Anti-BTLA Antibodies and Their Use in Cancer Treatment

Abstract

Anti-BTLA antibodies and antigen-binding fragments are provided, as are nucleic acids encoding the antibodies, compositions comprising the antibodies, and methods of producing the antibodies and methods of using the antibodies to treat or prevent cancer in a subject in need thereof.

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to PCT Patent Application No. PCT / CN2022 / 075097, filed January 29, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Technical field] The present invention relates to isolated anti-B and T lymphocyte attenuator (BTLA) monoclonal antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases, including cancer and / or related complications, are also provided.

[0003] [Reference to electronically submitted sequence listing] This application contains a Sequence Listing, which has been submitted electronically. The contents of the electronic Sequence Listing (065798.8WO2 Sequence Listing.xml; size: 148818 bytes; created Jan. 19, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] B- and T-lymphocyte attenuator (BTLA) belongs to the CD28 immunoglobulin superfamily. It is a co-inhibitory immune checkpoint molecule and shares structural and functional similarities with PD-1 and CTLA-4. BTLA expression is restricted to immune cells (T cells, B cells, DCs, NK cells, etc.). Upon binding of its ligand HVEM, the intracellular domain of BTLA signals through two phosphatases (SHP1 and SHP2) to inhibit downstream TCR and BCR signaling in T and B cells. BTLA has a distinct expression profile during T cell differentiation from other inhibitory checkpoints, suggesting that simultaneous or sequential blockade of multiple inhibitory molecules may improve T cell-based therapy. Therefore, BTLA is an ideal target for cancer immunotherapy to potentially treat and cure BTLA-positive cancers. Summary of the Invention

[0005] In one general aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to B and T lymphocyte attenuator (BTLA).

[0006] 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 153, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to B and T lymphocyte attenuator (BTLA), preferably human BTLA, and the monoclonal antibody or antigen-binding fragment thereof may not be of natural origin; An isolated monoclonal antibody or antigen-binding fragment thereof is provided. SEQ ID NO: 153 is represented by the amino acid sequence CAREDGYPYYTLDX1W, where X1 is an amino acid selected from C, A, S, T, or V.

[0007] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein said antibody or antigen-binding fragment thereof specifically binds to B and T lymphocyte attenuator (BTLA), preferably human BTLA, and said monoclonal antibody or antigen-binding fragment thereof may not be of natural origin.

[0008] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having a polypeptide sequence 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%, or at least 99% identical to SEQ ID NO: 15 or 13; or and a light chain variable region having a polypeptide sequence 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%, or at least 99% identical to SEQ ID NO: 16 or 14.

[0009] In one embodiment, the isolated anti-BTLA monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; or (2) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

[0010] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric or human or humanized.

[0011] In one embodiment, the humanized monoclonal antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; They have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 150, 10, 11, and 12, respectively; (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (3) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; (4) SEQ ID NOs: 7, 8, 149, 10, 11, and 12, respectively; (5) SEQ ID NOs: 7, 8, 151, 10, 11, and 12, respectively; or (6) SEQ ID NOs: 7, 8, 152, 10, 11, and 12, respectively.

[0012] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: SEQ ID NOs: 141, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119 , 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145, or 147; or SEQ ID NOs: 142, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 , 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, or 148.

[0013] In one embodiment, the humanized isolated anti-BTLA monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21, and a light chain variable region having the polypeptide sequence of SEQ ID NO:22; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:23, and a light chain variable region having the polypeptide sequence of SEQ ID NO:24; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:25, and a light chain variable region having the polypeptide sequence of SEQ ID NO:26; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:27, and a light chain variable region having the polypeptide sequence of SEQ ID NO:28; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:29, and a light chain variable region having the polypeptide sequence of SEQ ID NO:30; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:31, and a light chain variable region having the polypeptide sequence of SEQ ID NO:32; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 34; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:35, and a light chain variable region having the polypeptide sequence of SEQ ID NO:36; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:37, and a light chain variable region having the polypeptide sequence of SEQ ID NO:38; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 39, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 42; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 45, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 46; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:47, and a light chain variable region having the polypeptide sequence of SEQ ID NO:48; (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 49, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 50; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:51, and a light chain variable region having the polypeptide sequence of SEQ ID NO:52; (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53, and a light chain variable region having the polypeptide sequence of SEQ ID NO:54; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:55, and a light chain variable region having the polypeptide sequence of SEQ ID NO:56; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:57, and a light chain variable region having the polypeptide sequence of SEQ ID NO:58; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59, and a light chain variable region having the polypeptide sequence of SEQ ID NO:60; (24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 61, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 62; (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 63, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64; (26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:65, and a light chain variable region having the polypeptide sequence of SEQ ID NO:66; (27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 67, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68; (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:69, and a light chain variable region having the polypeptide sequence of SEQ ID NO:70; (29) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 73, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 74; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 75, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76; (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 77, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 78; (33) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 79, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80; (34) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 81, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 82; (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 83, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84; (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 87, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88; (38) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 89, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90; (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 91, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 92; (40) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 93, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 94; (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 95, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96; (42) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 97, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 98; (43) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 101, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 102; (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 103, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104; (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 105, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 106; (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 107, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108; (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 109, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 110; (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:111, and a light chain variable region having the polypeptide sequence of SEQ ID NO:112; (50) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 115, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 117, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 118; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 119, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 121, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 122; (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 123, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124; (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 125, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 126; (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 129, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 130; (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 131, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132; (60) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 133, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 134; (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 135, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 137, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 138; (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 139, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140; (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 146; or (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148.

[0014] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof specifically binds to cynomolgus BTLA.

[0015] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof has a K for human BTLA of less than about 25 nM, 20 nM, 15 nM, 5 nM, 2 nM, 1 nM, or 0.5 nM. D Combine with.

[0016] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an agonist of human BTLA and, upon binding to BTLA, activates downstream signaling from BTLA.

[0017] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting B cell proliferation and / or T cell and / or plasma cell activation. The T cells may, for example, be selected from the group consisting of CD4 T cells, CD8 T cells, Th1 T cells, THF T cells, αβ T cells, and γδ T cells.

[0018] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of blocking or abolishing the binding of BTLA to herpes virus entry mediator (HVEM).

[0019] In certain embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, is capable of binding to BTLA and increasing inflammatory cytokine production, such as interferon gamma (IFNγ), interleukin-2 (IL-2), CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), interleukin 1 beta (IL1β), and tumor necrosis factor alpha (TNFα); mediating the activity of a conjugated drug; and / or forming a bispecific antibody with another monoclonal antibody, or antigen-binding fragment thereof, that has cancer-killing effects.

[0020] Also provided is an isolated bispecific antibody or antigen-binding fragment thereof comprising the monoclonal antibody or antigen-binding fragment thereof of the invention.

[0021] Also provided is an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0022] Also provided is a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0023] Also provided is a host cell comprising a vector containing an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0024] In certain embodiments, a pharmaceutical composition is provided comprising an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the invention and a pharma- ceutically acceptable carrier.

[0025] Also provided is a method of specifically targeting B and T lymphocyte attenuator (BTLA) on the surface of cancer cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention.

[0026] Also provided is a method for treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the present invention. In one embodiment, the cancer is a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating effector T cells, more preferably a solid tumor with effector T cells expressing BTLA, and most preferably a solid tumor with infiltrating effector T cells expressing BTLA and with high expression of BTLA ligand HVEM in the tumor microenvironment. The cancer may be, for example, a BTLA-positive cancer. Examples of cancer may be selected from, for example, but are not limited to, melanoma, lung cancer, renal cell carcinoma, and liver cancer.

[0027] In some embodiments, the pharmaceutical composition further comprises a second anti-cancer therapeutic agent. The second anti-cancer therapeutic agent can be, for example, an anti-PD1 antibody or an antigen-binding fragment thereof.

[0028] Also provided is a method of producing a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, under conditions to produce the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cell or culture.

[0029] Also provided is a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising combining said monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof, with a pharma- ceutically acceptable carrier to obtain said pharmaceutical composition.

[0030] Also provided is a method for determining the level of BTLA in a subject. The method includes the steps of (a) obtaining a sample from the subject; (b) contacting the sample with an anti-BTLA monoclonal antibody or antigen-binding fragment thereof of the present invention; and (c) determining the level of BTLA in the subject. In some embodiments, the sample is a tissue sample. The tissue sample can be, for example, a cancer tissue sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample includes T cells (e.g., CD4, CD8, Th1, TFH, αβ, γδ), B cells, dendritic cells (DC), and natural killer (NK) cells.

[0031] [Brief description of the drawings] The foregoing summary and the following detailed description of preferred embodiments of the present application will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. [Brief description of the drawings]

[0032] [Figure 1A-1B] 1 shows a graph showing the identification of anti-BTLA blocking antibodies HFB6-2 and HFB6-3 through the HVEM-BTLA blocking reporter assay. [Figure 2A-2B] Graphs are shown demonstrating that HFB6-2 and HFB6-3 antibodies can bind to human and cynomolgus BTLA in FACS binding assays, HEK293 human BTLA (FIG. 2A) and Expi293 cynomolgus BTLA (FIG. 2B). [Figure 3A-3B] Graphs are shown demonstrating that HFB6-2 and HFB6-3 antibodies are anti-BTLA blocking agents in a cellular BTLA / soluble HVEM blocking assay (FIG. 3A) and a BTLA-HVEM blocking T cell reporter assay (FIG. 3B). [Figure 4A-4D] Graphs are shown showing that HFB6-2 and HFB6-3 antibodies bind to primary T and B cells. [Diagram 5] 1 shows a graph demonstrating that HFB6-2 and HFB6-3 antibodies reverse HVEM-mediated suppression of primary CD4+ T cell proliferation. [Figure 6] Graph showing that HFB6-3 increased IFNγ production and synergized with anti-PD1 antibody in a mixed lymphocyte reaction (MLR) assay. [Figure 7A-7G] Graph showing that HFB6-3 antibody, alone or in combination with anti-PD1 antibody, induced IFNγ and proinflammatory cytokine production in primary dissociated tumor cultures. [Figure 8A-8B] Graphs are shown demonstrating the desirable PK profiles of HFB6-2 and HFB6-3 antibodies in wild-type C57BL / 6 mice and hBTLA knock-in mice. [Figure 9] Graphs are presented showing functional profiling of the HFB6-2 humanized antibody in ELISA binding assays and HVEM-BTLA blocking reporter assays. [Figure 10A-10C]1 shows a graph depicting the results of humanized HFB6-3 antibody (containing cysteine ​​mutants of HCDR3 of humanized HFB6-3 antibody) in an HVEM-BTLA blocking reporter assay. [Figure 11A-11B] Graphs are shown showing that humanized HFB6-3 antibody variants can bind to human and cynomolgus BTLA in FACS binding assays in HEK293T human BTLA-expressing cells (FIG. 11A) and Expi293 cynomolgus BTLA-expressing cells (FIG. 11B). [Figure 12A-12B] Graphs are shown demonstrating that humanized HFB6-3 antibody variants can block the interaction of cellular BTLA with soluble HVEM in a FACS blocking assay (Figure 12A) and in a CHO.TCRa.hHVEM / Jurkat.hBTLA.NFAT-luc blocking reporter assay (Figure 12B). [Figure 13] 1 shows a graph demonstrating that humanized HFB6-3 antibody variants with cysteine ​​mutations were able to reverse HVEM-mediated suppression of primary CD4+ T cell proliferation. [Figures 14A-14C] Graph showing that humanized HFB6-3 antibody variants with cysteine ​​mutations induced IFNγ and proinflammatory cytokine production in primary dissociated tumor cultures. [Figures 15A-15C] Graph showing that humanized HFB6-3 antibody variants with cysteine ​​mutations induced IFNγ and proinflammatory cytokine production in primary dissociated tumor cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items form part of the prior art with respect to any invention(s) disclosed or claimed.

[0034] 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 invention belongs. If not, certain terms used herein have the meanings set forth herein.

[0035] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly indicates otherwise.

[0036] Unless otherwise stated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified in all cases by the term "about". Thus, numerical values ​​typically include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within that range, and includes integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0037] Unless otherwise indicated, the term "at least" preceding a series of elements is understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0038] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or other variations thereof, are understood to mean the inclusion of the recited element or group of elements, but not the exclusion of other elements or groups of elements, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied if any of the following are true: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), or both A and B are true (or exist).

[0039] As used herein, the conjunction "and / or" between multiple listed elements is understood to include both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any of these options are understood to be within the meaning and therefore meet the requirements of the term "and / or" as used herein. It is also understood that more than one of the options can be simultaneously applied and therefore meet the requirements of the term "and / or".

[0040] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," indicates that the specification and claims throughout include any recited element or group of elements, but that no additional element or group of elements may be added to the specified method, structure, or composition.

[0041] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," indicates that the specification and claims throughout may include any element or group of elements described, and may include any element or group of elements that do not materially alter the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0042] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably humans.

[0043] The words "right", "left", "lower" and "upper" designate directions in the drawings to which reference is made.

[0044] It should also be understood that terms such as "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or characteristics of preferred inventive components, indicate that the described dimensions / characteristics are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally identical or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not alter the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement errors or other systematic errors, manufacturing tolerances, and the like).

[0045] The term "identical" or percent "identical," in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-BTLA antibodies and the polynucleotides encoding them, BTLA polypeptides and the BTLA polynucleotides encoding them), refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence and determined using one of the following sequence comparison algorithms or by visual inspection, are the same or have a specified percentage of amino acid residues or nucleotides that are identical.

[0046] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on designated program parameters.

[0047] Optimal alignment for sequence comparison can be performed, for example, by the local homology algorithm of Smith & Waterman (Adv. Appl. Math. 1981; 2:482), the homology alignment algorithm of Needleman & Wunsch (J. Mol. Biol. 1970; 48:443), the search for similarity method of Pearson & Lipman (Proc. Nat'l. Acad. Sci. USA 1988; 85:2444), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by eye (generally as described in Current Protocols in Molecular Biology, F. M. Ausubel et al., (Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., 1995 Supplement (Ausubel).

[0048] Examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J. Mol. Biol. 1990; 215: 403-410 and Altschul et al., Nucleic Acids Res. 1997; 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet a certain positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0049] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (score for a pair of matching residues; always >0) and N (penalty for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score decreases by an amount X from its achieved maximum value; the cumulative score reaches zero or below due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989;89:10915).

[0050] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 1993; 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability of comparing the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0051] Another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with the polypeptide encoded by the second nucleic acid, as described below.Thus, for example, when two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide.Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0052] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, a peptide, or a protein) has been substantially separated, produced separately, or purified from other biological components of the organism in which it naturally originates, i.e., other chromosomal and non-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides, and proteins that are "isolated" therefore include nucleic acids and proteins that have been purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins are isolated while still part of a composition if the composition is not part of the natural environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, and chemically synthesized nucleic acids.

[0053] As used herein, the term "polynucleotide" is synonymous with "nucleic acid molecule," "nucleotides," or "nucleic acids," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, without limitation, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions, including RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases, and DNA or RNA whose backbones have been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Because various modifications can be made to DNA and RNA, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands, often referred to as oligonucleotides.

[0054] As used herein, the term "vector" is a replicon into which another nucleic acid segment can be operatively inserted so as to bring about the replication or expression of the segment.

[0055] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell that has been transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such a transfected cell. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome.

[0056] The term "expression" as used herein refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody may be present in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or anchored to the cell membrane.

[0057] As used herein, the term "peptide", "polypeptide", or "protein" refers to a molecule that contains amino acids and can be recognized as a protein by those of skill in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides that contain one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), and other modifications known in the art.

[0058] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomers of amino acids are known, it is the L-form of the amino acid that is represented unless otherwise specified.

[0059] [antibody] The present invention generally relates to isolated B and T lymphocyte attenuator (BTLA) antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, recombinant cells expressing the antibodies, and compositions containing the antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases such as cancer are also disclosed. The antibodies of the present invention have one or more desirable functional properties, including, but not limited to, high affinity binding to BTLA, high specificity for BTLA, ability to block binding of herpes virus entry mediator (HVEM) to BTLA, ability to inhibit B cell proliferation and / or T cell and / or plasma cell activation, ability to increase inflammatory cytokine production, and ability to inhibit tumor growth in subjects and animal models when administered alone or in combination with other anti-cancer therapies. Inflammatory cytokines may include, but are not limited to, interleukin 2 (IL-2), C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 1 beta (IL1 beta), tumor necrosis factor alpha (TNF alpha), and interferon gamma. T cells may include, but are not limited to, CD4 T cells, CD8 T cells, Th1 T cells, THF T cells, alpha beta T cells, and gamma delta T cells.

[0060] In a general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to B and T lymphocyte attenuator (BTLA).

[0061] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite antibodies, and chimeric antibodies, and antibody fragments that are monoclonal or polyclonal. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of antibodies is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibodies of the present invention can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domains. Thus, the antibody of the present invention may comprise a kappa or lambda light chain constant domain. According to a specific embodiment, the antibody of the present invention comprises a heavy and / or light chain constant region from a rat or human antibody. In addition to the heavy and light chain constant domains, the antibody comprises an antigen-binding region, which is composed of a light chain variable region and a heavy chain variable region, each of which comprises three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are also referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are also referred to as HCDR1, HCDR2, and HCDR3.

[0062] "Complementarity determining regions" (CDRs) are antibody regions that bind to antigens. CDRs can be defined using a variety of definition methods, including Kabat (Wu et al. J Exp Med 132: 211-50, 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. J Mol Biol 196: 901-17, 1987), IMGT (Lefranc et al. Dev Comp Immunol 27: 55-77, 2003), and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between various definition methods and variable region numbering has been described (see, for example, Lefranc et al. Dev Comp Immunol 27: 55-77, 2003; Honegger and Pluckthun, J Mol Biol 309:657-70, 2001; International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). Available programs such as abYsis by UCL Business PLC can be used to define CDRs. The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include CDRs defined by any of the above methods, i.e., Kabat, Chothia, IMGT or AbM, unless otherwise specified in the specification. Correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT proprietary numbering system, is well known to those of skill in the art (see, for example, Kabat; Chothia; Martin; Lefranc et al.).

[0063] JPEG2025504012000001.jpg57166

[0064] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTLA is substantially free of antibodies that do not bind BTLA). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in small amounts. The monoclonal antibodies of the invention may be made by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or by recombinant DNA methods. For example, monoclonal antibodies may be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse or rat, whose genome comprises a human heavy chain transgene and a light chain transgene.

[0066] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment binds. According to a specific embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain. According to other specific embodiments, the antigen-binding fragments include Fab and F(ab').

[0067] As used herein, the term "single-chain antibody" refers to a single-chain antibody conventionally known in the art, which comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids.

[0068] As used herein, the term "single domain antibody" refers to a single domain antibody as conventionally known in the art, which comprises a heavy chain variable region and a heavy chain constant region, or which comprises only a heavy chain variable region.

[0069] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. Human antibodies, within this definition, include complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0070] As used herein, the terms "humanized antibody" and / or "humanized antigen binding domain" refer to a non-human antibody that has been modified to increase sequence homology with human antibodies to maintain the antigen-binding properties of the antibody but reduce its antigenicity in the human body.

[0071] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of antibodies derived from another species of mammal (e.g., human) so as not to provoke an immune response in that species.

[0072] As used herein, the term "multi-specific antibody" refers to an antibody comprising multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are present on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are present on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0073] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds only up to two epitopes or up to two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are present on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are present on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a half antibody (or fragment thereof) having binding specificity for a first epitope and a half antibody (or fragment thereof) having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises an scFv (or fragment thereof) having binding specificity for a first epitope and an scFv (or fragment thereof) having binding specificity for a second epitope. In one embodiment, the first epitope is located on BTLA and the second epitope is located on PD-1, PD-L1, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD3, and / or other tumor-associated immunosuppressive factors or surface antigens.

[0074] As used herein, an antibody that "specifically binds to BTLA" is an antibody that binds to BTLA (preferably human BTLA) and has a KD of 1×10 -7 M or less, preferably 1×10 -8 M or less, preferably 5×10 -9 M or less, 1×10-9 M or less, 5×10 -10 M or less, or 1×10 -10 The term "KD" refers to an antibody and / or antigen-binding domain that is equal to or less than M. In one embodiment, the antibody and / or antigen-binding domain binds to cynomolgus BTLA. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using methods in the art in light of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, for example by using a biosensor system, such as a Biacore® system, or by using biolayer interference technology, such as an Octet RED96 system.

[0075] The smaller the K value of an antibody, the higher the affinity with which the antibody binds to a target antigen. In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, 20 nM, 15 nM, 5 nM, 2 nM, 1 nM, or 0.5 nM for human BTLA. D And then combine.

[0076] As used herein, "IC 50 The term IC refers to the half maximum inhibitory concentration of a monoclonal or bispecific antibody, or antigen-binding fragment thereof, of the invention. 50 is a measure of the potency of a monoclonal or bispecific antibody, or antigen-binding fragment thereof, of the invention to inhibit HVEM binding or to inhibit the function of BTLA in cells. In one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, or the bispecific antibody, or antigen-binding fragment thereof, has a potency of about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 Has a KD of less than M.

[0077] As used herein, "EC 50 The term EC refers to the half maximal effective concentration of a monoclonal or bispecific antibody, or antigen-binding fragment thereof, of the invention. 50 refers to the concentration of a monoclonal or bispecific antibody, or antigen-binding fragment thereof, to induce a biological response (i.e., cell death) that is exactly halfway between baseline and maximum over a particular exposure time. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, has an EC of less than about 1 μM, about 1000 nM to about 100 nM, about 100 nM to about 10 nM, about 10 nM to about 1 nM, about 1000 pM to about 500 pM, about 500 pM to about 200 pM, less than about 200 pM, about 200 pM to about 150 pM, about 200 pM to about 100 pM, about 100 pM to about 10 pM, or about 10 pM to about 1 pM. 50 has.

[0078] According to a particular embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, The monoclonal antibody or antigen-binding fragment or domain thereof comprises: heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 153, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein the antibody or antigen-binding fragment thereof or antigen-binding domain thereof specifically binds to B and T lymphocyte attenuator (BTLA), preferably human BTLA, and the monoclonal antibody or antigen-binding fragment thereof may not be of natural origin. SEQ ID NO: 153 is represented by the amino acid sequence CAREDGYPYYTLDX1W, where X1 is an amino acid selected from C, A, S, T, or V.

[0079] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein said antibody or antigen-binding fragment thereof specifically binds to B and T lymphocyte attenuator (BTLA), preferably human BTLA, and said monoclonal antibody or antigen-binding fragment thereof may not be of natural origin.

[0080] According to another specific embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15 or 13, or a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 or 14. According to a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14, respectively. According to a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16, respectively.

[0081] According to specific embodiments, the isolated anti-BTLA monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16; or (2) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

[0082] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14.

[0083] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16.

[0084] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof, of the present invention, wherein said monoclonal or bispecific antibody or antigen-binding fragment thereof is of chimeric type.

[0085] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof, of the present invention, wherein said monoclonal or bispecific antibody or antigen-binding fragment thereof is human or humanized.

[0086] According to another specific embodiment, the present invention relates to a humanized monoclonal antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, They have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 150, 10, 11, and 12, respectively; (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (3) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; (4) SEQ ID NOs: 7, 8, 149, 10, 11, and 12, respectively; (5) SEQ ID NOs: 7, 8, 151, 10, 11, and 12, respectively; or (6) SEQ ID NOs: 7, 8, 152, 10, 11, and 12, respectively; It relates to a humanized monoclonal antibody or an antigen-binding fragment thereof.

[0087] According to another specific aspect, the present invention relates to a humanized monoclonal antibody or antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising: SEQ ID NOs: 141, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119 , 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145, or 147; or SEQ ID NOs: 142, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 , 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, or 148.

[0088] According to another specific embodiment, the humanized anti-BTLA monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21, and a light chain variable region having the polypeptide sequence of SEQ ID NO:22; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:23, and a light chain variable region having the polypeptide sequence of SEQ ID NO:24; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:25, and a light chain variable region having the polypeptide sequence of SEQ ID NO:26; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:27, and a light chain variable region having the polypeptide sequence of SEQ ID NO:28; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:29, and a light chain variable region having the polypeptide sequence of SEQ ID NO:30; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:31, and a light chain variable region having the polypeptide sequence of SEQ ID NO:32; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 34; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:35, and a light chain variable region having the polypeptide sequence of SEQ ID NO:36; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:37, and a light chain variable region having the polypeptide sequence of SEQ ID NO:38; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 39, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 42; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 45, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 46; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:47, and a light chain variable region having the polypeptide sequence of SEQ ID NO:48; (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 49, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 50; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:51, and a light chain variable region having the polypeptide sequence of SEQ ID NO:52; (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53, and a light chain variable region having the polypeptide sequence of SEQ ID NO:54; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:55, and a light chain variable region having the polypeptide sequence of SEQ ID NO:56; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:57, and a light chain variable region having the polypeptide sequence of SEQ ID NO:58; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59, and a light chain variable region having the polypeptide sequence of SEQ ID NO:60; (24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 61, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 62; (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 63, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64; (26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:65, and a light chain variable region having the polypeptide sequence of SEQ ID NO:66; (27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 67, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68; (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:69, and a light chain variable region having the polypeptide sequence of SEQ ID NO:70; (29) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 73, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 74; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 75, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76; (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 77, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 78; (33) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 79, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80; (34) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 81, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 82; (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 83, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84; (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 87, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88; (38) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 89, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90; (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 91, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 92; (40) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 93, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 94; (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 95, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96; (42) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 97, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 98; (43) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 101, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 102; (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 103, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104; (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 105, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 106; (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 107, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108; (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 109, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 110; (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:111, and a light chain variable region having the polypeptide sequence of SEQ ID NO:112; (50) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 115, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 117, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 118; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 119, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 121, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 122; (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 123, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124; (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 125, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 126; (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 129, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 130; (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 131, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132; (60) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 133, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 134; (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 135, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 137, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 138; (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 139, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140; (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 146; or (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148.

[0089] In one embodiment, the invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a polypeptide that is at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, respectively. and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, respectively.

[0090] In one embodiment, the invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a polypeptide at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99, respectively. and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100, respectively.

[0091] In one embodiment, the present invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 149, 10, 11, and 12, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101, 109, 117, 125, 127, 129, 131, 133, or 135, respectively, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 102, 110, 118, 126, 128, 130, 132, 134, or 136, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 101, 109, 117, 125, 127, 129, 131, 133, or 135, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 102, 110, 118, 126, 128, 130, 132, 134, or 136, respectively.

[0092] In one embodiment, the present invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 150, 10, 11, and 12, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103, 111, 119, 137, 139, 141, 143, 145, or 147, respectively, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104, 112, 120, 138, 140, 142, 144, 146, or 148, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 103, 111, 119, 137, 139, 141, 143, 145, or 147, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104, 112, 120, 138, 140, 142, 144, 146, or 148, respectively.

[0093] In one embodiment, the invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 151, 10, 11, and 12, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 105, 113, or 121, respectively, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 106, 114, or 122, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 105, 113, or 121, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 106, 114, or 122, respectively.

[0094] In one embodiment, the invention relates to a humanized isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which have the polypeptide sequences of SEQ ID NOs: 7, 8, 152, 10, 11, and 12, respectively. In another embodiment, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 107, 115, or 123, respectively, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 108, 116, or 124, respectively. Preferably, the humanized isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 107, 115, or 123, respectively; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108, 116, or 124, respectively.

[0095] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an agonist of human BTLA and, upon binding to BTLA, activates downstream signaling from BTLA.

[0096] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting B cell proliferation and / or T cell and / or plasma cell activation. The T cells may, for example, be selected from the group consisting of CD4 T cells, CD8 T cells, Th1 T cells, THF T cells, αβ T cells, and γδ T cells.

[0097] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of blocking or abolishing the binding of BTLA to herpes virus entry mediator (HVEM).

[0098] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is capable of binding to BTLA and increasing inflammatory cytokine production, such as interferon gamma (IFNγ), interleukin-2 (IL-2), CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), interleukin 1β (IL1β), and tumor necrosis factor alpha (TNFα). In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is capable of increasing interferon gamma (IFNγ), interleukin-2 (IL-2), CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), interleukin 1β (IL1β), and / or tumor necrosis factor alpha (TNFα) production more than a control anti-BTLA antibody or antigen-binding fragment thereof. The control anti-BTLA antibody used herein may be an antibody capable of binding to BTLA and blocking the binding of BTLA to HVEM. Examples of control antibodies used herein may include, but are not limited to, the JS004 anti-BTLA antibody.

[0099] In another general aspect, the present invention relates to isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof and / or bispecific antibodies or antigen-binding fragments thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibodies or antigen-binding fragments thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0100] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. Any vector known to those skilled in the art, such as a plasmid, cosmid, phage vector, or viral vector, may be used in light of the present disclosure. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element for establishing the conventional function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter may be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to produce an antibody or antigen-binding fragment thereof in cells. Conventional cloning techniques or artificial gene synthesis may be used to generate recombinant expression vectors according to embodiments of the present invention.

[0101] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof, of the present invention. Any host cell known to those of skill in the art may be used for recombinant expression of an antibody or antigen-binding fragment thereof, in light of the present disclosure. In some embodiments, the host cell is an E. coli TG1 or BL21 cell (e.g., for expression of scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell, or a HEK293 cell (e.g., for expression of full-length IgG antibodies). According to specific embodiments, the recombinant expression vector is transformed into a host cell by conventional methods, such as chemical transfection, heat shock, or electroporation, where the recombinant nucleic acid is effectively expressed by stably integrating into the host cell genome.

[0102] In another general aspect, the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the invention, said method comprising culturing a cell comprising a nucleic acid encoding said monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof, under conditions to produce the monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention, and recovering said monoclonal and / or bispecific antibody or antigen-binding fragment thereof from said cell or cell culture (e.g. from the supernatant). The expressed monoclonal and / or bispecific antibody or antigen-binding fragment thereof may be harvested from the cells and purified according to conventional techniques known in the art and described herein.

[0103] [Pharmaceutical composition] In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, an isolated polynucleotide, and / or an isolated polypeptide of the invention, and a pharma- ceutically acceptable carrier.

[0104] As used herein, the term "pharmaceutical composition" refers to a product comprising an isolated polynucleotide of the invention, an isolated polypeptide of the invention, an anti-BTLA monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody of the invention, together with a pharma- ceutical acceptable carrier. The polynucleotides, polypeptides, anti-BTLA monoclonal antibodies or antigen-binding fragments thereof, and / or bispecific antibodies of the invention, and compositions comprising them, are also useful in the manufacture of medicaments for the therapeutic applications described herein.

[0105] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material that is widely known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration in a specific application. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the composition according to the present invention or the biological activity of the composition according to the present invention. According to specific embodiments, any pharmaceutical acceptable carrier suitable for use in antibody pharmaceutical compositions in light of the present disclosure may be used in the present invention.

[0106] Formulation of active pharmaceutical ingredients with pharmaceutically acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005), and later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, osmotic pressure adjusting agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used in formulating the pharmaceutical compositions of the present invention.

[0107] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation.Preferred examples of liquid formulations are aqueous formulations, i.e. formulations that contain water.Liquid formulations can include solutions, suspensions, emulsions, microemulsions, gels, etc.Aqueous formulations typically include at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0108] In one embodiment, the pharmaceutical composition may be formulated as an infusion and may be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection may be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0109] In another embodiment, the pharmaceutical composition is a solid formulation, such as a freeze-dried or spray-dried composition, which may be used as is or to which the physician or patient may add solvents and / or diluents before use.Solid dosage forms may include tablets, such as compressed tablets and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules).The pharmaceutical composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

[0110] The dosage forms may be of the immediate release type, in which case they may include a water soluble or dispersible carrier, or they may be of the delayed, sustained, or modified release type, in which case they may include a water insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneously.

[0111] In other embodiments, the pharmaceutical compositions may be delivered intranasally, intrabuccally, or sublingually.

[0112] The pH of the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0113] In another embodiment of the invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include: arginine, aspartic acid, bicine, citric acid, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)aminomethane, and mixtures thereof. The buffering agents, individually or collectively, may be present in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffering agents constitute alternative embodiments of the invention.

[0114] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservatives, individually or collectively, may be present in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments of the invention.

[0115] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonicity agent. Non-limiting examples of isotonicity agents include salts (e.g., sodium chloride), amino acids (e.g., glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditols (e.g., glycerol, 1,2-propanediol (propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Another example of an isotonicity agent includes sugars. Non-limiting examples of sugars may be monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans, such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonicity agent is a sugar alcohol, where the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactol, dulcitol, xylitol, and arabitol. The isotonicity agents, individually or collectively, may be present in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific isotonicity agents constitute alternative embodiments of the invention.

[0116] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agents, individually or collectively, may be present in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the invention.

[0117] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, non-limiting examples of which include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0118] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, said stabilizer being carboxy / hydroxycellulose and its derivatives (e.g., HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances (e.g., monothioglycerol), or thioglycolic acid. The stabilizers, individually or collectively, may be present in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the invention.

[0119] In a further embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion that comprises a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactants, individually or collectively, may be present in a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the invention.

[0120] In further embodiments of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA, and / or benzamidine hydrochloride (HCl). The protease inhibitors, individually or collectively, may be present in a concentration of from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0121] In another general aspect, the invention relates to a method of producing a pharmaceutical composition, said method comprising the step of combining a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the invention with a pharma- ceutical acceptable carrier to obtain said pharmaceutical composition.

[0122] [How to use] In another general aspect, the present invention relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-BTLA monoclonal and / or bispecific antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the cancer is a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating effector T cells, more preferably a solid tumor with effector T cells expressing BTLA, and most preferably a solid tumor with infiltrating effector T cells expressing BTLA and with high expression of the BTLA ligand HVEM in the tumor microenvironment. The cancer may be selected from, for example, but is not limited to, melanoma, lung cancer, renal cell carcinoma, and liver cancer.

[0123] In another general aspect, the invention relates to a method of targeting BTLA on the surface of cancer cells to achieve cell killing in a subject, the method comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof, that specifically binds to BTLA, or a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the invention. Binding of an anti-BTLA monoclonal or bispecific antibody or antigen-binding fragment to BTLA can block binding of herpes virus entry mediator (HVEM) to BTLA; upon binding to BTLA, can activate downstream signaling from BTLA; can inhibit B cell proliferation and / or T cell and / or plasma cell activation; and can increase inflammatory cytokine production, such as IL-2, CXCL9, CXCL10, IL1β, and TNFα. Monoclonal or bispecific antibodies or antigen-binding fragments thereof can, for example, serve to recruit conjugated drugs and / or form bispecific antibodies with another monoclonal antibody to mediate the death of targeted cancer cells.

[0124] Binding of an anti-BTLA monoclonal or bispecific antibody or antigen-binding fragment to BTLA can increase inflammatory cytokine production, such as IL-2, CXCL9, CXCL10, IL1β, and TNFα, more than a control anti-BTLA monoclonal or bispecific antibody or antigen-binding fragment thereof. The control anti-BTLA antibody used herein can be an antibody that can bind to BTLA and block the binding of BTLA to HVEM. Examples of control antibodies used herein can include, but are not limited to, the JS004 anti-BTLA antibody.

[0125] The functional activity of antibodies and antigen-binding fragments thereof that bind BTLA can be characterized by methods known in the art and described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind BTLA include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis, and detection of binding of antibodies and antigen-binding fragments to BTLA on cells (cells transfected with BTLA or cells naturally expressing BTLA) by FACS. According to specific embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind BTLA include those described below.

[0126] In another general aspect, the invention relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to BTLA, and / or a bispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the invention. The cancer may, for example, be selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, and liver cancer.

[0127] As used herein in connection with an anti-BTLA antibody or antigen-binding fragment thereof, a therapeutically effective amount refers to an amount of an anti-BTLA antibody or antigen-binding fragment thereof that modulates an immune response in a subject in need thereof. Also, as used herein in connection with an anti-BTLA antibody or antigen-binding fragment thereof, a therapeutically effective amount refers to an amount of an anti-BTLA antibody or antigen-binding fragment thereof that provides treatment for a disease, disorder, or condition; prevents or slows the progression of a disease, disorder, or condition; or reduces or completely alleviates symptoms associated with a disease, disorder, or condition.

[0128] According to a specific embodiment, the disease, disorder or condition to be treated is cancer, which may be, for example, melanoma, lung cancer, renal cell carcinoma, and liver cancer.

[0129] According to specific embodiments, a therapeutically effective amount refers to an amount of treatment sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition to be treated, or symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition to be treated, or symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition to be treated, or symptoms associated therewith; (iv) causing the regression of the disease, disorder, or condition to be treated, or symptoms associated therewith; (v) preventing the onset or onset of the disease, disorder, or condition to be treated, or symptoms associated therewith; (i) prevent the recurrence of the disease, disorder, or condition to be treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder, or condition to be treated, or symptoms associated therewith; (viii) reduce the length of hospitalization of a subject having the disease, disorder, or condition to be treated, or symptoms associated therewith; (ix) extend the survival of a subject having the disease, disorder, or condition to be treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder, or condition to be treated, or symptoms associated therewith in a patient; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0130] The therapeutically effective amount or dosage can vary depending on a variety of factors, such as: the disease, disorder, or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, weight, health status), whether the subject is human or animal, other administered drugs, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages are optimally titrated to optimize safety and efficacy.

[0131] According to specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein may be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0132] As used herein, the terms "treat", "treating", and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer, which may not necessarily be discernible in the subject, but may be discernible in the subject. The terms "treat", "treating", and "treatment" may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In specific embodiments, "treat", "treating", and "treatment" refer to alleviating, preventing the onset or onset, or reducing the duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor, or more preferably a cancer. In specific embodiments, "treat", "treating", and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In specific embodiments, "treat", "treating", and "treatment" refer to prolonging the survival of a subject having a disease, disorder, or condition. In specific embodiments, "treat", "treating", and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0133] According to specific embodiments, compositions are provided for use in the treatment of cancer. For cancer therapy, the compositions provided may be used in combination with other treatments, including but not limited to chemotherapy, anti-CD20 mAb, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD33 mAb, anti-CD47 mAb, anti-CD73 mAb, anti-PD-1 mAb, anti-PD-L1 mAb, anti-CTLA mAb, anti-TNFR2 mAb, anti-OX40 mAb, other immuno-oncology drugs, anti-angiogenic agents, radiation therapy, antibody drug conjugates (ADC), targeted therapy, other anti-cancer drugs, and / or treatments targeting immune-modulating targets, including but not limited to PD1 and PD-L1. Antibodies to BTLA can be used to construct bispecific antibodies with partner mAbs to PD-1, PD-L1, CTLA-4, CTLA, TNFR2, OX40, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, and / or CD3. Two antibodies that recognize two different epitopes on BTLA can also be used to construct bispecific antibodies to treat cancers / tumors that express BTLA.

[0134] As used herein, the term "in combination" refers to the use of multiple therapies in the context of the administration of two or more therapies to a subject. The use of the term "in combination" does not restrict the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapy to a subject.

[0135] In another general aspect, the invention relates to a method of determining the level of BTLA in a subject, the method comprising the steps of: (a) obtaining a sample from the subject; (b) contacting the sample with a monoclonal antibody or antigen-binding fragment thereof of the invention; and (c) determining the level of BTLA in the subject.

[0136] As used herein, "sample" refers to a biological sample isolated from a subject, and may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy (e.g., cancer tissue), lymph, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine, or other secretions, excretions, or other bodily fluids. "Blood sample" refers to whole blood or any fraction thereof, including blood cells, serum, and plasma. The sample may include, for example, Treg cells.

[0137] In one embodiment, the level of BTLA in a subject can be determined using an assay selected from, but not limited to, Western blot assay, immunohistochemistry (IHC), and ELISA. Relative protein levels can be determined by using Western blot analysis and IHC, and absolute protein levels can be determined by using ELISA. When determining the relative level of BTLA, the level of BTLA can be determined between at least two samples, for example, between samples from the same subject at different time points, between samples from different tissues in the same subject, and / or between samples from different subjects. Alternatively, when determining the absolute level of BTLA by ELISA or the like, the absolute level of BTLA in a sample can be determined by making a standard for ELISA before testing the sample. Those skilled in the art will understand which analytical technique should be used to determine the level of BTLA in a sample from a subject using the antibody or antigen-binding fragment thereof of the present invention.

[0138] The method of determining the level of BTLA in a sample from a subject can lead to the diagnosis of abnormal (elevated, decreased or insufficient) BTLA levels in disease and to appropriate therapeutic decisions.Such diseases can include cancer.Furthermore, by monitoring the level of BTLA in a subject, the risk of developing such diseases can be determined based on knowledge of the level of BTLA in a particular disease and / or during the progression of a particular disease.

[0139] [Embodiment] The present invention also provides the following non-limiting embodiments.

[0140] Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising: heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 153, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to BTLA, preferably specifically binds to human BTLA, and the monoclonal antibody or antigen-binding fragment thereof may not be of natural origin; An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0141] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising: heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; or (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to BTLA, preferably specifically binds to human BTLA, and the monoclonal antibody or antigen-binding fragment thereof may not be of natural origin; An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0142] Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment thereof described in embodiment 1 or 2, A heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 15 or 13; or A light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 16 or 14. Including, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0143] Embodiment 4 is an isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1 to 3, comprising: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16; or (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14; Including, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0144] Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, The antibody or antigen-binding fragment thereof is chimeric and / or human or humanized. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0145] Embodiment 6 is a humanized isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 5, the isolated monoclonal antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; They have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 150, 10, 11, and 12, respectively; (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (3) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; (4) SEQ ID NOs: 7, 8, 149, 10, 11, and 12, respectively; (5) SEQ ID NOs: 7, 8, 151, 10, 11, and 12, respectively; or (6) SEQ ID NOs: 7, 8, 152, 10, 11, and 12, respectively; An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0146] Embodiment 7 is a humanized isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 6, The isolated monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of SEQ ID NOs: 141, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 9, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145, or 147; or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 142, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, or 148. Including, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0147] Embodiment 8 is a humanized isolated monoclonal antibody or antigen-binding fragment thereof described in embodiment 7, The isolated monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21, and a light chain variable region having the polypeptide sequence of SEQ ID NO:22; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:23, and a light chain variable region having the polypeptide sequence of SEQ ID NO:24; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:25, and a light chain variable region having the polypeptide sequence of SEQ ID NO:26; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:27, and a light chain variable region having the polypeptide sequence of SEQ ID NO:28; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:29, and a light chain variable region having the polypeptide sequence of SEQ ID NO:30; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:31, and a light chain variable region having the polypeptide sequence of SEQ ID NO:32; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 34; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:35, and a light chain variable region having the polypeptide sequence of SEQ ID NO:36; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:37, and a light chain variable region having the polypeptide sequence of SEQ ID NO:38; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 39, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 42; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (16) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 45, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 46; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:47, and a light chain variable region having the polypeptide sequence of SEQ ID NO:48; (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 49, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 50; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:51, and a light chain variable region having the polypeptide sequence of SEQ ID NO:52; (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53, and a light chain variable region having the polypeptide sequence of SEQ ID NO:54; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:55, and a light chain variable region having the polypeptide sequence of SEQ ID NO:56; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:57, and a light chain variable region having the polypeptide sequence of SEQ ID NO:58; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59, and a light chain variable region having the polypeptide sequence of SEQ ID NO:60; (24) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 61, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 62; (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 63, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64; (26) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:65, and a light chain variable region having the polypeptide sequence of SEQ ID NO:66; (27) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 67, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68; (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:69, and a light chain variable region having the polypeptide sequence of SEQ ID NO:70; (29) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 73, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 74; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 75, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76; (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 77, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 78; (33) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 79, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80; (34) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 81, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 82; (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 83, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84; (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 87, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88; (38) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 89, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90; (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 91, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 92; (40) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 93, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 94; (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 95, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96; (42) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 97, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 98; (43) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 101, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 102; (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 103, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104; (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 105, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 106; (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 107, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108; (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 109, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 110; (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO:111, and a light chain variable region having the polypeptide sequence of SEQ ID NO:112; (50) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 115, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 117, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 118; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 119, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 121, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 122; (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 123, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124; (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 125, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 126; (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 129, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 130; (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 131, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132; (60) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 133, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 134; (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 135, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 137, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 138; (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 139, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140; (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 146; or (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148; An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0148] Embodiment 9 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, The monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, 20 nM, 15 nM, 5 nM, 2 nM, 1 nM, or 0.5 nM for human BTLA. D Join with An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0149] Embodiment 10 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, The monoclonal antibody or antigen-binding fragment thereof is an agonist of human BTLA and, upon binding to BTLA, activates downstream signaling from BTLA. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0150] Embodiment 11 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, The monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting B cell proliferation and / or T cell and / or plasma cell activation. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0151] Embodiment 12 is an isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 11, The T cells are selected from the group consisting of CD4 T cells, CD8 T cells, Th1 T cells, THF T cells, αβ T cells, and γδ T cells. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0152] Embodiment 13 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, The monoclonal antibody or antigen-binding fragment thereof is capable of blocking or eliminating binding of BTLA to herpes virus entry mediator (HVEM). An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0153] Embodiment 14 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, The monoclonal antibody or antigen-binding fragment thereof is capable of increasing inflammatory cytokine production; and / or mediating the mobilization of a conjugated drug; and / or forming a bispecific antibody with another mAb or antigen-binding fragment thereof that has a cancer-killing effect. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0154] Embodiment 15 is an isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 14, the inflammatory cytokine is selected from interferon gamma (IFNγ), interleukin-2 (IL-2), C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 1 beta (IL1β), and / or tumor necrosis factor alpha (TNFα); An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0155] Embodiment 16 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 15, The monoclonal antibody or antigen-binding fragment thereof specifically binds to cynomolgus monkey BTLA. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0156] Embodiment 17 is an isolated bispecific antibody or antigen-binding fragment thereof comprising the monoclonal antibody or antigen-binding fragment thereof described in any one of Embodiments 1 to 16.

[0157] Embodiment 18 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any one of embodiments 1-16.

[0158] Embodiment 19 is an isolated nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof described in embodiment 17.

[0159] Embodiment 20 is a vector comprising the isolated nucleic acid of embodiment 18 or 19.

[0160] Embodiment 21 is a host cell comprising the vector described in embodiment 20.

[0161] Embodiment 22 is a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof described in any one of Embodiments 1 to 16, or a bispecific antibody or antigen-binding fragment thereof described in Embodiment 17, and a pharma- ceutically acceptable carrier.

[0162] Embodiment 23 is a method of targeting BTLA on a cancer cell surface and / or treating cancer in a subject in need thereof, comprising: 23. The method of claim 22, further comprising administering to said subject in need thereof the pharmaceutical composition of embodiment 22. This is the method.

[0163] Embodiment 24 is the method of embodiment 23, further comprising: The cancer is a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating effector T cells, more preferably a solid tumor with effector T cells expressing BTLA, and most preferably a solid tumor with infiltrating effector T cells expressing BTLA and with high expression of the BTLA ligand HVEM in the tumor microenvironment. This is the method.

[0164] Embodiment 25 is the method of embodiment 23 or 24, comprising: The cancer is selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, and liver cancer; This is the method.

[0165] Embodiment 26 is the method according to any one of embodiments 23 to 25, The subject comprises BTLA-expressing T cells, B cells, dendritic cells (DCs), or natural killer (NK) cells. This is the method.

[0166] Embodiment 27 is the method according to any one of embodiments 23 to 26, The pharmaceutical composition further comprises a second anti-cancer agent. This is the method.

[0167] Embodiment 28 is the method of embodiment 27, further comprising: the second anti-cancer agent is an anti-PD-1 antibody or an antigen-binding fragment thereof; This is the method.

[0168] Embodiment 29 is a method for producing a monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, or a bispecific antibody or antigen-binding fragment thereof according to embodiment 17, comprising the steps of: Culturing a cell comprising a nucleic acid encoding the monoclonal antibody, or antigen-binding fragment thereof, or the bispecific antibody, or antigen-binding fragment thereof, under conditions to produce the monoclonal antibody, or antigen-binding fragment thereof, or the bispecific antibody, or antigen-binding fragment thereof; and Recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cell or culture. Including, This is the method.

[0169] Embodiment 30 is a method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 16, or a bispecific antibody or an antigen-binding fragment thereof according to embodiment 17, comprising: combining said monoclonal antibody or antigen-binding fragment thereof, or said bispecific antibody or antigen-binding fragment thereof, with a pharma- ceutically acceptable carrier to obtain said pharmaceutical composition; This is the method.

[0170] Embodiment 31 is a method for determining a level of BTLA in a subject, comprising: The method includes: a. obtaining a sample from said subject; b. contacting the sample with an isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-16; and c. determining the level of BTLA in the subject; Including, This is the method.

[0171] Embodiment 32 is the method of embodiment 31, further comprising: the sample is a tissue sample or a blood sample; The tissue sample may be a cancer tissue sample. This is the method.

[0172] Embodiment 33 is the method of embodiment 31, further comprising: The sample comprises T cells, B cells, dendritic cells (DCs), or natural killer (NK) cells. This is the method. EXAMPLES

[0173] Example 1: Identification of anti-human BTLA blocking antibodies that cross-react with cynomolgus monkey BTLA. This example describes the identification of two murine lead antibody candidates (HFB6-2 and HFB6-3, sequences set forth in Tables 2-5 below) based on their ability to reverse HVEM-mediated T cell suppression, as well as a benchmark anti-BTLA blocking antibody (referred to herein as JS004).

[0174] JPEG2025504012000002.jpg30166

[0175] JPEG2025504012000003.jpg35166

[0176] JPEG2025504012000004.jpg57166

[0177] JPEG2025504012000005.jpg62166

[0178] In the HVEM-BTLA blocking reporter assay, a CHO cell line engineered to express a TCR activator (TCRa) and HVEM (the ligand for BTLA) was co-cultured with a Jurkat reporter cell line expressing BTLA and a luciferase reporter driven by an NFAT-binding promoter. A panel of anti-BTLA antibody candidates was screened based on their ability to ablate the HVEM-BTLA interaction and restore the luciferase reporter signal (Figures 1A-1B). HFB6-2 and HFB6-3 were able to restore TCR activation to the same extent as the benchmark JS004 in this reporter assay.

[0179] In Figures 2A-2B, HEK293 cells overexpressing human BTLA and Expi293 cells overexpressing cynomolgus BTLA were incubated with different concentrations of anti-BTLA parental antibodies. Binding of anti-BTLA antibodies to target cells was quantified by FACS using an AF647-labeled anti-human IgG Fc secondary antibody. HFB6-2 and HFB6-3 bind human BTLA and cynomolgus BTLA with single-digit nM and sub-nM EC 50 It was shown to bind to the α-terminal amino acid sequence (Table 6).

[0180] JPEG2025504012000006.jpg45166

[0181] [Example 2: HFB6-2 and HFB6-3 are anti-BTLA blocking agents.] This example shows that HFB6-2 and HFB6-3 blocked the interaction of recombinant HVEM soluble protein with cellular BTLA and eliminated HVEM-mediated TCR inhibition in T cells.

[0182] In Figure 3A, recombinant HVEM soluble protein was labeled with DyLight 650 (DL650) fluorescent dye. HEK293 cells overexpressing human BTLA (HEK293.BTLA) were preincubated with increasing concentrations of HFB6-2 and HFB6-3 for 30 min at 4 °C and then with DL650-labeled HVEM for an additional 30 min. After washing, soluble HVEM that remained bound to BTLA on HEK293.BTLA cells was measured by FACS. HFB6-2 and HFB6-3 detected the interaction of soluble HVEM with cellular BTLA with IC in the single-digit nM. 50 and blocked it.

[0183] In Figure 3B, the functional blocker activity of HFB6-2 and HFB6-3 was evaluated in an HVEM-BTLA blockade reporter assay. In this assay, a CHO cell line engineered to express a TCR activator (TCRa) and HVEM (the ligand for BTLA) was co-cultured with a Jurkat reporter cell line expressing BTLA and a luciferase reporter driven by an NFAT-binding promoter. Application of HFB6-2 and HFB6-3 restored the luciferase reporter signal in a dose-dependent manner, similar to the benchmark JS004.

[0184] JPEG2025504012000007.jpg44166

[0185] [Example 3: HFB6-2 and HFB6-3 bind to primary T and B cells.] This example demonstrates that HFB6-2 and HFB6-3 each have an EC 50 and EC 50 and show binding to primary T and B cells.

[0186] In Figures 4A-4D, primary T and B cells were isolated from human PBMCs using the EasySep™ Human T Cell Isolation Kit and B cell isolation kit (STEMCELL Technologies; Vancouver, Canada). Isolated T or B cells were incubated with different concentrations of anti-BTLA parent antibodies. Binding of anti-BTLA antibodies to target cells was quantified by FACS using an AF647-labeled anti-human IgG Fc secondary antibody.

[0187] [Example 4: HFB6-2 and HFB6-3 reverse HVEM-mediated suppression of primary CD4+ T cell proliferation.] In Figures 5A-5B, cell culture plates were coated overnight at 4°C with anti-CD3 antibody together with recombinant HVEM-Fc fusion protein or control Fc protein (control Fc). + T cells were isolated from human PBMCs using the EasySep™ Human CD4+ T Cell Isolation Kit (STEMCELL Technologies) and labeled with CFSE. CD4+ T cells were added onto anti-CD3 and HVEM-Fc coated plates and incubated with anti-CD28 and anti-BTLA blocking antibodies and an isotype-matched control antibody (MGO53). After 4 days, CD4 + T cell proliferation is related to unstimulated CD4 + Quantitated by T cell associated CFSE dilution (%CFSE low). HFB6-2 and HFB6-3 reversed HVEM-mediated suppression of primary CD4+ T cell proliferation.

[0188] [Example 5: HFB6-3 increased IFNγ production and exerted a synergistic effect with anti-PD1 antibody in a mixed lymphocyte reaction (MLR) assay.] In the MLR assay in Figure 6, CD14 +Monocytes were isolated from one PBMC donor using the EasySep™ Human Monocyte Isolation Kit and differentiated into immature dendritic cells with GM-CSF and IL-4 for 5 days, then into mature dendritic cells (moDCs) with LPS for another 2 days. The moDCs were then mixed with primary T cells from a second donor at a 1:10 ratio in the presence of anti-BTLA and / or anti-PD1 antibodies. After 5 days, IFNγ in the supernatants was measured by AlphaLISA. In both moDC:T cell pairs tested, HFB6-3 alone enhanced IFNγ production compared to the isotype control (MGO53). HFB6-3 in combination with anti-PD1 further enhanced IFNγ production.

[0189] [Example 6: HFB6-3 alone or in combination with anti-PD1 induced IFNγ and proinflammatory cytokine production in primary dissociated tumor cultures.] Primary tumor tissues from lung cancer and melanoma patients were dissociated into single cell suspensions using a Human Tumor Dissociation Kit (Miltenyi Biotec; Bergisch Gladbach, Germany). BTLA and PD1 expression on tumor infiltrating lymphocytes (TILs) was measured by FACS. 1 × 10 5 Dissociated tumor cells were cultured with HFB6-3 and / or anti-PD1 (10 μg / ml) in the presence of anti-CD3 (10 ng / ml). Supernatants were collected on day 3 post-treatment and analyzed using the Human Cytokine / Chemokine 48-Plex Discovery Assay® (HD48) (Eve Technologies; Calgary, Canada). Supernatants were also collected on day 7 post-treatment and analyzed using an IFNγ ELISA (R&D Systems; Minneapolis, MN). HFB6-3 showed single agent efficacy and synergistic effects with anti-PD1 to increase the expression of multiple inflammatory cytokines in multiple lung and melanoma primary tumor cultures.

[0190] In Figures 7A-7G, dissociated tumor tissue from lung patient TIL320 has a higher % of BTLA+CD4+T cells than TIL619. In TIL320, HFB6-3 treatment alone increased IL-2, CXCL9, IL1β, and IFNγ production. Combination of HFB6-3 with anti-PD1 further enhanced IL-2, CXCL10, TNFα, and IFNγ production compared to either single agent treatment (Figures 7B, 7D, 7E, and 7G). In TIL619, HFB6-3 showed no single agent effect. However, HFB6-3 in combination with anti-PD1 further enhanced IL-2, CXCL9, CXCL10, TNFα, and IFNγ production compared to anti-PD1 treatment alone (Figures 7B, 7C, 7D, 7E, and 7G).

[0191] [Example 7: Favorable PK profile of HFB6-2 and HFB6-3 in wild-type C57BL / 6 mice and human BTLA knock-in mice.] In this experiment (Figures 8A and 8B), a single dose (10 mg / kg) of HFB6-2, HFB6-3, and benchmark JS004 was injected into wild-type C57BL / 6 mice (intravenous route, Figure 8A) and human BTLA knock-in mice (intraperitoneal route, hBTLA KI, Figure 8B). Blood was collected from wild-type C57BL / 6 mice after 1, 24, and 72 hours. Blood was collected from hBTLA KI mice after 0.5, 6, 24, 48, 72, 96, and 168 hours. Plasma concentrations of selected antibodies were measured by ELISA.

[0192] A single intravenous or intraperitoneal dose of HFB6-2 or HFB6-3 (10 mg / kg) in wild-type C57BL / 6 or hBTLA KI mice resulted in plasma concentrations of >40 μg / ml for at least 72 hours (Figures 8A and 8B); this supports dosing no more frequently than every 3 days for efficacy evaluation.

[0193] [Example 8: Functional profiling of HFB6-2 and HFB6-3 humanized antibodies] Twenty-one humanized variants of HFB6-2 (e.g., HFB6-2hz1-hz21) were generated based on standard humanization protocols, and the resulting humanized variants were tested to identify those with high affinity binding while maintaining the blocking activity of the parent HFB6-2.

[0194] In Figure 9, the binding of humanized HFB6-2 variants to recombinant human BTLA was evaluated in an ELISA binding assay. All tested HFB6-2 humanized variants maintained the antibody binding affinity of parental HFB6-2 in ELISA assay. Among them, HFB6-2hz16, HFB6-2hz18, and HFB6-2hz20 showed the closest functional blocking activity to parental HFB6-2 in HVEM-BTLA blocking reporter assay (Figure 9 and Table 8).

[0195] JPEG2025504012000008.jpg112166

[0196] Forty-five humanized variants of HFB6-3 containing cysteine ​​mutations on HCDR3 (e.g., HFB6-3hz1-hG4P to HFB6-3hz21-hG4P, HFB6-3hz20-hG4P-C109A / S / T / V) were generated based on standard humanization protocols. Because of the presence of an unpaired cysteine ​​on HCDR3 of HFB6-3, C109 on some humanized variants was mutated to A / S / T / V. In HVEM-BTLA blocking reporter assays (Figures 10A-10C), multiple humanized HFB6-3 variants, including but not limited to: HFB6-3hz6-hG4P, HFB6-3hz13-hG4P, HFB6-3hz20-hG4P, HFB6-3hz20-hG4P-C109A, HFB6-3hz24-hG4P-C109A, HFB6-3hz25-hG4P-C109A, HFB6-3hz24-hG4P-C109S, HFB6-3hz25-hG4P-C109S, restored TCR activation close to levels of parental HFB6-3.

[0197] The humanized antibody sequences are provided in Tables 9-12 below.

[0198] JPEG2025504012000009.jpg220166JPEG2025504012000010.jpg224166JPEG2025504012000011.jpg223166 JPEG2025504012000012.jpg223166JPEG2025504012000013.jpg222166JPEG2025504012000014.jpg132166

[0199] JPEG2025504012000015.jpg221166JPEG2025504012000016.jpg220166JPEG2025504012000017.jpg223166 JPEG2025504012000018.jpg225166JPEG2025504012000019.jpg222166JPEG2025504012000020.jpg132166

[0200] JPEG2025504012000021.jpg183166

[0201] JPEG2025504012000022.jpg52166

[0202] [Example 9: Binding and cross-reactivity of HFB6-3 humanized variants with cysteine ​​mutations.] In Figures 11A and 11B, HEK293 cells overexpressing human BTLA (Figure 11A) and Expi293 cells overexpressing cynomolgus BTLA (Figure 11B) were incubated with different concentrations of HFB6-3 humanized variants with selected cysteine ​​mutations. Binding of anti-BTLA antibodies to target cells was quantified by FACS using an AF647-labeled anti-human IgG Fc secondary antibody. HFB6-3hz24-hG4P-C109A, HFB6-3hz25-hG4P-C109A, HFB6-3hz24-hG4P-C109S, and HFB6-3hz25-hG4P-C109S, like the parental HFB6-3, bind human BTLA and cynomolgus BTLA with single-digit nM and sub-nM EC 50 (Figures 11A and 11B and Table 13).

[0203] JPEG2025504012000023.jpg76166

[0204] [Example 10: Blocking activity of HFB6-3 humanized variants with cysteine ​​mutations.] This example shows that HFB6-3 humanized variants with selected cysteine ​​mutations blocked the interaction of recombinant HVEM soluble protein with cellular BTLA and eliminated HVEM-mediated TCR inhibition in T cells comparable to the parent HFB6-3 antibody.

[0205] In Figure 12A, recombinant HVEM soluble protein was labeled with DyLight 650 (DL650) fluorescent dye. HEK293 cells overexpressing human BTLA (HEK293.BTLA) were preincubated with increasing concentrations of HFB6-3 humanized variants with selected cysteine ​​mutations or parental HFB6-3 for 30 min at 4°C, then with DL650-labeled HVEM for another 30 min. After washing, soluble HVEM remaining bound to BTLA on HEK293.BTLA cells was measured by FACS. HFB6-3hz24-hG4P-C109A, HFB6-3hz25-hG4P-C109A, HFB6-3hz24-hG4P-C109S, and HFB6-3hz25-hG4P-C109S inhibited the interaction of soluble HVEM with cellular BTLA with single-digit nM IC , comparable to the parental HFB6-3. 50 (Figure 12A and Table 14).

[0206] JPEG2025504012000024.jpg76166

[0207] In Figure 12B, the functional blocking activity of HFB6-3 humanized variants with selected cysteine ​​mutations was evaluated in the HVEM-BTLA blocking reporter assay. In this assay, a CHO cell line engineered to express TCR activator (TCRa) and HVEM (the ligand for BTLA) was co-cultured with a Jurkat reporter cell line expressing BTLA and a luciferase reporter driven by an NFAT-binding promoter. Application of HFB6-3hz24-hG4P-C109A, HFB6-3hz25-hG4P-C109A, HFB6-3hz24-hG4P-C109S, and HFB6-3hz25-hG4P-C109S restored the luciferase reporter signal in a dose-dependent manner, similar to the parental HFB6-3 (Figure 12B and Table 15).

[0208] JPEG2025504012000025.jpg56166

[0209] [Example 11: HFB6-3 humanized variants with cysteine ​​mutations reverse mediated suppression of primary CD4+ T cell proliferation.] In Figure 13, HFB6-3 humanized variants with selected cysteine ​​mutations were evaluated in the same assay as in Example 4. HFB6-3hz24-hG4P-C109A, HFB6-3hz25-hG4P-C109A, HFB6-3hz24-hG4P-C109S, and HFB6-3hz25-hG4P-C109S reversed HVEM-mediated suppression of primary CD4+ T cell proliferation, similar to the parental HFB6-3 antibody.

[0210] [Example 12: HFB6-3 humanized variants with cysteine ​​mutations induced IFNγ and proinflammatory cytokine production in primary dissociated tumor cultures.] In Figures 14A-14C and 15A-15C, the assays are the same as in Example 6. TIL F and TIL O are dissociated tumor samples from lung cancer patients. In Figures 14A-14C, treatment of HFB6-3hz24-hG4P-C109S in combination with anti-PD1 enhanced CXCL9, IL2, IL1β, TNFα, IFNγ, GM-CSF, and IL-17A production in TIL F compared to monotherapy alone. In Figures 15A-15C, monotherapy of HFB6-3hz24-hG4P-C109S enhanced IL1β, TNFα, and IFNγ production more strongly in TIL O compared to anti-PD1 or benchmark JS004.

[0211] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is therefore understood that the invention is not limited to the particular embodiments disclosed, but that it is intended to cover modifications within the scope of the invention as defined herein.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; These have the following polypeptide sequences: (1) SEQ ID NOs: 7, 8, 153, 10, 11, and 12, respectively; (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (3) SEQ ID NOs: 7, 8, 150, 10, 11, and 12, respectively; (4) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (5) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; (6) SEQ ID NOs: 7, 8, 149, 10, 11, and 12, respectively; (7) SEQ ID NOs: 7, 8, 151, 10, 11, and 12, respectively; or (8) SEQ ID NOs: 7, 8, 152, 10, 11, and 12, respectively; wherein the antibody or antigen-binding fragment thereof specifically binds to B- and T-lymphocyte attenuator (BTLA), preferably human or cynomolgus monkey BTLA, and the monoclonal antibody or antigen-binding fragment thereof may not be of natural origin; An isolated monoclonal antibody or antigen-binding fragment thereof.

2. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, The isolated monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of SEQ ID NOs: 15, 13, 141, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 139, 145, 146, 147, 148, 149, 150, 151, 152, 153, a heavy chain variable region having a polypeptide sequence at least 95% identical to any of the sequences of any of the sequences of the invention, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 16, 14, 142, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, or 148. Including, An isolated monoclonal antibody or antigen-binding fragment thereof.

3. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, Including: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 22; (7) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 23, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 24; (8) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 26; (9) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 27, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 28; (10) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (11) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 31, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 32; (12) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 34; (13) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 35, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 36; (14) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 37, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 38; (15) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 39, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 42; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (18) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 45, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 46; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 47, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 48; (20) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 49, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 50; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 51, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 52; (22) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 53, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 54; (23) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 55, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 56; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (25) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 59, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 60; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 61, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 62; (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 63, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64; (28) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 65, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 66; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 67, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68; (30) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 69, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 70; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (32) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 73, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 74; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 75, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76; (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 77, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 78; (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 79, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80; (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 81, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 82; (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 83, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84; (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 87, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88; (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 89, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90; (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 91, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 92; (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 93, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 94; (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 95, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96; (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 97, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 98; (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 101, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 102; (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 103, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104; (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 105, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 106; (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 107, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108; (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 109, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 110; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 111, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 112; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 115, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 117, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 118; (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 119, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120; (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 121, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 122; (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 123, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 125, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 126; (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 129, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 130; (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 131, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 133, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 134; (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 135, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136; (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 137, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 138; (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 139, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140; (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 146; or (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148; An isolated monoclonal antibody or antigen-binding fragment thereof.

4. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, The monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, 20 nM, 15 nM, 5 nM, 2 nM, 1 nM, or 0.5 nM for human BTLA. Ｄ Combine with An isolated monoclonal antibody or antigen-binding fragment thereof.

5. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, The monoclonal antibody or antigen-binding fragment thereof: (1) is an agonist of human BTLA and, upon binding to BTLA, activates downstream signaling from BTLA; (2) capable of inhibiting B cell proliferation and / or T cell and / or plasma cell activation; wherein said T cells may be selected from the group consisting of CD4 T cells, CD8 T cells, Th1 T cells, TFH T cells, ab T cells, and gd T cells; (3) can block or abolish the binding of BTLA to herpes virus entry mediator (HVEM); and / or (4) capable of increasing inflammatory cytokine production; mediating the mobilization of conjugated drugs; and / or forming a bispecific antibody with another mAb or antigen-binding fragment thereof having a carcinocidal effect, wherein the inflammatory cytokine may be selected from interferon g (IFN g), interleukin-2 (IL-2), C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 1b (IL1b), and / or tumor necrosis factor a (TNF a); An isolated monoclonal antibody or antigen-binding fragment thereof.

6. A bispecific antibody or antigen-binding fragment thereof, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof according to claim 6.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof according to claim 6, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, For use as a medicine, Pharmaceutical compositions.

12. The pharmaceutical composition of claim 11, for use as a pharmaceutical for targeting BTLA on the surface of cancer cells and / or for treating cancer in a subject; where, optionally: (a) the cancer may be a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating effector T cells, more preferably a solid tumor with effector T cells that express BTLA, and most preferably a solid tumor with infiltrating effector T cells that express BTLA and high expression of the BTLA ligand HVEM in the tumor microenvironment; or (b) the cancer may be selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, and liver cancer; The subject may comprise BTLA-expressing T cells, B cells, dendritic cells (DCs), or natural killer (NK) cells. Pharmaceutical compositions.

13. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof according to claim 6, comprising: culturing a cell comprising nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, under conditions to produce the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof; and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cell or culture. Including, method.

14. 10. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof of claim 6, comprising: combining the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition; method.

15. 1. An in vitro method for determining the level of BTLA in a subject, comprising: The method comprises: (a) contacting an isolated sample from a subject with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 5; and (b) determining the level of BTLA in said subject; Including, where, optionally: (i) the sample may comprise T cells, B cells, dendritic cells (DCs), or natural killer (NK) cells; and / or (ii) the sample may be a tissue sample or a blood sample, preferably the tissue sample may be a cancer tissue sample; method.

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