acid sequence as set forth 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 an amino acid sequence as set forth 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 an amino acid sequence as set forth in SEQ ID NO:352; (3) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth 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 an amino acid sequence as set forth in SEQ ID NO:353; (4) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:305. (5) 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 set forth in SEQ ID NO: 306, 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 set forth in SEQ ID NO: 356; (6) 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 set forth in SEQ ID NO: 307, 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 set forth in SEQ ID NO: 357; (7) 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 set forth in SEQ ID NO: 308, 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 set forth in SEQ ID NO: 309. (8) 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 as set forth in SEQ ID NO: 309, 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 as set forth in SEQ ID NO: 359; (9) 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 as set forth in SEQ ID NO: 310, 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 as set forth in SEQ ID NO: 360; (10) 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 as set forth in SEQ ID NO: 311, 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 as set forth in SEQ ID NO: 361;(11) 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 as set forth in SEQ ID NO: 312, 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 as set forth in SEQ ID NO: 362; (12) 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 as set forth in SEQ ID NO: 313, 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 as set forth in SEQ ID NO: 363; (13) 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 as set forth in SEQ ID NO: 314, 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 as set forth in SEQ ID NO: 364; (14) 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 as set forth in SEQ ID NO: 315, 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 set forth in SEQ ID NO: 364; (15) 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 set forth in SEQ ID NO: 316, 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 set forth in SEQ ID NO: 366; (16) 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 set forth in SEQ ID NO: 317, 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 set forth in SEQ ID NO: 367; (17) 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 set forth in SEQ ID NO: 318, 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 set forth in SEQ ID NO: 368;(18) 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 set forth in SEQ ID NO: 319, 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 set forth in SEQ ID NO: 369; (19) 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 set forth in SEQ ID NO: 320, and the light chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 370; (20) 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 set forth in SEQ ID NO: 321, 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 set forth in SEQ ID NO: 370; (21) 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 set forth in SEQ ID NO: 322, 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 set forth in SEQ ID NO: 372; (22) 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 set forth in SEQ ID NO: 324, 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 set forth in SEQ ID NO: 374; (23) the heavy chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 306. (24) 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 as set forth in SEQ ID NO: 326, 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 as set forth in SEQ ID NO: 376; (25) 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 as set forth in SEQ ID NO: 327, 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 as set forth in SEQ ID NO: 377; (26) 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 as set forth 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 the amino acid sequence as set forth in SEQ ID NO: 15;(27) 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 as set forth 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 the amino acid sequence as set forth in SEQ ID NO:19; (28) 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 as set forth in SEQ ID NO:330, 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 as set forth in SEQ ID NO:380; (29) 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 as set forth in SEQ ID NO:331, 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 as set forth in SEQ ID NO:381; (30) 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 as set forth in SEQ ID NO:382, (31) 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 as set forth in SEQ ID NO: 384, 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 as set forth in SEQ ID NO: 385; (32) 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 as set forth in SEQ ID NO: 389, 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 as set forth in SEQ ID NO: 383; (33) 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 as set forth in SEQ ID NO: 390, 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 as set forth in SEQ ID NO: 385;or (34) the antibody or antigen-binding fragment thereof, wherein 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 set forth in SEQ ID NO: 390, 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 set forth in SEQ ID NO: 378;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0130] In other embodiments, the heavy chain variable region polypeptide is selected from the group consisting of 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, 382, ​​384, 389, or 390.

[0131] 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 to the sequence disclosed in SEQ ID NO:14.

[0132] 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 to the sequence disclosed in SEQ ID NO:15.

[0133] 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 to 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 to a sequence disclosed in SEQ ID NO: 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] According to another variation of the first aspect of the invention, there is provided an isolated antibody or antigen-binding fragment thereof having a primary VH domain, a primary VL domain, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any of the antibody clones as set forth in Table 1. In certain embodiments, provided herein is an isolated antibody selected from the antibody clones as set forth in Table 1.

[0136] Table 1. Exemplary BTLA agonistic antibodies

[0137] [Table 1]

[0138] In certain embodiments, the heavy or light chain also comprises a constant region. If the molecule is a full-length IgG type antibody molecule, the heavy chain may comprise three constant domains. In certain embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA has a molecular weight of up to about 10×10 -9 K for binding of M to human BTLA D In certain embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA has a concentration of up to about 4x10 -9 K for binding of M to human BTLA D In certain embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to human BTLA has a concentration of up to about 1x1 0 -9 K for binding of M to human BTLA D Shows.

[0139] In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) has a specific activity of about 10 nM (1x10 -8 suitably not exceeding about 1 nM K D more preferably, the antibody binds to human BTLA at approximately 500 pM (5x10 -10 M), K not exceeding 200pM, 100pM, 50pM, 20pM, 10pM, 5pM or even 2pM D The term "about" as used in this context means + / - 10%.

[0140] In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) has a cytoplasmic affinity of at least 1.0x10 at 37°C. 5 (1 / Ms). In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an on-rate of at least 2.0x10 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 with an on-rate of (1 / Ms).

[0141] In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) has a cytoplasmic affinity of 1.0x10 at 37°C. -3 (1 / s). In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an off rate of no more than 3.0x10 -4 (1 / s). In certain embodiments, an isolated antibody of the invention (e.g., a humanized antibody) binds to human BTLA with an off rate of no more than 2.0x10 -4 (1 / s) or 1.0x10 -4 (1 / s) binds to human BTLA with an off rate no greater than or less than (1 / s).

[0142] In certain embodiments of the first aspect of the invention, provided herein is an agonistic antibody or antigen-binding fragment thereof that specifically binds to human B and T lymphocyte attenuation 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, and which binds to cynomolgus BTLA with a KD of less than 20 nM, as determined at 37° C. by surface plasmon resonance (SPR) using a method such as that described in Example 2; does not inhibit binding of BTLA to herpes virus entry mediator (HVEM), as determined, e.g., by surface plasmon resonance (SPR), using a method such as that described in Example 4; and inhibits T cell proliferation in vitro, as determined, e.g., by a mixed lymphocyte reaction assay, using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof has a molecular weight of at least 5.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. 5(1 / Ms) to human B and T lymphocyte attenuation factor (BTLA). In some embodiments, the antibody or antigen-binding fragment thereof binds to human B and T lymphocyte attenuation factor (BTLA) with an on-rate of 3.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 In some embodiments, the antibody or antigen-binding fragment thereof binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than 3.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -4 (1 / s)~1.0x10 -3 (1 / s) to human B and T lymphocyte attenuation factor (BTLA). In some embodiments, the antibody or antigen-binding fragment thereof 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 mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. The antigen-binding fragment binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: H68. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: N65 and A64.

[0143] Methods for characterizing the properties of the antibodies or antigen-binding fragments thereof 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 a test antibody / fragment thereof inhibits the binding of BTLA to herpes virus entry mediator (HVEM) is described in Example 4; this also uses surface plasmon resonance (SPR). A suitable method for determining whether a test antibody / fragment thereof 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 an antibody / fragment thereof to BTLA may utilize X-ray crystallography or flow cytometry of mutant receptors, such as by the method described in Example 5.

[0144] In certain embodiments of the first aspect of the invention, the ion exchange rate is at least 5.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. 5 Provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that specifically binds human B and T lymphocyte attenuation factor (BTLA) with an on-rate of 3.0x10 at 37°C 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 using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof has an on-rate of 3.0x10 at 37°C as determined by surface plasmon resonance (SPR) using a method such as that described in Example 2. -4(1 / s) or less. In some embodiments, the antibody or antigen-binding fragment thereof binds to human B and T lymphocyte attenuator (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 antigen-binding fragment thereof binds to cynomolgus monkey BTLA with a KD of less than 20 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 antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 as determined by X-ray crystallography of mutant receptors or by flow cytometry using a method such as that described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0145] In certain embodiments of the first aspect of the invention, the solubility of ... -4 (1 / s)~1.0x10 -3 2. An isolated agonistic antibody or antigen-binding fragment thereof that specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of 1 / s, wherein the antibody is capable of being assayed using a method such as that described in Example 4, e.g., by surface plasmon resonance ( Provided herein is an antibody or antigen-binding fragment thereof that does not inhibit binding of BTLA to herpes virus entry mediator (HVEM) as determined by surface plasmon resonance (SPR); and inhibits T cell proliferation in vitro as 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 antigen-binding fragment thereof binds to human B and T lymphocyte attenuation 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 antigen-binding fragment thereof binds to cynomolgus monkey BTLA with a KD of less than 20 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 antigen-binding fragment thereof binds to cynomolgus monkey BTLA with a KD of at least 5.0×10 as determined at 37° C. by surface plasmon resonance (SPR), using a method such as that described in Example 2. 5 (1 / Ms) to human B and T lymphocyte attenuation factor (BTLA). In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92, as determined by X-ray crystallography of mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to BTLA residues selected from: N65 and A64.

[0146] In certain embodiments of the first aspect of the invention, each has a molecular weight of 1.0x10 as measured by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -3 (1 / s) and an off rate of at least 5.0x10 5Provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that specifically binds human B and T lymphocyte attenuation factor (BTLA) with an on-rate of (1 / Ms), wherein the antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM) as determined, e.g., by surface plasmon resonance (SPR) using a method such as that described in Example 4; and inhibits T cell proliferation in vitro as determined, e.g., by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof binds human B and T lymphocyte attenuation 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 antigen-binding fragment thereof binds to cynomolgus monkey BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37° C. using methods such as those described in Example 2. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 as determined by x-ray crystallography of mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0147] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonistic antibody or antigen-binding fragment thereof 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 binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof has a KD of less than 1.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. 6 (1 / Ms)) at 37° C., as determined by surface plasmon resonance (SPR) using a method such as that described in Example 2. -3(1 / s) or less. In some embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey B and T lymphocyte attenuator (BTLA) with a KD of less than 10 nM as determined by surface plasmon resonance (SPR) at 37° C. using methods such as those described in Example 2. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 as determined by X-ray crystallography of mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45, and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0148] In certain embodiments of the first aspect of the invention, the ELISA assay is more than 1x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -3Provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that specifically binds human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than (1 / s), wherein the antibody inhibits binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus B and T lymphocyte attenuation 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 antigen-binding fragment thereof 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 methods such as those described in Example 2. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 as determined by X-ray crystallography of mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0149] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonistic antibody or antigen-binding fragment thereof 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; and inhibits binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof 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 methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0150] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that specifically binds 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 the antibody does not inhibit binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof 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 methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0151] In certain embodiments of the first aspect of the invention, an isolated agonistic antibody or antigen-binding fragment thereof 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 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 4. Provided herein is an antibody or antigen-binding fragment thereof that does not inhibit binding of BTLA to the rupesvirus entry mediator (HVEM); and inhibits T cell proliferation in vitro as 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 antigen-binding fragment thereof inhibits T cell proliferation in vitro as determined by surface plasmon resonance (SPR) at 37° C., using a method such as that described in Example 2, by at least 2.0×10 5 (1 / Ms). In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with an on-rate of 10.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s) or less. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 as determined by X-ray crystallography of mutant receptors or by flow cytometry using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to BTLA residues selected from: N65 and A64.

[0152] In certain embodiments of the first aspect of the invention, the ion exchange rate is 1.7x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 (1 / Ms)~2.5x10 5Provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that specifically binds human B and T lymphocyte attenuation factor (BTLA) with an on-rate of 3.0x10 at 37°C as determined by surface plasmon resonance (SPR) using a method such as that described in Example 4; and wherein the antibody does not inhibit binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof has an on-rate of 3.0x10 at 37°C as determined by surface plasmon resonance (SPR) using a method such as that described in Example 2. -1 In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with an off rate of less than 3.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s)~5.0x10 -1(1 / s). In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with a KD of at least 150 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 antigen-binding fragment thereof binds to human BTLA with a KD of 150 nM to 1500 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 antigen-binding fragment thereof binds to an epitope that blocks binding of the 286 antibody. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106, and E92 as determined by x-ray crystallography of mutant receptors or by flow cytometry using a method such as that described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to a human BTLA residue selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In one embodiment, the antibody or antigen-binding fragment thereof binds to a BTLA residue selected from: N65 and A64.

[0153] In certain embodiments of the first aspect of the invention, provided herein is an isolated agonistic antibody or antigen-binding fragment thereof that 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 the antibody does not inhibit binding of BTLA to herpes virus 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, for example, by a mixed lymphocyte reaction assay using a method such as that described in Example 9. In some embodiments, the antibody or antigen-binding fragment thereof has a KD of at least 1.0×10 as determined by surface plasmon resonance (SPR) at 37° C. using a method such as that described in Example 2. 5 (1 / Ms). In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with an on-rate of 1.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. 5 (1 / Ms)~10x10 5 (1 / Ms). In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with an on-rate of 6.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA with an off rate of less than 6.0x10 as determined by surface plasmon resonance (SPR) at 37°C using a method such as that described in Example 2. -1 (1 / s)~10.0x10 -2(1 / s). In some embodiments, the antibody or antigen-binding fragment thereof 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 using methods such as those described in Example 5. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibody or antigen-binding fragment thereof binds to human BTLA residues selected from: D35, T78, K81, S121 and L123. In some embodiments, the antibody or antigen-binding fragment thereof binds to residue H68 of human BTLA. In some embodiments, the antibody or antigen-binding fragment thereof binds to BTLA residues selected from: N65 and A64.

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

[0155] In certain embodiments, the antigen-binding fragment of the present invention is: scFv, sc(Fv) 2 , dsFv, Fab, Fab', (Fab')2 and diabody. In certain embodiments, the heavy and light chain molecules that form the antigen-binding fragment are linked by a flexible linker. There are many flexible linkers in common use, and the selection of the linker can be made by one of skill in the art.

[0156] The peptide linker linking the scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of another variable domain without significantly compromising the fidelity of the VH-VL pairing and antigen binding site. The peptide linker may vary from 10 to 25 amino acids in length and typically, but not always, contains a glycine residue. The linker is composed of hydrophilic amino acids such as cyclic amines (G) and serine (S). The linker is similar to those found in natural multidomain proteins (e.g., Argos P. J Mol Biol. 211:943-958, 1990; and Heringa G. Protein Eng. 15:871-879, 2002) or adapted therefrom.

[0157] 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 The length of this GS linker may be modified by adjusting the copy number "n" to achieve proper separation of functional domains or to maintain the required inter-domain 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 commercially available recombinant phage antibody system (RPAS kit) 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 present inventors have mapped the epitopes on BTLA bound by the potent 2.8.6 and 11.5.1 agonists and other antibodies disclosed herein. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention binds to human BTLA residues 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 certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106, E92.

[0160] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least two residues of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106, E92.

[0161] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least three residues of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

[0162] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least five residues of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

[0163] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to all of the human BTLA residues selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

[0164] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention binds to a human BTLA residue selected from: Y39, K41, R42, Q43, E45 and S47. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least two residues of human BTLA selected from: Y39, K41, R42, Q43, E45 and S47.

[0165] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention binds to all of the human BTLA residues selected from: Y39, K41, R42, Q43, E45 and S47. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention binds to human BTLA residues selected from: D35, T78, K81, S121 and L123.

[0166] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least two residues of human BTLA selected from: D35, T78, K81, S121 and L123.

[0167] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to residue H68 of human BTLA. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention binds to human BTLA residues selected from: N65 and A64.

[0168] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to both the N65 and A64 residues of human BTLA. Residue numbering, e.g., K41, refers to the amino acid at position 41 (K; lysine); where the numbering refers to the position in the human BTLA polypeptide as disclosed in SEQ ID NO:23.

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

[0170] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are fully human antibodies. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention act as agonists to induce signaling through the BTLA receptor.

[0171] The antibodies (or antigen-binding fragments thereof) of the invention are particularly potent agonists. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention have an EC50 of no more than 1 nM.

[0172] The agonistic antibodies (or antigen-binding fragments thereof) of the invention have particularly high potency. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention inhibit T cell proliferation by at least 20%, suitably by at least 30%, and more suitably by at least 40%.

[0173] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention inhibit T cell IFN-gamma production in an in vitro mixed lymphocyte reaction by at least 50%, suitably by at least 75%, and more suitably by at least 95%, as measured, for example, by ELISA of supernatants.

[0174] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention inhibit T cell IL-2 production in an in vitro mixed lymphocyte reaction by at least 50%, suitably at least 75%, and more suitably at least 95%, as measured, for example, by ELISA of supernatants.

[0175] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are In lymphocyte responses, it inhibits T cell IL-17 production by at least 50%, suitably at least 75%, more suitably at least 95%, as measured, for example, by ELISA of supernatants.

[0176] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention reduce mortality in a murine GVHD model by at least 50%, suitably at least 75%, and more suitably at least 95%, using methods such as those described in Example 12.

[0177] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention reduce weight loss in a murine T-cell colitis model by at least 50%, suitably at least 75%, and more suitably at least 95%, using methods such as those described in Example 11.

[0178] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention reduce colonic inflammation in a murine T cell colitis model by at least 50%, suitably at least 75%, and more suitably at least 95%, using methods such as those described in Example 11.

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

[0180] nucleic acid molecule The antibody or antigen-binding fragment of the present invention will be encoded by a nucleic acid. The antibody or antigen-binding fragment may be encoded by a single nucleic acid molecule, or may be encoded by two or more nucleic acid molecules. For example, since an 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 and light chain) polypeptide regions may be encoded by separate nucleic acid molecules. Alternatively, for example in the case of scFv, they may be encoded by the same nucleic acid molecule.

[0181] According to a second aspect of the invention there is provided one or more nucleic acid molecules encoding an antibody or antigen-binding fragment thereof according to the first aspect of the invention. From the primary amino acid sequence of the polypeptide(s) encoding an antibody or antigen-binding fragment thereof of the invention, one of skill in the art can determine the appropriate nucleotide sequence(s) encoding the polypeptide(s), and, if desired, one that has been codon-optimized (see, e.g., Mauro and Chappell. Trends Mol Med. 20(11):604-613, 2014).

[0182] In this specification, where reference is made to previous aspects of the invention, for example "in accordance with a first (e.g. second etc.) aspect of the invention", this is understood to include any variant of the aspect referred to (e.g. a variant of the first (or second etc.) aspect).

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

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

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

[0186] For example, the nucleic acid molecule(s) encoding an antibody or antigen-binding fragment thereof of the invention according to the first aspect of the invention may be or be part of a vector (e.g., a plasmid, cosmid or viral vector, or an artificial chromosome), which may include other functional regions (elements), such as one or more promoters, one or more origins of replication, one or more selectable marker(s), and one or more other elements typically found in expression vectors. Cloning and expression of nucleic acids encoding proteins, including antibodies, is well established and well within the skill of one of ordinary skill in the art.

[0187] According to a third aspect of the invention there is provided a vector comprising a nucleic acid of the second aspect of the invention, hi particular embodiments the vector is a plasmid vector, a cosmid vector, a viral vector or an artificial chromosome.

[0188] The nucleic acids of the invention, including vector nucleic acids comprising a nucleotide sequence encoding a polypeptide capable of forming an antibody or antigen-binding fragment thereof of the invention, may be in purified / isolated form. The isolated / purified nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention may be used in the preparation of the antibody or antigen-binding fragment thereof in a manner similar to that described above, but which is not limited to the nucleic acid itself. If recombinant DNA techniques are performed in vivo, they will be free or substantially free from other proteins or nucleic acids with which they are found in the environment in which they are prepared (eg, cell culture).

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

[0190] The nucleic acids and / or vectors of the invention may be introduced into a host cell. Introduction may use any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or for insect cells, baculovirus. Introduction of nucleic acids in host cells, particularly eukaryotic cells, may use virus- or plasmid-based systems. Plasmid systems may be maintained episomally or may be incorporated into the host cell or into artificial chromosomes. Incorporation may occur at single or multiple loci. The vector may be introduced into the host cell by either random or targeted integration of one or more copies in the host cell. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection with bacteriophage.

[0191] In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of the host cell. Integration may be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

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

[0193] The host cell may be of any species, for example bacterial or yeast origin, but suitably the host cell is a mammalian cell, for example a human cell or a rodent cell, for example a HEK293T cell or a CHO-K1 cell.

[0194] Thus, according to a fourth aspect of the invention there is provided a host cell comprising a nucleic acid according to the second aspect of the invention, or a vector according to the third aspect of the invention. Host cells may be treated to cause or permit expression of a protein of the invention from a nucleic acid, for example by culturing the host cells under conditions for expression of the encoding nucleic acid. Purification of the expressed product may be achieved by methods known to those of skill in the art.

[0195] Thus, the nucleic acid of the present invention, including a vector nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of forming an antibody or antigen-binding fragment thereof of the present invention, may be present in an isolated host cell. The host cell is typically part of a clonal population of host cells. Reference herein to a host cell also includes a clonal population of said cells. A clonal population is one that has been propagated from a single parent host cell. The host cell may be from any suitable organism. Suitable host cells include bacterial, fungal or mammalian cells.

[0196] The host cell may serve to support the amplification of vector nucleic acid (e.g., using a plasmid) or may serve as a biological factory to express the polypeptide(s) of the invention that form the BTLA antibody or antigen-binding fragment thereof of the invention. Suitable hosts for amplifying vector nucleic acid may be bacterial or fungal cells, such as Escherichia coli cells or Saccharomyces cerevisiae cells. Suitable hosts for expressing the proteins of the invention (i.e., the polypeptides that constitute the human BTLA binding antibodies or antigen-binding fragments thereof of the invention) would 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 cells.

[0197] A variety of host-expression vector systems may be utilized to express BTLA binding molecules as described herein (see, e.g., U.S. Patent No. 5,807,715). For example, mammalian cells, such as Chinese hamster ovary cells (CHO), in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for CEA protein (Foecking et al., Gene, 45:101 (1986); and Cockett et al., Bio / Technology, 8:2 (1990)). Different host cells have different post-translational processing of proteins and gene products. Eukaryotic host cells have characteristic and specific mechanisms for glycosylation and modification. Appropriate cell lines or host systems may be selected to ensure correct modification and processing of the proteins of the present disclosure. To this end, eukaryotic host cells may be used that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. 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 According to a fifth aspect of the invention there is provided a method of producing an antibody or antigen-binding fragment thereof according to the first aspect of the invention, the method comprising culturing a host cell according to the fourth aspect of the invention under conditions for the production of said antibody or antigen-binding fragment thereof, and optionally isolating and / or purifying said antibody or antigen-binding fragment thereof.

[0199] According to a variant of the fifth aspect of the invention, there is provided a method for producing an antibody or antigen-binding fragment thereof that binds human BTLA, comprising culturing a host cell comprising nucleic acid encoding a polypeptide(s) that form an antibody or antigen-binding fragment thereof that binds human BTLA under conditions for the production of said antibody or antigen-binding fragment thereof, and optionally further comprising the step of isolating / purifying said antibody or antigen-binding fragment thereof.

[0200] By isolated / purified, we mean that the antibody or antigen-binding fragment thereof of the present invention, or the polypeptides constituting these molecules, will be free or substantially free of other proteins or nucleic acids with which it is naturally associated, e.g., found in its natural environment, or in the environment in which it is prepared if such preparation is by recombinant DNA techniques carried out in vitro or in vivo (e.g., cell culture).

[0201] According to a variant of the fifth aspect of the invention, there is provided a method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human BTLA, comprising: a) providing a host cell containing one or more nucleic acid molecules encoding amino acid sequences of heavy chain variable domains and light chain variable domains which, when expressed, are capable of combining to produce a human BTLA-binding molecule; b) culturing a host cell that expresses the encoded amino acid sequence; and c) isolating the antibody or antigen-binding fragment molecule The method includes the steps of:

[0202] The one or more nucleic acid molecules are as described above that encode a polypeptide capable of forming an antibody or antigen-binding fragment thereof of the invention that specifically binds to human BTLA. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: i) a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:1, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:2, and CDRH3 has the amino acid sequence as set forth 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 as set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:6. Includes.

[0203] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: i) three CDRs: CDRH1 a heavy chain variable region comprising CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:7, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:8, and CDRH3 has the amino acid sequence as set forth 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 as set forth in SEQ ID NO: 10, CDRL2 has the amino acid sequence as set forth in SEQ ID NO: 11 and CDRL3 has the amino acid sequence as set forth in SEQ ID NO: 12. Includes.

[0204] In certain embodiments, the antibody or antigen-binding fragment thereof is: i) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having at least 90% sequence identity thereto; and ii) a light chain variable region comprising the amino acid sequence disclosed in SEQ ID NO: 14, or a sequence having at least 90% sequence identity thereto; Includes.

[0205] In various embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2, and CDH3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (1) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively. and SEQ ID NO:36; (2) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively; (3) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively, and CDRL (4) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:56, and SEQ ID NO:57, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, respectively; (5) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, respectively. (6) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:69, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:72, respectively;(7) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:76, SEQ ID NO:47, and SEQ ID NO:78, respectively; (8) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84, respectively; (9) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, respectively; (10) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:61, 92, and 93, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:94, 95, and 96, respectively; (11) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:97, 98, and 99, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:100, 101, and 102, respectively; (12) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:103, 104, and 105, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:64, 107, and 10 (13) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:109, SEQ ID NO:110, and SEQ ID NO:111, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:114, respectively; (14) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:85, SEQ ID NO:110, and SEQ ID NO:117, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:114, respectively; (15) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:123, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, respectively;(16) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 128, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 34, 35, and 36, respectively; (17) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 133, 134, and 135, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 136, 137, and 138, respectively; (18) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:139, 140, and 141, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:142, 143, and 144, respectively; (19) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:145, 146, and 147, respectively, (20) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:148, SEQ ID NO:149, and SEQ ID NO:150, respectively; (21) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:148, SEQ ID NO:149, and SEQ ID NO:150, respectively; (21) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, respectively;(23) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth 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 as set forth in SEQ ID NO:64 and SEQ ID NO:6, respectively; (24) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:31, 182, and 183, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:136, 137, and 186, respectively; (25) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:187, 188, and 189, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:190, 191, and 192, respectively; (26) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:193, 194, and 195, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:196, 197, and 12, respectively. (27) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:199, SEQ ID NO:200, and SEQ ID NO:201, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:6, respectively; (28) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:205, SEQ ID NO:206, and SEQ ID NO:207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:142, SEQ ID NO:209, and SEQ ID NO:210, respectively; (29) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:211, SEQ ID NO:212, and SEQ ID NO:213, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:214, SEQ ID NO:35, and SEQ ID NO:216, respectively;(30) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 386, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 34, 35, and 36, respectively; (31) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 205, 206, and 207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 142, 209, and 210, respectively; (32) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 388, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: (33) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:205, SEQ ID NO:387, and SEQ ID NO:207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:142, SEQ ID NO:209, and SEQ ID NO:210, respectively; or (34) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:205, SEQ ID NO:387, and SEQ ID NO:207, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NO:142, SEQ ID NO:143, and SEQ ID NO:210, respectively; 0 to 3 amino acid modifications may be present in any CDR / SEQ ID NO.;

[0206] Conditions for producing the antibodies or antigen-binding fragments of the present invention and for purifying said molecules are well known in the art. One way to address this is to prepare a clonal population of cells capable of expressing an antibody of the invention or a fragment thereof, and culture these in an appropriate growth medium for a period of time and at a temperature that is conducive to allowing proliferation / growth of the cell population and expression of the protein(s) of interest. If the protein(s) of interest (e.g., an antibody of the invention) is expressed within a host cell, the cells can be lysed (e.g., using a mild detergent or sonication) and the contents of the cell (and thus the protein(s) of interest) can be isolated from the periphery of the host cell. The cells may be released into the surrounding medium (which may be culture medium or another medium in which the cells are reconstituted), and this medium is then subjected to a purification process. If the protein(s) of interest (e.g., an antibody of the present invention) are secreted into the growth medium, the medium is subjected to a purification process. Antibody purification involves isolation of the antibody from, for example, the culture medium or from the culture supernatant of the hybridoma cell line, using well-established methods that typically involve chromatography (e.g., using affinity chromatography, anion and / or cation exchange chromatography, size exclusion chromatography or other separation techniques) to separate the protein of interest from undesired host-derived proteins and other cellular contaminants (e.g., nucleic acids, carbohydrates, etc.).

[0207] The purified protein may also be subjected to a viral inactivation step. Finally, the purified protein of interest may be, for example, lyophilized or formulated for storage, transportation and subsequent use. Preferably, the protein of interest (e.g., the antibody or antigen-binding fragment thereof of the present invention) will be substantially free of contaminating proteins originally present in the medium after expression or cell lysis.

[0208] In certain embodiments, the antibodies or antigen-binding fragments thereof of the 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 of the present invention) may be formulated into a suitable composition. composition Although the BTLA-binding molecule may be administered alone, in certain embodiments, the administration is of a pharmaceutical composition in which the BTLA-binding molecule is formulated with at least one pharma- ceutically acceptable excipient. The excipient may be a suitable pharmaceutical carrier solute. Such carriers are well known in the art and include phosphate buffered saline solution, water, liposomes, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers may be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to subjects in appropriate doses. Dosage regimens will be determined by the attending physician and clinical factors.

[0210] According to a sixth aspect of the invention there is provided a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the first aspect of the invention, or that produced according to the fifth aspect, hi a particular embodiment the composition comprises phosphate buffered saline.

[0211] A "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered. A pharmaceutical composition will include one or more pharma-ceutically acceptable excipients. The term excipient, in this context, refers to any additive, such as a filler, solubilizer, carrier, vehicle, additive, etc.

[0212] Pharmaceutical compositions may contain one or more pharma- ceutically acceptable excipients, including, for example, water, ion exchange agents, proteins, buffer substances, and salts. Preservatives and other additives may also be present. Excipients may be solvents or dispersion media. Suitable formulations for use in the therapies disclosed herein are described in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980).

[0213] A "pharmaceutical acceptable" excipient is one that may be reasonably administered to a target mammal to provide an effective dosage of the active ingredient employed. The pharmaceutical compositions of the present invention are prepared for storage by mixing the composition with an optional pharmaceutical acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Acceptable excipients are nontoxic to recipients at the dosages and concentrations employed, and include, for example, buffers such as phosphate, 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; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable surfactants include: serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). A lyophilized HER2 antibody formulation is described in WO97 / 04801.

[0214] Pharmaceutical compositions to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes. The route of administration of a BTLA-binding moiety, e.g., an antibody or antigen-binding fragment thereof, may be, for example, oral, parenteral, inhalation, or topical. The term parenteral, as used herein, 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 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), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like. In addition, the composition may include a proteinaceous carrier, such as serum albumin or immunoglobulin, in certain embodiments of human origin. For intravenous injection, or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity, and stability. A person skilled in the art is well able to prepare suitable solutions, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, using isotonic vehicles. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as required. As mentioned above, these are all referred to herein as excipients.

[0216] Injectable compositions may be administered with medical devices known in the art, such as a hypodermic needle. Needle-free injection devices, such as those disclosed in U.S. Patent Nos. 6,620,135 and 5,312,335, may also be utilized.

[0217] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, liquid or semi-solid form. 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 solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, may be included as appropriate.

[0218] The antibodies or antigen-binding fragments of the present invention may be formulated in liquid, semi-solid or solid form depending on the physicochemical properties of the molecule and the route of delivery. The formulation may include excipients, or combinations of excipients, such as: sugars, amino acids and surfactants. Liquid formulations may include a wide range of antibody concentrations and pH. Solid formulations may be produced, for example, by freeze-drying, spray-drying, or drying by supercritical fluid techniques.

[0219] Pharmaceutical compositions may be administered as a single dose, multiple doses, or by infusion over an established period of time. Dosage regimens may also be adjusted to provide the optimum desired response (e.g., 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, for example, once a day, or "as needed."

[0220] Dosage Amounts of BTLA-binding molecules, or pharmaceutical formulations containing such molecules, that will be therapeutically effective may be determined by standard clinical techniques, for example, through dose-ranging clinical trials. Additionally, in vitro assays may be optionally used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Dosages of the compositions to be administered may be determined by those skilled in the art without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making these determinations include the condition or conditions to be treated, the choice of the composition to be administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms. For example, the actual patient weight may be used to calculate the dose of the formulation in milliliters (mL) to be administered. There may be no downward adjustment to the "ideal" weight. In such circumstances, the appropriate dose may be calculated by the following formula: Dose (ml) = [patient weight (kg) x dose level (mg / kg) / drug concentration (mg / mL)] The therapeutically effective dose of pharmaceutical compositions for treating BTLA-associated diseases or disorders as discussed herein will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, the weight of the patient, the sex of the patient, the age of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. The therapeutically effective dose is likely to have been determined from clinical trials and can be determined by the attending physician using treatment guidelines. Usually, the patient is a human, but non-human mammals may also be treated. To optimize safety and efficacy, the therapeutic dosage may be titrated using routine methods known to those skilled in the art.

[0221] In various embodiments, the BTLA binding molecule is administered at a concentration of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg.

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

[0223] According to a seventh aspect of the invention there is provided a method of preparing a pharmaceutical composition comprising the step of formulating an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or one produced according to the fifth aspect of the invention, into a composition comprising at least one further component, in a particular embodiment the at least one further component is a pharma- ceutically acceptable excipient.

[0224] kit Additionally, the product (e.g., a BTLA binding molecule or pharmaceutical composition thereof) may be packaged and sold in the form of a kit. Such articles of manufacture may have a label or package insert indicating the product and directions for the proper use of the product for the treatment of a subject suffering from or prone to a disease or disorder.

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

[0226] therapy / medical use An antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, may be used in therapy, typically as a medicament.

[0227] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, may be used to treat or prevent any disease or condition in a subject in need thereof.

[0228] BTLA is involved in downregulating immune responses, and there are many diseases or conditions that can be treated 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-associated diseases". BTLA-associated diseases include inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation.

[0229] Specific BTLA-associated diseases that may be treated with the BTLA binding molecules of the 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 polyendocrine syndrome, Behcet'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-Barre syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis of the liver), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, and inflammatory fibrosis. These include: 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] According to an eighth aspect of the invention there is provided an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, for use in therapy.

[0231] In certain embodiments, the therapy is the treatment or prevention of a BTLA-associated disease. In certain embodiments, the BTLA-associated disease is one caused by a decrease in BTLA expression and / or activity in a 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 antibodies or antigen-binding fragments of the invention.

[0232] In one embodiment, the BTLA-associated disease is an inflammatory disease (eg, rheumatoid arthritis), an autoimmune disease or disorder (eg, graft versus host), or a proliferative disease or disorder (eg, cancer).

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

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

[0235] In a variation of this aspect, there is provided an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the 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-associated disease. In certain embodiments, the method is for treating or preventing inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation.

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

[0237] In one embodiment, the therapy is for treating a BTLA-associated disease. Suitably, the BTLA-associated disease is an inflammatory disease (e.g., asthma), an autoimmune disease or disorder (e.g., rheumatoid arthritis), or an immunoproliferative disease or disorder (e.g., lymphoma).

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

[0239] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is used in the treatment of: 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 polyendocrine syndrome, Behcet'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, The present invention is for treating or preventing a disease or condition in a subject in need thereof selected from the group consisting of hidradenitis suppurativa, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and liver 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, Sjogren'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 a particular embodiment, the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is used to treat or prevent a disease or condition in a subject in need thereof selected from the group consisting of: GVHD, colitis, rheumatoid arthritis, psoriasis and MS. In one embodiment, the immunoproliferative disease is cancer. Suitably, the cancer is leukemia or lymphoma.

[0241] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is for use in the prevention or treatment of transplant rejection.

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

[0243] In other embodiments, the antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is for use in the treatment of diabetes, such as type 1 diabetes.

[0244] In another embodiment, the antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is for use in the treatment of psoriasis. In another embodiment, the antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is for use in the treatment of multiple sclerosis.

[0245] In another embodiment, the antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is for use in the treatment of colitis. The term "effective amount" or "therapeutically effective amount" refers to a dosage or amount of a drug that is sufficient to ameliorate symptoms or achieve a desired biological outcome in a patient, such as, for example, in cancer, increasing tumor cell death, decreasing tumor size, increasing progression-free or overall survival, etc. As disclosed elsewhere herein, an effective amount will typically be assessed through thorough human clinical studies.

[0246] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context otherwise requires. In particular, when using the indefinite article, the specification shall be understood to contemplate the plural as well as the singular unless the context otherwise requires.

[0247] It is to be understood that features, integers, properties, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. All of the features disclosed herein (including any accompanying claims, abstract and figures) and / or all of the steps of any method or process thus disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one or any novel combination of features disclosed herein (including any accompanying claims, abstract 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 in connection with this specification, submitted contemporaneously with or prior to this specification and open to public inspection hereof, and the contents of all such articles and documents are incorporated herein by reference.

[0249] The invention will now be further described with reference to the following non-limiting examples and the accompanying figures. EXAMPLES

[0250] In the Examples that follow, antibodies 11.5.1 and 2.8.6 are shown to bind human BTLA with high affinity. Using transgenic mice expressing the human receptor, these antibodies are shown to be able to inhibit T cell responses in vitro and in vivo following binding to BTLA, and to ameliorate disease in murine models of inflammatory bowel disease and graft-versus-host disease. While their agonistic 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 domain (BTLA K31~R151 Antibodies recognizing the human immune cell receptor BTLA were generated by BioGenes GmbH by immunizing mice with 11.5.1.) Spleen cells from immunized mice were fused with Sp2 / 0-Ag14 myeloma cells and the resulting hybridomas were selected for reactivity with human BTLA by ELISA of the supernatants in combination with dilution cloning. Antibodies were isotyped from hybridoma supernatants using a rapid mouse isotyping kit (RayBiotech). Both antibodies produced by clones 2.8.6 and 11.5.1 were found to be IgG1k.

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

[0253] Then, using primers targeting conserved regions of the immunoglobulin locus 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, identification of functional light chains was complicated by the abundance of non-functional kappa light chain cDNA from fused myeloma cell lines, and to resolve this, primers specific for the non-functional chain CDR3 were added in excess to force excision of the aberrant chain products, using a technique previously described (Yuan et al. J Immunol Methods. 294:39553-61, 2005).

[0254] The variable domain sequences were evaluated and the CDR positions determined using the NCBI IgBlast tool. Example 2. Binding to soluble human and cynomolgus BTLA The binding affinity and kinetics of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to human or cynomolgus monkey BTLA were determined by surface plasmon resonance using a Biacore T200 (GE Healthcare). A series S CM5 sensor chip (GE Healthcare) was 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. Various concentrations of monomeric soluble human BTLA extracellular domain (BTLA) were then assayed in a single cycle kinetic analysis. K31~R151 ) (derived from SEQ ID NO: 23) or a soluble cynomolgus monkey BTLA extracellular domain (BTLA K31~R151 ) (derived from SEQ ID NO:24) in 10 mM buffer Injections were performed over immobilized antibody in Hepes, 150 mM NaCl, 0.005% v / v surfactant P20, pH 7.4 (HBS-P) at 37° C. (FIG. 1a). After reference and blank subtraction, association and dissociation rates were fitted and dissociation constants were calculated using BiaEvaluation software (GE Healthcare) (FIG. 1b). Clone 2.8.6 binds human BTLA with a KD of 0.65 nM and cynomolgus BTLA with a KD of 7.89 nM. Clone 11.5.1 binds human BTLA with a KD of 0.75 nM and cynomolgus BTLA with a KD of 0.99 nM. In a separate experiment on human BTLA alone, clone 2.8.6 bound human BTLA with a KD of 0.37 nM and clone 11.5.1 bound 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 invention (2.8.6 and 11.5.1) to bind to human or cynomolgus BTLA expressed on the cell surface was assessed by flow cytometry. Full-length human or cynomolgus BTLA was expressed in Jurkat T cell lines using a lentiviral transfection system. 1x10 per well 5 Cells were plated in 96-well U-bottom plates. BTLA antibody binding to mIgG1 isotype control (clone MOPC-21, Biolegend #400165) was assessed at 12 concentrations in 1:3 serial dilutions in FACS buffer (PBS, 2% FCS, 0.05% sodium azide) starting at a concentration of 90 μg / ml. Non-specific 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 before staining 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 EC50s for receptor binding were calculated by nonlinear curve fitting using aphPad Prism software. 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 invention (2.8.6 and 11.5.1) to block natural ligand binding to BTLA was assessed by surface plasmon resonance using a Biacore T200 (GE Healthcare). Amine coupling was used to block binding of the human BTLA extracellular domain (BTLA 31K~151R ) was covalently coupled to a CM5 sensor chip. Human HVEM extracellular domain fused to mouse IgG1 Fc was then injected over the immobilized hBTLA in HBS-P buffer at 37° C. and allowed to dissociate completely. A saturating amount of anti-BTLA antibody (2.8.6 or 11.5.1) was then injected immediately followed by a second injection of human HVEM-mFc at the same concentration as the first injection (FIG. 2a). After saturation of BTLA with antibody, equilibrium HVEM binding (resonance units) was expressed as a percentage of binding before antibody injection (FIG. 2b). An antibody was considered non-blocking if HVEM binding after saturation with antibody was >90% of binding before antibody injection.

[0257] Example 5. Binding epitope of antibody 11.5.1 on human BTLA The functional epitope of antibody 11.5.1 on human BTLA was determined by flow cytometric assessment of binding to a panel of single residue mutants of the receptor expressed on the cell surface. Constructs encoding the human extracellular domain of BTLA and the transmembrane and intracellular domains of murine CD28 were cloned into the bicistronic 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). Mock and non-transfected controls were included in each experiment. Cells were harvested at 48 hours and stained with 10 μg / ml fluorochrome-conjugated anti-BTLA antibody in PBS, 0.05% azide, 2% FCS (FACS buffer) for 1 hour at 4° C. along with live / dead markers. Cells were washed, pelleted, and resuspended in 200 μl FACS buffer before analysis on a BD FACScan on a flow cytometer. GFP-positive (transfected) viable cells were gated and analyzed for anti-BTLA antibody binding (an example of the binding analysis of clone 11.5.1 is shown in FIG. 3a). For each mutant, the geometric mean of anti-BTLA antibody binding to transfected cells was expressed as a percentage of binding to the wild-type receptor (FIG. 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 hypothesized that such mutations led to dramatic changes in protein folding or expression rather than revealing antibody epitopes. Mutations Y39R and K41E completely abolished binding of antibody 11.5.1, whereas 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 in Figure 3c (black residues). Residues required for HVEM binding (Gln37, Arg42, Pro59, His127; from patent publication WO2017004213) are also mapped onto the structure in grey, indicating that 11.5.1 binds to an epitope very close to the HVEM binding site.

[0258] Example 6. Crystal structure of the 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 the antibody Fab in complex with the human BTLA extracellular domain. The heavy and light chain variable domains of antibody 2.8.6 were cloned into pOPINVH and pOPINVL expression vectors (Addgene), which encode the first constant domain of mouse IgG1 heavy chain (including a 6x histidine tag) and the constant domain of mouse Ig kappa chain, respectively. These vectors were transiently co-transfected into HEK293T cells to produce the Fab' fragment of anti-BTLA 2.8.6, which was purified by Ni-NTA purification. The human BTLA Ig-V set domain (BTLA S33~D135) was cloned into the pGMT7 vector and expressed in BL21(DE3)pLysS E. coli cells (Novagen) to produce inclusion bodies. Inclusion bodies were isolated from the cell pellet by sonication and washed repeatedly with a washing solution containing 0.5% Triton X-100. The purified BTLA inclusion bodies were solubilized in a denaturing solution containing 6 M guanidine hydrochloride. The solubilized protein solution was slowly diluted in refolding buffer [0.1 M Tris-HCl (pH 8.0), 0.6 M L-arginine, 2 mM ethylenediaminetetraacetic acid, 3.73 mM cystamine, and 6.73 mM cysteamine] to a final protein concentration of 1-2 μM and then stirred at 4 °C for 48 h. The BTLA refolding mixture was then concentrated with 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 collection were obtained by hanging drop vapor diffusion in 0.2 M calcium acetate, 0.1 M imidazole pH 8.0, 10% (w / v) PEG8000 at 293°K. The final data set was collected on the Photon Factory and the structure was determined by molecular replacement using the structures of BTLA (PDB ID; 2AW2 chain A) and anti-PD1-Fab (PDB ID: 5GGS chains C, D) as search probes.

[0260] The residues on BTLA at the interface with antibody 2.8.6 are A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, 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 containing the human extracellular domain and the murine transmembrane and signaling domains. 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 exon-intron junction sequences at the start of mouse exon 2 and the end of mouse exon 4 were left intact 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 invention (2.8.6 and 11.5.1) to inhibit antigen-specific T cell proliferation in vivo was assessed using a sensitive T cell transfer assay (Figure 6a). In this assay, purified OTII (TCR transgenic) CD4 T cells specific for ovalbumin (OVA) from mice expressing homozygous human BTLA (hBTLA) and from OT-II mice expressing the wild-type murine BTLA receptor (The Jackson Laboratory) were used to assess the ability of the BTLA agonist antibodies of the invention (2.8.6 and 11.5.1) to inhibit antigen-specific T cell proliferation in vivo (Figure 6a). + Contains a mixture of T cells Mu5x10 5T cells were transferred into non-transgenic C57BL / 6 recipients. The CD45.2 (versus CD45.1) allotypic marker was used to distinguish the transferred cells from the host cells. Wild-type donor cells also expressed green fluorescent protein under the control of the human ubiquitin C promoter, allowing them to be distinguished from the humanized donor cells by flow cytometry. The day after T cell transfer, recipient mice were immunized with 100 μg ovalbumin (Sigma-Aldrich) in 100 μl PBS mixed with 100 μl Imject Alum (ThermoFisher) to induce T cell proliferation. On day 2, mice were administered 200 μg of antibody intraperitoneally. Eight days after the initial transfer of T cells, the ratio of humanized BTLA-expressing and wild-type OVA-specific T cells in the spleen was determined by flow cytometry. In this manner, it was possible to follow the proliferation or loss of humanized cells that bound the anti-human BTLA antibodies compared to a wild-type control that did not bind to the anti-human BTLA antibodies. Antibodies 2.8.6 and 11.5.1 both led to decreased proliferation of humanized BTLA cells compared to the wild-type control, indicating that these antibodies induce signaling through the inhibitory BTLA receptor, which leads to decreased T cell proliferation (Figure 6b).

[0263] Example 9. Inhibition of T cell proliferation in the mixed lymphocyte reaction A mixed lymphocyte reaction (MLR) was used to evaluate the ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit proliferation of primary T cells from humanized mice in vitro. Spleen cells from Balb / c mice were treated with mitomycin C for 30 minutes at 37°C, then washed and used as stimulator cells. T cells were purified from the spleens of humanized BTLA mice by negative selection using magnetic activated cell sorting (Mojosort Mouse CD3 T Cell Isolation Kit, Biolegend #480023) and stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher) to be used as responder cells. 4x10 per well 5 Stimulator cells and 2x105 Responding cells were mixed with various concentrations of anti-BTLA or isotype control antibody (clone MOPC-21, Biolegend #400165) in 96-well U-bottom plates. Serial 1:3 dilutions of antibody were evaluated starting at a concentration of 1 μg / ml for a total of 10 concentrations. 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 agonist antibodies. After 96 hours, dilution of CellTrace Violet in responding cells was evaluated by flow cytometry as a proliferation marker. Proliferation in the presence of anti-BTLA antibody or isotype control was compared to proliferation in the absence of antibody. CD4 + and CD8 + Populations were gated and analyzed separately. Antibodies 2.8.6 and 11.5.1 both reduced 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 maximal 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 maximal 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 A BTLA-transfected reporter T cell line was used to assess the ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit NFκB signaling. A Jurkat T cell line stably transfected with an expression cassette containing an NFκB-responsive transcription element upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences #TR850A-1) was used as a reporter cell line for NFκB signaling. A lentiviral transfection system was used to express full-length human or cynomolgus BTLA in this reporter cell line. These cells were mixed with a stimulator cell line composed 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 stimulator cell line was also transfected with murine FcγRIIB to provide Fc receptors for presentation of the agonist BTLA antibody. 5x10 cells per well were transfected with 10% IgG4-specific IgG4 antibodies. 4 Reporter cells were cultured at 5x10 in the presence of various concentrations of BTLA antibody or isotype control (clone MOPC-21, Biolegend #400165). 4Stimulator cells were mixed in a 96-well U-bottom plate. After 24 hours of incubation at 37°C, cells were pelleted and stained for flow cytometry with a viability dye (Zombie Aqua, Biolegend #423101) and mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830) to separate stimulator (murine) from responder (human) cells. For each antibody concentration, the geometric mean of GFP expression was evaluated and normalized to GFP expression in the absence of antibody. Clone 2.8.6 inhibited human BTLA-transfected cells with an IC50 of 0.06 nM and cynomolgus 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 Humanized mice were used to evaluate the ability of the BTLA agonist antibody 2.8.6 to ameliorate a T cell-driven model of colitis. This T cell transfer model has previously been described as a murine model of inflammatory bowel disease (Ostanin et al., Am J Physiol Gastrointest Liver Physiol. 296:G135-46, 2009). CD45RB sorted from spleens and lymph nodes of humanized BTLA mice. hi CD25-CD4+ T cells were administered at 5x10 per mouse. 5 At the dose of cells, Rag1 KO recipients (Rag1 tm1Mom ; The Jackson The recipient mice were injected intraperitoneally into the colon of the 2.8.6-treated mice (Laboratory). The transferred T cells cause an inflammatory colitis that develops after approximately 3 weeks and leads to diarrhea and weight loss. Rag1 KO caged mice that did not receive T cell transfer serve as non-diseased controls. 7, 21, and 35 days after T cell transfer, recipient mice were injected intraperitoneally with 200 μg of 2.8.6 or isotype control antibody. All mice were weighed periodically, and at 8 weeks, colons were weighed and 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 colonic inflammatory infiltration (Figure 8b). Colon inflammation in diseased mice led to an increase in colon weight:length ratio that was not seen 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 non-lethal parent-to-F1 GVHD model. Bone marrow cells (BMC) and spleen cells were cultured from humanized BTLA donor mice (C57BL / 6 background; H2 b ) was taken from 2x10 7 BMC and 107 spleen cells were cultured in CB6F1(H2 b / d ) 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 injected intraperitoneally with 200 μg anti-BTLA antibody or isotype control. Mice were weighed periodically and GVHD was monitored by calculating relative weight loss and by clinical observation. Mice were culled 5 weeks after immune cell transfer or when a humane end point was reached (including >20% weight loss compared to starting weight in the first 14 days or >15% weight loss at any other time point). At the time of death, colons were weighed and measured, and colon weights were calculated. Mass:length ratios were calculated as a marker of colonic inflammation, 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 colonic inflammation (Figure 9b).

[0267] Example 13. 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 previously described to significantly reduce Fc receptor binding (Clynes et al., Nat Med. 6:443-446, 2000). This mutant antibody was evaluated in the T cell transfer assay described in Example 8. The parental 11.5.1 antibody inhibited proliferation of humanized T cells such that its net effect was agonism of the BTLA receptor. However, the FcR-null D265A mutation led to enhanced proliferation of humanized T cells, suggesting that the FcR-null mutation removes the agonistic effect of the antibody, leaving only the effect of receptor blockade (Figure 10a).

[0268] The D265A mutant 11.5.1 antibody was also assayed in vitro as described in Example 9. The parental 11.5.1 antibody was also evaluated in an MLR assay. Again, the parental 11.5.1 antibody inhibited proliferation of humanized T cells such that its net effect was agonism of the BTLA receptor. The FcR-null D265A mutation removed the agonist effect of the antibody, and therefore the antibody had no effect in this assay (Figure 10b). The FcR-null 11.5.1 antibody did not enhance 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 do not fix complement in vitro Spleen cells from humanized mice were incubated with 10% baby rabbit complement (BioRad) and 20 μg / ml of anti-BTLA antibody (or isotype control or positive control depleting anti-CD20 antibody; clone SA271G2 from Biolegend) for 15 min at 37°C. Anti-CD20 antibody was B220 + While depleting the majority of B cells, B220 + or CD4 + Although both cells stained positive for BTLA, anti-BTLA antibodies did not deplete either of these populations (FIG. 11).

[0270] Example 15. Antibodies 2.8.6 and 11.5.1 do not induce ADCC in vitro Total spleen cells (including bone marrow effector cells) from humanized mice were incubated with 20 μg / ml of anti-BTLA antibody (or isotype control or depleting anti-CD20 antibody SA271G2) for 24 hours at 37°C. Anti-CD20 antibodies were B220 + While it depletes most of the cells, B220 + or CD4 + Although both cells stained positive for BTLA, anti-BTLA antibodies did not deplete either of these populations (FIG. 12).

[0271] Example 16. Antibodies 2.8.6 and 11.5.1 do not deplete BTLA-expressing cells in vivo Humanized BTLA mice were injected intraperitoneally with 200 μg anti-BTLA antibody or isotype control. 24 hours later, spleens were harvested and the frequency of different cell populations was identified by flow cytometry. Anti-BTLA antibody did not affect the frequency or absolute number of B or T cells in the spleen or the number of B cell precursors in the bone marrow (FIG. 13).

[0272] Example 17. Antibodies 2.8.6 and 11.5.1 stabilize BTLA expression on immune cells in vivo Humanized mice were injected intraperitoneally with 10 mg / kg of antibody 2.8.6 or 11.5.1. Six days after injection, mice were humanely sacrificed and spleens were harvested and processed into single cell suspensions for evaluation by flow cytometry. Cells were stained with antibody cocktails that identify immune cell subsets and with a fluorescently conjugated anti-BTLA antibody that has an epitope that is non-competitive with the injected antibody. After in vivo incubation with anti-BTLA antibody, the geometric mean of BTLA staining was normalized to the geometric mean of BTLA staining (using the same staining antibody) after incubation with an isotype control. 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 immunosuppressive purposes, agonistic antibodies that stabilize receptor expression offer the advantage of allowing prolonged high levels of inhibitory signaling through the pathway, compared to downregulatory antibodies.

[0273] Example 18. Tolerability and side effects in animal models No tolerability issues or side effects were observed in any animal studies with antibodies 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 homologous human germline framework regions (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 assessed by SPR. Humanized 2.8.6 had a K of 0.73 nM.D and bound to monomeric BTLA.

[0275] Example 20. Characterization of exemplary BTLA antibodies Described in this Example is the characterization of exemplary mIgG1 BTLA antibodies provided herein in addition to 2.8.6 and 11.5.1. Various clones listed in Table 1 were evaluated for their binding affinity to BTLA and efficacy in inhibiting lymphocytes (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 BTLA, were measured according to the methods described in Example 2, and curves were fitted for injection of the BTLA extracellular domain at a single concentration.

[0276] The inhibitory efficiency of each antibody against T cells was also evaluated at a single concentration of 10 μg / ml. For each individual antibody, MLR assays were performed (two biological replicates as shown in Table 3) according to the method described in Example 9; anti-CD3 assays were performed (two biological replicates, Table 3) according to the method described below; and the inhibition of NFκB signaling in human BTLA-transfected Jurkat T cell lines by each antibody was determined (Table 3) according to the method described in Example 10. The average inhibition of T cells in the various in vitro stimulation assays for each exemplary antibody 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 assessed as follows: Spleen cells from humanized BTLA mice were processed into single cell suspensions and treated with ACK buffer to lyse red blood cells. The cells were incubated with CFS The cells were stained with E (Biolegend Cat. No. 423801) to allow tracking of cell proliferation. 2x10 cells per well 5Cells were plated in 96-well U-bottom plates with soluble anti-CD3 (clone 145.2C11; Biolegend #100339) and anti-CD28 (clone 37.51; Biolegend #102115) antibodies at 50 ng / ml each, and soluble anti-BTLA antibodies or isotype controls at a concentration of 10 μg / ml. 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 surface expressed activation markers ("anti-CD3 / CD28 (CD69+ CD4 T cells)"). Percent inhibition was calculated for each BTLA antibody compared to the isotype control antibody.

[0278] Additionally, for each BTLA antibody, its ligand blocking ability, e.g., competition with HVEM for binding to BTLA, was assessed according to the methods described in Example 4, and the results are indicated as "yes" for greater than 90% inhibition of HVEM-BTLA binding and "no" for less than 10% inhibition of HVEM-BTLA binding. The functional epitope of each BTLA antibody was also determined according to the methods described in Example 5. The "Epitope" column in Table 2 summarizes the epitopes bound by each individual BTLA antibody. 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 first epitope (referred to in the table as "Epitope 1") that includes at least one critical 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 the ligand HVEM for binding to BTLA. Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, 26F3 all bind to a different second epitope ("epitope 2") that includes at least one critical residue selected from the list: Y39, K41, R42, Q43, E45, and S47. Antibodies that bind to epitope 2 compete with the ligand HVEM for binding to BTLA. Antibody 26B1 binds to a third epitope ("epitope 3") that includes at least one critical residue selected from the list: D35, T78, K81, S121, and L123. Antibodies that bind to epitope 3 compete with the ligand HVEM for binding to BTLA. Antibodies 24H7, 4B1, 8B4, 4H4 all bind to a different fourth epitope ("epitope 4") that includes the critical residue H68. Antibodies that bind to epitope 4 do not compete with the ligand HVEM for binding to BTLA. Antibody 21C7 binds to a fifth, distinct epitope ("epitope 5") that includes at least one critical residue selected from the list: N65 and A64. Antibodies that bind to epitope 5 do not compete with the ligand HVEM for binding to BTLA.

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

[0280] It may also be possible to substitute specific residues in the CDRs of an antibody to remove undesirable properties without significantly affecting target binding. CDRH2 of humanized antibody 6.2 was modified with D54E and N56Q substitutions (SEQ ID NO: 387) to remove deamidation potential (the engineered humanized VH sequence of 6.2 is provided in SEQ ID NO: 390). Similarly, the CDRH2 of humanized antibody 3E8, as determined by Lonza's Epibase analysis, ... 2 was modified with an N57Q substitution (SEQ ID NO:386) to eliminate deamidation potential and a K63S substitution to reduce expected immunogenicity (the engineered humanized VH sequence of 3E8 is provided in SEQ ID NO:389).

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

[0282] [Table 2]

[0283] Table 3. Inhibitory Efficacy Assay Results of Exemplary Antibodies

[0284] [Table 3]

[0285] Sequence Listing

[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 antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VH CDR having an amino acid sequence as set forth in any of 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, comprising 0-3 amino acid modifications.

2. 0-3 amino acid modifications, and 136, 137, 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 antigen-binding fragment thereof that specifically binds to human BTLA, comprising at least one VL CDR having an amino acid sequence as set forth 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 as set forth in SEQ ID NO: 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 or antigen-binding fragment thereof that specifically binds to human BTLA, comprising the CDRs.

5. 1. An isolated human antibody or antigen-binding fragment thereof that specifically binds to B and T lymphocyte attenuator (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, wherein (i) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, containing 0-3 amino acid modifications; or (ii) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, containing 0-3 amino acid modifications.

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

7. 1. An isolated human antibody or antigen-binding fragment thereof 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, CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:4, 5, and 6, respectively, containing 0-3 amino acid modifications; or (ii) CDRL1, CDRL2, CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:10, 11, 12, respectively, containing 0-3 amino acid modifications.

8. An isolated human antibody or antigen-binding fragment thereof 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 CDRL1, CDRL2, CDRL3 are selected from the group consisting of: (1) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, each of which comprises 0-3 amino acid modifications; (2) SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, each of which comprises 0-3 amino acid modifications; (3) SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, each of which comprises 0-3 amino acid modifications; (4) SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, each of which comprises 0-3 amino acid modifications; (5) SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, each of which comprises 0-3 amino acid modifications; (6) SEQ ID NO:66, each of which comprises 0-3 amino acid modifications. (7) SEQ ID NO:76, SEQ ID NO:47, and SEQ ID NO:78, each of which contains 0 to 3 amino acid modifications; (8) SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84, each of which contains 0 to 3 amino acid modifications; (9) SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, each of which contains 0 to 3 amino acid modifications; (10) SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, each of which contains 0 to 3 amino acid modifications; (11) SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:102, each of which contains 0 to 3 amino acid modifications; (12) SEQ ID NO:64, SEQ ID NO:107, and SEQ ID NO:108, each of which contains 0 to 3 amino acid modifications; (13) SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:114, each of which contains 0 to 3 amino acid modifications; (14) SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, each of which contains 0 to 3 amino acid modifications; (15) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, each of which contains 0 to 3 amino acid modifications; (16) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:138, each of which contains 0 to 3 amino acid modifications; (17) SEQ ID NO:142, SEQ ID NO:143, and SEQ ID NO:144, each of which contains 0 to 3 amino acid modifications; (18) SEQ ID NO:142, SEQ ID NO:143, and SEQ ID NO:144, each of which contains 0 to 3 amino acid modifications. (19) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:162, each of which contains 0 to 3 amino acid modifications; (20) SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, each of which contains 0 to 3 amino acid modifications; (21) SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, each of which contains 0 to 3 amino acid modifications; (22) SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:180, each of which contains 0 to 3 amino acid modifications; (23) SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:186, each of which contains 0 to 3 amino acid modifications; (24) SEQ ID NO:190, SEQ ID NO:191, and SEQ ID NO:192, each of which contains 0 to 3 amino acid modifications; (25) SEQ ID NO:196, SEQ ID NO:197, and SEQ ID NO:12, each of which contains 0 to 3 amino acid modifications; (26) SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:6, each of which contains 0 to 3 amino acid modifications; (27) SEQ ID NO:14, each of which contains 0 to 3 amino acid modifications. (28) SEQ ID NO:214, SEQ ID NO:35, and SEQ ID NO:216, each of which contains 0 to 3 amino acid modifications; (29) SEQ ID NO:10, 11, or 12, each of which contains 0 to 3 amino acid modifications; (30) SEQ ID NO:4, 5, or 6, each of which contains 0 to 3 amino acid modifications; or (31) the antibody or antigen-binding fragment thereof having the amino acid sequence as set forth in SEQ ID NO:142, 143, or 210, each of which contains 0 to 3 amino acid modifications.

9. An isolated human antibody or antigen-binding fragment thereof 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:1, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:2, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:3, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:

6.

10. An isolated human antibody or antigen-binding fragment thereof 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, wherein CDRH1 has the amino acid sequence as set forth in SEQ ID NO:7, CDRH2 has the amino acid sequence as set forth in SEQ ID NO:8, and CDRH3 has the amino acid sequence as set forth in SEQ ID NO:9, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has the amino acid sequence as set forth in SEQ ID NO:10, CDRL2 has the amino acid sequence as set forth in SEQ ID NO:11, and CDRL3 has the amino acid sequence as set forth in SEQ ID NO:

12.

11. 1. An isolated human antibody or antigen-binding fragment thereof 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, wherein (1) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:31, 32, and 33, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:34, 35, and 36, respectively, containing 0-3 amino acid modifications. (2) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:37, 38, and 39, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:40, 41, and 42, respectively, with 0-3 amino acid modifications; (3) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:43, 44, and 45, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:6, 47, and 48, respectively, with 0-3 amino acid modifications; (4) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:43, 56, and 57, respectively, with 0-3 amino acid modifications. (5) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:61, 62, and 63, respectively, with zero to three amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:64, 65, and 66, respectively, with zero to three amino acid modifications; (6) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:31, 32, and 69, respectively, with zero to three amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:34, 35, and 72, respectively, with zero to three amino acid modifications;(7) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:73, 74, and 75, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:76, 47, and 78, respectively, with 0-3 amino acid modifications; or (8) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:79, 80, and 81, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:79, 80, and 81, respectively, with 0-3 amino acid modifications. (9) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 85, 86, and 87, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 88, 89, and 90, respectively, containing 0-3 amino acid modifications; (10) C (10) DRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:61, 92, and 93, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:94, 95, and 96, respectively, with 0-3 amino acid modifications; or (11) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:97, 98, and 99, respectively, with 0-3 amino acid modifications, and CDRL1 (11) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:100, 101, and 102, respectively, with 0-3 amino acid modifications; (12) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:103, 104, and 105, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:64, 107, and 108, respectively, with 0-3 amino acid modifications;(13) A polypeptide as set forth in SEQ ID NOs: 88, 89, and 114, in which CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 109, 110, and 111, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 88, 89, and 114, respectively ... and (14) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:85, 110, and 117, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:88, 89, and 114, respectively, containing 0-3 amino acid modifications; (15) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:85, 110, and 117, respectively, containing 0-3 amino acid modifications; (15) having an amino acid sequence as set forth in SEQ ID NO:121, SEQ ID NO:122, and SEQ ID NO:123, each of which contains 0-3 amino acid modifications, and wherein CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, each of which contains 0-3 amino acid modifications; or (16) having an amino acid sequence as set forth in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, each of which contains 0-3 amino acid modifications. and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:34, 35, and 36, respectively, with 0-3 amino acid modifications; or (17) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:133, 134, and 135, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each contain 0-3 amino acid modifications. (18) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:139, 140, and 141, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:142, 143, and 144, respectively, containing 0-3 amino acid modifications;(19) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 145, 146, and 147, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 148, 149, and 150, respectively, containing 0-3 amino acid modifications; or (20) CDRH1, CDRH2, and CDRH3 each contain 0-3 amino acid modifications. (21) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:31, 32, and 33, respectively, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:148, 149, and 150, respectively, containing 0-3 amino acid modifications; or (22) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:31, 32, and 159, respectively, containing 0-3 amino acid modifications; (21) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 136, 137, and 162, respectively, containing 0-3 amino acid modifications; or (22) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 169, 170, and 171, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 each contain 0-3 amino acid modifications. (23) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:61, 62, and 63, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:64, 65, and 180, respectively, containing 0-3 amino acid modifications;(24) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:31, 182, and 183, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:136, 137, and 186, respectively, with 0-3 amino acid modifications; (25) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:187, 188, and 189, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3, respectively; (26) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:193, 194, and 195, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:196, 197, and 12, respectively, containing 0-3 amino acid modifications. (27) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:199, 200, and 201, respectively, containing 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:202, 203, and 6, respectively, containing 0-3 amino acid modifications; (28) CDRH1, CDRH2, CDRH3 each contain 0-3 amino acid modifications. (29) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 142, 209, and 210, respectively, containing 0-3 amino acid modifications; or (30) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 211, 212, and 213, respectively, containing 0-3 amino acid modifications; (30) CDRH1, CDRH2, CDRH3 have the amino acid sequences as set forth in SEQ ID NOs: 127, 386, and 129, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 34, 35, and 36 ...5, respectively, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 35, 35, and 35, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 36, 38, and 39, respectively, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs: 37, 38, and 39, respectively, and (31) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:205, 206, and 207, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:142, 209, and 210, respectively, with 0-3 amino acid modifications; (32) CDRH1, CDRH2, CDRH3 have amino acid sequences as set forth in SEQ ID NOs:127, 388, and 129, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have amino acid sequences as set forth in SEQ ID NOs:34, 35, and 36, respectively, with 0-3 amino acid modifications. (33) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:205, 387, and 207, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:142, 209, and 210, respectively, with 0-3 amino acid modifications; or (34) CDRH1, CDRH2, and CDRH3 have the amino acid sequences as set forth in SEQ ID NOs:205, 387, and 207, respectively, with 0-3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have the amino acid sequences as set forth in SEQ ID NOs:142, 143, and 210, respectively, with 0-3 amino acid modifications.

12. An isolated human antibody or antigen-binding fragment thereof that specifically binds to 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 set forth in SEQ ID NO:13, or a sequence having at least 90% identity to said sequence.

13. An isolated human antibody that specifically binds to BTLA, comprising a heavy chain and a light chain, or an antigen-binding A fragment of the antibody or antigen-binding fragment thereof, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence having at least 90% identity to said sequence.

14. The antibody or antigen-binding fragment thereof of 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 as set forth 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 as set forth in SEQ ID NO: 14, and the antibody binds to BTLA.

15. An isolated human antibody or antigen-binding fragment thereof that specifically binds to 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 set forth in SEQ ID NO: 17 or 21, or a sequence having at least 90% identity to said sequence.

16. An isolated human antibody or antigen-binding fragment thereof that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15, 19, or a sequence having at least 90% identity to said sequence.

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

18. 1. An isolated human antibody or antigen-binding fragment thereof that specifically binds to 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 set forth in SEQ ID NO: 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 with at least 90% identity thereto.

19. 1. An isolated human antibody or antigen-binding fragment thereof that specifically binds to BTLA, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 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 with at least 90% identity to said sequence.

20. (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:301, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:351; (2) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:302, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:352; (3) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:303, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:353; (4) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:305, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:355; (5) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:306, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:

356. (6) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 307, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 357; (7) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 308, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 358; (8) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 309, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:

359. (9) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:310, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:360; (10) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:311, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:361; (11) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:312, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:

362. (12) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 313, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 363; (13) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 314, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 364; (14) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 315, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:

364. (15) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:316, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:366; (16) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:317, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:367; (17) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:318, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:368;(18) The heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 319, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 369; (19) The heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 320, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 370; (20) The heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 321, and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 370; (21) (21) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 322, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 372; (22) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 324, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 374; (23) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 306, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 375; (24) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 322, and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 372; (25) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 327 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 377; (26) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 17 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15; (27) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21 (29) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:330 and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:380; (30) the heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:331 and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:381, and the antibody specifically binds to BTLA;(31) The heavy chain comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 382, ​​and the light chain comprises a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 383; (32) The heavy chain comprises SEQ ID NO: 3; 84 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 385; (33) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 389 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 383; (34) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 390 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 385; or (35) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 390 and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 378, and the antibody binds to BTLA, or an antigen-binding fragment thereof.

21. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the heavy or light chain also comprises a constant region.

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

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

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

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

27. 10. The antibody of any of the preceding claims, selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody and a multispecific antibody (e.g. a bispecific antibody).

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

29. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, which is monoclonal.

30. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, which agonizes human BTLA expressed on the surface of an immune cell, wherein the immune cell may be a T cell.

31. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein binding of the antibody or antigen-binding fragment thereof to human BTLA expressed on the surface of an immune cell reduces proliferation of the 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 thereof of claim 31 , wherein the decrease in cell proliferation is measured by the assay described in Example 8 or Example 9.

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

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

35. 34. The antibody or antigen-binding fragment thereof of any one of claims 31 to 33, wherein the decrease in cell proliferation is about 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. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, comprising a domain that binds to an Fc receptor.

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

38. The antibody or antigen-binding fragment thereof of claim 37, wherein the immune cell is an antigen-presenting cell.

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

40. 40. The antibody or antigen-binding fragment thereof of any one of claims 36 to 39, which binds to human BTLA expressed on the surface of a T cell.

41. 41. The antibody or antigen-binding fragment thereof of claim 40, wherein the binding to the Fc receptor expressed on the surface of the immune cell and the binding of the antibody or antigen-binding fragment thereof to human BTLA on the surface of the T cell results in the cell surface of the immune cell and the cell surface of the T cell being within 250 Å, 200 Å, 150 Å, or 100 Å.

42. 41. The antibody or antigen-binding fragment thereof of claim 40, wherein said binding to the Fc receptor expressed on the surface of the immune cell and said binding of said antibody or antigen-binding fragment thereof to human BTLA on the surface of the T cell results in a cell surface of the immune cell and a cell surface of the T cell 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 thereof of any one of claims 36 to 42, wherein the Fc receptor is FcγRIIB.

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

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

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

47. 46. ​​The antibody or antigen-binding fragment thereof of claim 44 or 45, wherein the decrease in NFκB signaling of the immune cell 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 thereof of any of the preceding claims, wherein binding of the antibody or antigen-binding fragment thereof to human BTLA expressed on the surface of an immune cell reduces dephosphorylation of the cytoplasmic domain of the human BTLA.

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

50. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof 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, and the antibody binds to cynomolgus BTLA with a KD of less than 20 nM as determined by surface plasmon resonance (SPR) at 37°C; does not inhibit binding of BTLA to herpes virus 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 51. The antibody or antigen-binding fragment thereof of claim 50, which binds to human B and T lymphocyte attenuation factor (BTLA) with an on-rate of (1 / Ms).

52. At 37°C, 3.0 x 10 －４ 52. The antibody or antigen-binding fragment thereof of claim 50 or 51, which binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than (1 / s).

53. 3.0x10 －４ (1 / s) ~1.0x10 －３ 53. The antibody or antigen-binding fragment thereof of any one of claims 50 to 52, which binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of (1 / s).

54. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has a molecular weight of at least 5.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C. ５ The antibody or antigen-binding fragment thereof, which specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an on-rate of (1 / Ms), wherein the antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and the antibody inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

55. 3.0x10 －４ 55. The antibody or antigen-binding fragment thereof of claim 54, which binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than (1 / s).

56. 56. The antibody or antigen-binding fragment thereof of claim 54 or 55, 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.

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

58. 10. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has a molecular weight of 3.0 x 10 as measured by surface plasmon resonance (SPR) at 37°C. －４ (1 / Ms) ~1.0x10 －３ The antibody or antigen-binding fragment thereof, which specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of (1 / Ms), wherein the antibody does not inhibit binding of BTLA to herpes virus 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 thereof of claim 58, 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.

60. 60. The antibody or antigen-binding fragment thereof 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 61. The antibody or antigen-binding fragment thereof of any one of claims 58 to 60, which binds to human B and T lymphocyte attenuation factor (BTLA) with an on-rate of (1 / Ms).

62. 10. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has a molecular weight of 1.0 x 10, as measured by surface plasmon resonance (SPR) at 37°C, respectively. －３ (1 / Ms) and an off-rate of at least 5.0x10 ５ The antibody or antigen-binding fragment thereof, which specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an on-rate of (1 / Ms), wherein the antibody does not inhibit binding of BTLA to herpes virus 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 thereof 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. 64. The antibody or antigen-binding fragment thereof 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. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof 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, wherein the antibody inhibits binding of BTLA to herpes virus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

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

67. 1.0 x 10 as determined by surface plasmon resonance (SPR) at 37°C. －３ 67. The antibody or antigen-binding fragment thereof of claim 65 or 66, which binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than (1 / s).

68. 68. The antibody or antigen-binding fragment thereof of any one of claims 65 to 67, which binds to cynomolgus 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. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has a concentration of 1x10 －３ The antibody or antigen-binding fragment thereof specifically binds to human B and T lymphocyte attenuation factor (BTLA) with an off rate of less than (1 / s), wherein the antibody inhibits binding of BTLA to herpes virus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

70. The antibody or antigen-binding fragment thereof of claim 69, which binds to cynomolgus 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. 71. The antibody or antigen-binding fragment thereof of claim 69 or 70, which 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.

72. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof 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; and the antibody inhibits binding of BTLA to herpes virus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

73. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof 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; and the antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and inhibits T cell proliferation in vitro as determined by a mixed lymphocyte reaction assay.

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

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

76. 10.0×10 as measured by surface plasmon resonance (SPR) at 37° C. －１ 76. The antibody or antigen-binding fragment thereof of claim 74 or 75, which binds to human BTLA with an off rate of less than (1 / s).

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

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

79. 3.0 x 10 as measured by surface plasmon resonance (SPR) at 37°C －１ 79. The antibody or antigen-binding fragment thereof of claim 78, which binds to human BTLA with an off rate of less than (1 / s).

80. 3.0 x 10 as measured by surface plasmon resonance (SPR) at 37°C －１ (1 / s) ~5.0x10 －１ 80. The antibody or antigen-binding fragment thereof of claim 78 or 79, which binds to human BTLA with an off rate of (1 / s).

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

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

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

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

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

86. 1.0 x 10 as measured by surface plasmon resonance (SPR) at 37°C ５ (1 / Ms) ~10x10 ５ 86. The antibody or antigen-binding fragment thereof of claim 84 or 85, which binds to human BTLA with an on-rate of (1 / Ms).

87. 6.0 x 10 as measured by surface plasmon resonance (SPR) at 37 °C －１ 87. The antibody or antigen-binding fragment thereof of any one of claims 84 to 86, which binds to human BTLA with an off rate of less than (1 / s).

88. 6.0 x 10 as measured by surface plasmon resonance (SPR) at 37 °C －１ (1 / s) ~10.0x10 －２ 88. The antibody or antigen-binding fragment thereof of any one of claims 84 to 87, which binds to human BTLA with an off rate of (1 / s).

89. 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 one of claims 1-88.

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

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

92. 1. A method for preparing a human antibody or antigen-binding fragment thereof that specifically binds to BTLA, comprising: 1) providing a host cell containing one or more nucleic acid molecules encoding heavy and light chain amino acid sequences that, when expressed, are capable of combining to produce an antibody or antigen-binding molecule thereof; 2) culturing a host cell that expresses the encoded amino acid sequence; and 3) Isolating the antibody or antigen-binding molecule of any one of claims 1 to 88. The method comprises the steps of:

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

94. An antibody or antigen-binding fragment thereof according to any 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 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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