Anti-human vista antibodies and use thereof

Abstract

To provide antagonistic and agonistic anti-human VISTA antibodies and antibody fragments.SOLUTION: These antagonist antibodies and antibody fragments may be used to inhibit or block VISTA's suppressive effects on T cell immunity and thereby promote T cell immunity. These agonist antibodies and antibody fragments may be used to enhance or mimic VISTA's suppressive effects on T cell immunity and thereby suppress T cell immunity. These antagonist antibodies and antibody fragments are especially useful in the treatment of cancer and infectious conditions. These agonist antibodies and antibody fragments are especially useful in the treatment of autoimmunity, allergy, inflammatory conditions, GVHD, sepsis and transplant recipients. Screening assays for identifying these agonists are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application Nos. 62 / 323,193, filed April 15, 2016; 62 / 343,355, filed May 31, 2016; 62 / 372,362, filed August 9, 2016; 62 / 385,627, filed September 9, 2016; 62 / 425,184, filed November 22, 2016; and 62 / 425,184, filed July 19, 2016. No. 62 / 363,929 filed on July 21, 2016, No. 62 / 365,085 filed on September 9, 2016, No. 62 / 385,805 filed on July 19, 2016, No. 62 / 363,931 filed on July 21, 2016, No. 62 / 365,102 filed on July 21, 2016, No. 62 / 385,871 filed on September 9, 2016, No. 62 / 363,917 filed July 19, 2016, No. 62 / 365,081 filed July 21, 2016, No. 62 / 385,888 filed September 9, 2016, No. 62 / 364,073 filed July 19, 2016, No. 62 / 365,166 filed July 21, 2016, No. 62 / 365,166 filed September 9, 2016, This application claims priority to PCT Application Nos. 85,893, filed July 19, 2016, 62 / 363,925, filed July 21, 2016, 62 / 365,087, filed July 9, 2016, 62 / 385,785, filed September 9, 2016, and 62 / 406,632, filed October 11, 2016, each and every one of which is incorporated herein by reference. This application is related to PCT Application No. _______________ entitled "ANTI-HUMAN VISTA ANTIBODIES AND USE THEREOF," filed April ___, 2017 (Attorney Docket No. 43260.2214), which is incorporated by reference and also claims priority.

[0002] The present invention relates to the identification of novel anti-human VISTA antibodies and antibody fragments, i.e., anti-human VISTA (immunoglobulin V region-containing suppressor of T cell activation (1)) ("VISTA") antibodies and antibody fragments. More specifically, the present application provides novel human VISTA agonists, i.e., anti-human VISTA antibodies and antibody fragments that stimulate or enhance the suppressive effects of human VISTA on immunity, particularly T cell immunity. The present invention also provides novel anti-human VISTA antibodies and antibody fragments that stimulate or enhance the suppressive effects of human VISTA on immunity, particularly T cell immunity. + or CD8 + T cell proliferation, CD4 + or CD8 + The invention also relates to the use of such agonists to enhance or mimic the immune suppressive effects of VISTA, such as the suppressive effects of VISTA on T cell activation and on the production of immune cytokines, particularly pro-inflammatory cytokines. The invention also relates to the specific use of these agonistic antibodies and antibody fragments as prophylactic or therapeutic agents, particularly in the treatment of conditions in which the prevention or inhibition of T cell immunity and pro-inflammatory cytokine expression is therapeutically beneficial, such as autoimmune, inflammatory, allergic disorders, sepsis, GVHD, or in reducing the inflammatory side effects of some conditions, such as cancer.

[0003] The present application also provides novel antagonists, i.e., anti-human VISTA antibodies and antibody fragments, that antagonize or inhibit the immune suppressive effects of human VISTA, particularly the effects of VISTA on T cell immunity. The present invention also provides novel antagonists, i.e., anti-human VISTA antibodies and antibody fragments that antagonize or inhibit the immune suppressive effects of VISTA, i.e., the effects of VISTA on CD4 + or CD8 + T cell proliferation, CD4 + or CD8 + The invention also relates to the use of such novel antagonists to block or inhibit the suppressive effects of VISTA on T cell activation and immune cytokine production. The invention also relates to the specific use of these antagonistic antibodies and antibody fragments as prophylactic or therapeutic agents, particularly in the treatment of conditions in which the promotion of T cell immunity is therapeutically beneficial, such as the treatment of cancer and infectious diseases. [Background technology]

[0004] The immune negative checkpoint regulator (NCR) pathway has proven to be a special clinical target in the treatment of human immune-related diseases. Blockade of two NCRs, CTLA-4 and PD-1, using monoclonal antibodies (mAbs) to enhance tumor immunity has revolutionized the treatment of cancer and established these pathways as clinically validated targets for human disease. Soluble versions of NCR ligands that drive the NCR pathway have also been deployed in the clinic as immunosuppressive drugs to treat autoimmunity (i.e., AMP-110 / B7-H4-Ig for rheumatoid arthritis).

[0005] VISTA (see reference 1) is an NCR ligand whose closest phylogenetic relative is PD-L1. VISTA shares homology with PD-L1 but displays a unique expression pattern that is restricted to the hematopoietic compartment. Specifically, VISTA is a marker for CD11b high Constitutively and highly expressed on myeloid cells, CD4 + T cells and CD8 + It is expressed at lower levels on T cells. Like PD-L1, VISTA is a potent immunosuppressive ligand (Ref. 1), and like PD-L1, VISTA blockade can confer therapeutic immunity against cancer in preclinical oncology models (Ref. 2). VISTA blockade suppresses immune responses, particularly CD8 + and CD4 + Although VISTA enhances T cell-mediated immunity, treatment with a soluble Ig fusion protein of the extracellular domain of VISTA (VISTA-Ig) has been shown to suppress immunity and halt progression in multiple mouse models of autoimmune disease.

[0006] Clear scientific evidence has demonstrated that VISTA is a ligand that induces strong T cell suppression. Numerous antagonistic anti-human VISTA antibodies have been reported by different groups, including Dartmouth College and Jannsen. These antibodies are useful in the treatment of conditions, such as cancer and infection, in which suppression of the immunosuppressive effects of VISTA on T cell immunity is desirable. However, to the best of the inventors' knowledge, no anti-human VISTA antibodies or antibody fragments that stimulate the action of human VISTA have been identified to date. Such agonistic anti-human VISTA antibodies and antibody fragments would be desirable for the treatment of conditions in which suppression of immunity, particularly T cell immunity, is desirable, and / or in which VISTA expression is abnormally downregulated. Summary of the Invention

[0007] It is an object of the present invention to provide novel antibodies and antibody fragments that specifically bind to human VISTA and its variants, for example, chimeric, human, humanized, or multispecific anti-human VISTA antibodies that specifically bind to human VISTA and enhance or mimic the immune effects of human VISTA.

[0008] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof that comprises an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment binds to the same or overlapping epitope as any one of the anti-human VISTA antibodies having the CDRs and variable heavy and variable light polypeptides shown in Figure 4.

[0009] It is a specific object of the present invention to provide an isolated antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human IgV domain suppressor of T cell activation (human VISTA), wherein the antibody or antibody fragment stimulates or enhances one or more of the immune effects of VISTA, e.g., a human IgG2 constant region or a human IgG2 Fc region that optionally binds to an Fc gamma receptor, wherein the human IgG2 constant region or Fc region comprises human CD32A and / or native human IgG2 that binds to an Fc gamma receptor and / or an IgG2 that binds to an FcyR comprising one or more of hFcγRI (CD64), FcyRIIA or hFcyRIIB, (CD32 or CD32A), and FcyRIIIA (CD16A), or FcyRIIIB (CD16B).

[0010] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment binds to a VISTA epitope that includes or overlaps with the epitope bound by any of the anti-human VISTA antibodies having the sequence of Figure 4.

[0011] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof that comprises an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment binds to or interacts with one of more residues of an epitope that includes residues LLDSGLYCCLVVEIRHHHSEHRVH.

[0012] It is a specific object of the present invention to provide agonistic antibodies or antibody fragments thereof that comprise an antigen binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment binds to or interacts with one of more residues of an epitope that includes one or more of the following residues: 79EVQTCSERRPIR90, 48NVTLTCRLLGPV60, 153HHHSEHRVHGAM164, 52LTCRLLGPV60, 56LLGPVDKGHDVTFYK70, 113LAQRHGLESASDHHG127, 153HHHSEHRVHGAM164, 93TFQDLHLHHGGHQAA107, 146CLVVEIRHHHSEH158, 53TCRLLGPVDKG63, 123SDHHG127, and / or 153HHHSEHRVHGAM164.

[0013] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof that comprises an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment binds to or interacts with one of more residues of an epitope that includes one or more residues of 79EVQTCSERRPIR90.

[0014] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, which antibody or antibody fragment promotes or enhances the inhibitory effect of human VISTA on at least one of the immune effects of human VISTA, for example, induction of T cell immunity, monocyte activation, T cell proliferation; induction or suppression of cytokine expression, increased monocyte survival, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in cell-expressed VISTA; and induction of antibody-dependent cellular phagocytosis (ADCP) in cell-expressed VISTA.

[0015] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment thereof comprises an antigen-binding region that specifically binds to human VISTA, and the antibody or antibody fragment comprising a variable heavy sequence and a variable light sequence has a CDR polypeptide identical to any one of the anti-human VISTA antibodies having the CDRs and variable heavy and variable light polypeptides shown in Figure 4, with the proviso that when the antibody or fragment comprises an antagonist anti-human VISTA antibody or antibody fragment, the antibody or antibody fragment does not comprise the same CDR as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0016] In the case of antibodies, including agonistic or antagonistic anti-human VISTA antibodies or fragments, it is a specific object of the present invention to provide agonistic antibodies or antibody fragments thereof that comprise an antigen-binding region that specifically binds to human VISTA, wherein the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0017] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment comprises a variable heavy polypeptide and / or a variable light polypeptide having at least 90% sequence identity to the sequence of an anti-human VISTA antibody selected from any one of VSTB49 to VSTB116, the variable heavy polypeptide sequence and variable light polypeptide sequence being as shown in Figure 4, with the proviso that when the antibody or fragment comprises an antagonist anti-human VISTA antibody or fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0018] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment comprises a variable heavy polypeptide and / or variable light polypeptide having at least 95% sequence identity to the sequence of an anti-human VISTA antibody selected from any one of VSTB49 to VSTB116, the variable heavy polypeptide sequence and variable light polypeptide sequence being as shown in Figure 4, with the proviso that when the antibody or fragment comprises an antagonist anti-human VISTA antibody or fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0019] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment comprises a variable heavy polypeptide and / or a variable light polypeptide having at least 96-99% sequence identity to the sequence of an anti-human VISTA antibody selected from any one of VSTB49 to VSTB116, the variable heavy polypeptide sequence and variable light polypeptide sequence being as shown in Figure 4, with the proviso that when the antibody or fragment comprises an antagonist anti-human VISTA antibody or antibody fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0020] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment comprises a variable heavy polypeptide and / or a variable light polypeptide that is identical to the sequence of an anti-human VISTA antibody selected from one of VSTB49 to VSTB116, the variable heavy polypeptide sequence and variable light polypeptide sequence being as shown in Figure 4, with the proviso that when the antibody or fragment comprises an antagonist anti-human VISTA antibody or antibody fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0021] It is a specific object of the present invention to provide an antagonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, which antagonizes or blocks at least one immune effect of human VISTA.

[0022] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, which stimulates or promotes at least one immune effect of human VISTA.

[0023] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen binding region that specifically binds to human VISTA as defined in any of the foregoing, which comprises a human constant domain.

[0024] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, for example comprising a human constant domain selected from IgG1, IgG2, IgG3, and IgG4, optionally modified by deletion, substitution, or addition mutations, or by any combination of the foregoing.

[0025] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described in any of the foregoing, wherein the antibody fragment comprises or is a Fab, F(ab')2, or scFv antibody fragment.

[0026] It is a specific object of the present invention to provide an antagonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to any of the above-described human VISTA, which blocks or inhibits at least one of the immune effects of human VISTA selected from, for example, the inhibitory effect of human VISTA on T cell immunity, monocyte activation, or T cell proliferation; induction or inhibition of cytokine expression, increased monocyte survival, inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) of cell-expressed VISTA; and inhibition of antibody-dependent cellular phagocytosis (ADCP) of cell-expressed VISTA.

[0027] For example, it is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to any of the above-described human VISTA, which promotes or enhances at least one of the immune effects of human VISTA selected from the inhibitory effects of human VISTA: inhibition of T cell immunity, monocyte activation, or T cell proliferation; induction or inhibition of cytokine expression, increased monocyte survival, inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) in cell-expressed VISTA; and inhibition of antibody-dependent cellular phagocytosis (ADCP) in cell-expressed VISTA.

[0028] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, which comprises a human IgG2 constant region or an Fc region.

[0029] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to any of the above-described human VISTA, which promotes or enhances the immune suppressive effects of human VISTA on any one or more of the following: T cell immunity, monocyte activation, T cell proliferation; cytokine expression, monocyte survival, antibody-dependent cell-mediated cytotoxicity (ADCC) in cell-expressed VISTA; and antibody-dependent cellular phagocytosis (ADCP) in cell-expressed VISTA.

[0030] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, which inhibits T cell immunity and / or pro-inflammatory cytokine expression.

[0031] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described in any of the above, which is a human antibody, humanized antibody, or chimeric antibody comprising a human Fc region, for example, human IgG1, IgG2, IgG3, and IgG4, or a chimera of any of the above.

[0032] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, which is chimeric, human, or humanized.

[0033] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, comprising a human IgG2 constant domain or Fc region that can potentially be mutated.

[0034] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen binding region which specifically binds to human VISTA as defined in any of the foregoing, comprising a human IgG2 constant domain or fragment thereof, or hlgG1, hIgG3, hIgG4, IgA, IgD, IgE, or IgM, wherein the entire hinge or substantially the entire hinge and CH1 domain, and optionally the entire light chain constant region or substantially the entire light chain constant region of the antibody has been replaced with the corresponding entire light chain and hinge and CH1 domain of hlgG2 ("H2 region" or "H2 domain").

[0035] (i) an IgG2 Fc region in which either or both of the heavy chain cysteine ​​residue at position 127 and the light chain cysteine ​​residue at position 214 (numbering according to Kabat) have been deleted or changed to a different amino acid residue, resulting in increased agonistic properties of the resulting modified antibody compared to an antibody in which those residues are not altered; and (ii) the cysteine ​​residue at position 214 in the H2 region of the antibody has been mutated or replaced with another amino acid, and / or the cysteine ​​residues at positions 127, 232, or 233 of the heavy chain have been deleted or changed to a different amino acid residue. It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising: (i) a human IgG2 constant domain in which one or more of the following residues are deleted or substituted with another amino acid; and (ii) at least one cysteine ​​residue is deleted or changed to another amino acid; and (iii) an antigen-binding region that specifically binds to human VISTA as described above, which competes with or binds to the same epitope on human VISTA as VSTB95 (the variable heavy and variable light sequences shown in Figure 4).

[0036] (i) V of SEQ ID NOs: 100, 101, and 102 H CDRs and V of SEQ ID NOs: 103, 104, and 105 L containing CDRs, (ii) V of SEQ ID NOs: 110, 111, and 112 HCDRs and V of SEQ ID NOs: 113, 114, and 115 L containing CDRs, (iii) V of SEQ ID NOs: 120, 121, and 122 H CDRs and V of SEQ ID NOs: 123, 124, and 125 L containing CDRs, (iv) V of SEQ ID NOs: 130, 131, and 132 H CDRs and V of SEQ ID NOs: 133, 134, and 135 L containing CDRs, (v) V of SEQ ID NOs: 140, 141, and 142 H CDRs and V of SEQ ID NOs: 143, 144, and 145 L containing CDRs, (vi) V of SEQ ID NOs: 150, 151, and 152 H CDRs and V of SEQ ID NOs: 153, 154, and 155 L containing CDRs, (vii) V of SEQ ID NOs: 160, 161, and 162 H CDRs and V of SEQ ID NOs: 163, 164, and 165 L containing CDRs, (viii) V of SEQ ID NOs: 170, 171, and 172 H CDRs and V of SEQ ID NOs: 173, 174, and 175 L containing CDRs, (ix) V of SEQ ID NOs: 180, 181, and 182 H CDRs and V of SEQ ID NOs: 183, 184, and 185 L containing CDRs, (x) V of SEQ ID NOs: 190, 191, and 192 H CDRs and V of SEQ ID NOs: 193, 194, and 195 L containing CDRs, (xi) V of SEQ ID NOs: 200, 201, and 202 H CDRs and V of SEQ ID NOs: 203, 204, and 205 L containing CDRs, (xii) V of SEQ ID NOs: 210, 211, and 212 HCDRs and V of SEQ ID NOs: 213, 214, and 215 L containing CDRs, (xiii) V of SEQ ID NOs: 220, 221, and 222 H CDRs and V of SEQ ID NOs: 223, 224, and 225 L containing CDRs, (xiv) V of SEQ ID NOs: 230, 231, and 232 H CDRs and V of SEQ ID NOs: 233, 234, and 235 L containing CDRs, (xv) V of SEQ ID NOs: 240, 241, and 242 H CDRs and V of SEQ ID NOs: 243, 244, and 245 L containing CDRs, (xvi) V of SEQ ID NOs: 250, 251, and 252 H CDRs and V of SEQ ID NOs: 253, 254, and 255 L containing CDRs, (xvii) V of SEQ ID NOs: 260, 261, and 262 H CDRs and V of SEQ ID NOs: 263, 264, and 265 L containing CDRs, (xviii) V of SEQ ID NOs: 270, 271, and 272 H CDRs and V of SEQ ID NOs: 273, 274, and 275 L containing CDRs, (xix) V of SEQ ID NOs: 280, 281, and 282 H CDRs and V of SEQ ID NOs: 283, 284, and 285 L containing CDRs, (xx) V of SEQ ID NOs: 290, 291, and 292 H CDRs and V of SEQ ID NOs: 293, 294, and 295 L containing CDRs, (xxi) V of SEQ ID NOs: 300, 301, and 302 H CDRs and V of SEQ ID NOs: 303, 304, and 305 L containing CDRs, (xxii) V of SEQ ID NOs: 310, 311, and 312 HCDRs and V of SEQ ID NOs: 313, 314, and 315 L containing CDRs, (xxiii) V of SEQ ID NOs: 320, 321, and 322 H CDRs and V of SEQ ID NOs: 323, 324, and 325 L containing CDRs, (xxiv) V of SEQ ID NOs: 330, 331, and 332 H CDRs and V of SEQ ID NOs: 333, 334, and 335 L containing CDRs, (xxv) V of SEQ ID NOs: 340, 341, and 342 H CDRs and V of SEQ ID NOs: 343, 344, and 345 L containing CDRs, (xxvi) V of SEQ ID NOs: 350, 351, and 352 H CDRs and V of SEQ ID NOs: 353, 354, and 355 L containing CDRs, (xxvii) V of SEQ ID NOs: 360, 361, and 362 H CDRs and V of SEQ ID NOs: 363, 364, and 365 L containing CDRs, (xxviii) V of SEQ ID NOs: 370, 371, and 372 H CDRs and V of SEQ ID NOs: 373, 374, and 375 L containing CDRs, (xxix) V of SEQ ID NOs: 380, 381, and 382 H CDRs and V of SEQ ID NOs: 383, 384, and 385 L containing CDRs, (xxx) V of SEQ ID NOs: 390, 391, and 392 H CDRs and V of SEQ ID NOs: 393, 394, and 395 L containing CDRs, (xxxi) V of SEQ ID NOs: 400, 401, and 402 H CDRs and V of SEQ ID NOs: 403, 404, and 405 L containing CDRs, (xxxii) V of SEQ ID NOs: 410, 411, and 412 HCDRs and V of SEQ ID NOs: 413, 414, and 415 L containing CDRs, (xxxiii) V of SEQ ID NOs: 420, 421, and 422 H CDRs and V of SEQ ID NOs: 423, 424, and 425 L containing CDRs, (xxxiv) V of SEQ ID NOs: 430, 431, and 432 H CDRs and V of SEQ ID NOs: 433, 434, and 435 L containing CDRs, (xxxv) V of SEQ ID NOs: 440, 441, and 442 H CDRs and V of SEQ ID NOs: 443, 444, and 445 L containing CDRs, (xxxvi) V of SEQ ID NOs: 450, 451, and 452 H CDRs and V of SEQ ID NOs: 453, 454, and 455 L containing CDRs, (xxxvii) V of SEQ ID NOs: 460, 461, and 462 H CDRs and V of SEQ ID NOs: 463, 464, and 465 L containing CDRs, (xxxviii) V of SEQ ID NOs: 470, 471, and 472 H CDRs and V of SEQ ID NOs: 473, 474, and 475 L containing CDRs, (xxxix) V of SEQ ID NOs: 480, 481, and 482 H CDRs and V of SEQ ID NOs: 483, 484, and 485 L containing CDRs, (xl) V of SEQ ID NOs: 490, 491, and 492 H CDRs and VL CDR polypeptides of SEQ ID NOs: 493, 494, and 495, (xli) V of SEQ ID NOs: 500, 501, and 502 H CDRs and VL CDR polypeptides of SEQ ID NOs: 503, 504, and 505, (xlii) V of SEQ ID NOs: 510, 511, and 512 HCDRs and VL CDR polypeptides of SEQ ID NOs: 513, 514, and 515, (xliii) V of SEQ ID NOs: 520, 521, and 522 H CDRs and VL CDR polypeptides of SEQ ID NOs: 523, 524, and 525, (xliv) V of SEQ ID NOs: 530, 531, and 532 H CDRs and VL CDR polypeptides of SEQ ID NOs: 533, 534, and 535, (xlv) V of SEQ ID NOs: 540, 541, and 542 H CDRs and VL CDR polypeptides of SEQ ID NOs: 543, 544, and 545, (xlvi) V of SEQ ID NOs: 550, 551, and 552 H CDRs and VL CDR polypeptides of SEQ ID NOs: 553, 554, and 555, (xlvii) V of SEQ ID NOs: 560, 561, and 562 H CDRs and V of SEQ ID NOs: 563, 564, and 565 L containing CDRs, (xlviii) V of SEQ ID NOs: 570, 571, and 572 H CDRs and V of SEQ ID NOs: 573, 574, and 575 L containing CDRs, (xlix) V of SEQ ID NOs: 580, 581, and 582 H CDRs and V of SEQ ID NOs: 583, 584, and 585 L containing CDRs, (l) V of SEQ ID NOs: 590, 591, and 592 H CDRs and V of SEQ ID NOs: 593, 594, and 595 L containing CDRs, (li) V of SEQ ID NOs: 600, 601, and 602 H CDRs and V of SEQ ID NOs: 603, 604, and 605 L containing CDRs, (lii) V of SEQ ID NOs: 610, 611, and 612 H CDRs and V of SEQ ID NOs: 613, 614, and 615L containing CDRs, (liii) V of SEQ ID NOs: 620, 621, and 622 H CDRs and V of SEQ ID NOs: 623, 624, and 625 L containing CDRs, (liv) V of SEQ ID NOs: 630, 631, and 632 H CDRs and V of SEQ ID NOs: 633, 634, and 635 L containing CDRs, (lv) V of SEQ ID NOs: 640, 641, and 642 H CDRs and V of SEQ ID NOs: 643, 644, and 645 L containing CDRs, (lvi) V of SEQ ID NOs: 650, 651, and 652 H CDRs and V of SEQ ID NOs: 653, 654, and 655 L containing CDRs, (lvii) V of SEQ ID NOs: 660, 661, and 662 H CDRs and V of SEQ ID NOs: 663, 664, and 665 L containing CDRs, (lviii) V of SEQ ID NOs: 670, 671, and 672 H CDRs and V of SEQ ID NOs: 673, 674, and 675 L containing CDRs, (lix) V of SEQ ID NOs: 680, 681, and 682 H CDRs and V of SEQ ID NOs: 683, 684, and 685 L containing CDRs, (lx) V of SEQ ID NOs: 690, 691, and 692 H CDRs and V of SEQ ID NOs: 693, 694, and 695 L containing CDRs, (lxi) V of SEQ ID NOs: 700, 701, and 702 H CDRs and V of SEQ ID NOs: 703, 704, and 705 L containing CDRs, (lxii) V of SEQ ID NOs: 710, 711, and 712 H CDRs and V of SEQ ID NOs: 713, 714, and 715L containing CDRs, (lxiii) V of SEQ ID NOs: 720, 721, and 722 H CDRs and V of SEQ ID NOs: 723, 724, and 725 L containing CDRs, (lxiv) V of SEQ ID NOs: 730, 731, and 732 H CDRs and V of SEQ ID NOs: 733, 734, and 735 L containing CDRs, (lxv) V of SEQ ID NOs: 740, 741, and 742 H CDRs and V of SEQ ID NOs: 743, 744, and 745 L containing CDRs, (lxvi) V of SEQ ID NOs: 750, 751, and 752 H CDRs and V of SEQ ID NOs: 753, 754, and 755 L containing CDRs, (lxvii) V of SEQ ID NOs: 760, 761, and 762 H CDRs and V of SEQ ID NOs: 763, 764, and 765 L containing CDRs, (lxviii) V of SEQ ID NOs: 770, 771, and 772 H CDRs and V of SEQ ID NOs: 773, 774, and 775 L containing CDRs, (lxix) V of SEQ ID NOs: 780, 781, and 782 H CDRs and V of SEQ ID NOs: 783, 784, and 785 L containing CDRs, (lxx) V of SEQ ID NOs: 790, 791, and 792 H CDRs and V of SEQ ID NOs: 793, 794, and 795 L containing CDRs, (lxxi) V of SEQ ID NOs: 800, 801, and 802 H CDRs and V of SEQ ID NOs: 803, 804, and 805 L containing CDRs, (lxxii) V of SEQ ID NOs: 810, 811, and 812 H CDRs and V of SEQ ID NOs: 813, 814, and 815L It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen binding region comprising the CDRs that specifically binds to human VISTA as described above.

[0037] (i) V of SEQ ID NO: 106 H Polypeptide and V of SEQ ID NO: 108 L comprising a polypeptide, (ii) V of SEQ ID NO: 116 H Polypeptide and V of SEQ ID NO: 118 L comprising a polypeptide, (iii) V of SEQ ID NO: 126 H Polypeptide and V of SEQ ID NO: 128 L comprising a polypeptide, (iv) V of SEQ ID NO: 136 H Polypeptide and V of SEQ ID NO: 138 L comprising a polypeptide, (v) V of SEQ ID NO: 146 H Polypeptide and V of SEQ ID NO: 148 L comprising a polypeptide, (vi) V of SEQ ID NO: 156 H Polypeptide and V of SEQ ID NO: 158 L comprising a polypeptide, (vii) V of SEQ ID NO: 166 H Polypeptide and V of SEQ ID NO: 168 L comprising a polypeptide, (viii) V of SEQ ID NO: 176 H Polypeptide and V of SEQ ID NO: 178 L comprising a polypeptide, (ix) V of SEQ ID NO: 186 H Polypeptide and V of SEQ ID NO: 188 L comprising a polypeptide, (x) V of SEQ ID NO: 196 H Polypeptide and V of SEQ ID NO: 198 L comprising a polypeptide, (xi) V of SEQ ID NO: 206 H Polypeptide and V of SEQ ID NO: 208 Lcomprising a polypeptide, (xii) V of SEQ ID NO: 216 H Polypeptide and V of SEQ ID NO: 218 L comprising a polypeptide, (xiii) V of SEQ ID NO: 226 H Polypeptide and V of SEQ ID NO: 228 L comprising a polypeptide, (xiv) V of SEQ ID NO: 236 H Polypeptide and V of SEQ ID NO: 238 L comprising a polypeptide, (xv) V of SEQ ID NO: 246 H Polypeptide and V of SEQ ID NO: 248 L comprising a polypeptide, (xvi) V of SEQ ID NO: 256 H Polypeptide and V of SEQ ID NO: 258 L comprising a polypeptide, (xvii) V of SEQ ID NO: 266 H Polypeptide and V of SEQ ID NO: 268 L comprising a polypeptide, (xviii) V of SEQ ID NO: 276 H a polypeptide and a VL polypeptide of SEQ ID NO: 278, (xix) V of SEQ ID NO: 286 H Polypeptide and V of SEQ ID NO: 288 L comprising a polypeptide, (xx) V of SEQ ID NO: 296 H Polypeptide and V of SEQ ID NO: 298 L comprising a polypeptide, (xxi) V of SEQ ID NO: 306 H Polypeptide and V of SEQ ID NO: 308 L comprising a polypeptide, (xxii) V of SEQ ID NO: 316 H Polypeptide and V of SEQ ID NO: 318 L comprising a polypeptide, (xxiii) V of SEQ ID NO: 326 H Polypeptide and V of SEQ ID NO: 328 L comprising a polypeptide, (xxiv) V of SEQ ID NO: 336 H Polypeptide and V of SEQ ID NO: 338 L comprising a polypeptide, (xxv) V of SEQ ID NO: 346 H Polypeptide and V of SEQ ID NO: 348 L comprising a polypeptide, (xxvi) V of SEQ ID NO: 356 H Polypeptide and V of SEQ ID NO: 358 L comprising a polypeptide, (xxvii) V of SEQ ID NO: 366 H Polypeptide and V of SEQ ID NO: 368 L comprising a polypeptide, (xxviii) V of SEQ ID NO: 376 H Polypeptide and V of SEQ ID NO: 378 L comprising a polypeptide, (xxix) V of SEQ ID NO: 386 H Polypeptide and V of SEQ ID NO: 388 L comprising a polypeptide, (xxx) V of SEQ ID NO: 396 H Polypeptide and V of SEQ ID NO: 398 L comprising a polypeptide, (xxxi) V of SEQ ID NO: 406 H Polypeptide and V of SEQ ID NO: 408 L comprising a polypeptide, (xxxii) V of SEQ ID NO: 416 H Polypeptide and V of SEQ ID NO: 418 L comprising a polypeptide, (xxxiii) V of SEQ ID NO: 426 H Polypeptide and V of SEQ ID NO: 428 L comprising a polypeptide, (xxxiv) V of SEQ ID NO: 436 H Polypeptide and V of SEQ ID NO: 438 L comprising a polypeptide, (xxxv) V of SEQ ID NO: 446 H Polypeptide and V of SEQ ID NO: 448 L comprising a polypeptide, (xxxvi) V of SEQ ID NO: 456 H Polypeptide and V of SEQ ID NO: 458 L comprising a polypeptide, (xxxvii) V of SEQ ID NO: 466 H Polypeptide and V of SEQ ID NO: 468 L comprising a polypeptide, (xxxviii) V of SEQ ID NO: 476 H Polypeptide and V of SEQ ID NO: 478 L comprising a polypeptide, (xxxix) V of SEQ ID NO: 486 H Polypeptide and V of SEQ ID NO: 488 L comprising a polypeptide, (xl) V of SEQ ID NO: 496 H Polypeptide and V of SEQ ID NO: 498 L comprising a polypeptide, (xli) V of SEQ ID NO: 506 H Polypeptide and V of SEQ ID NO: 508 L comprising a polypeptide, (xlii) V of SEQ ID NO: 516 H Polypeptide and V of SEQ ID NO: 518 L comprising a polypeptide, (xliii) V of SEQ ID NO: 526 H Polypeptide and V of SEQ ID NO: 528 L comprising a polypeptide, (xliv) V of SEQ ID NO: 536 H Polypeptides and V of SEQ ID NOs: 533, 534, and 535 L comprising a polypeptide, (xlv) V of SEQ ID NO: 546 H Polypeptide and V of SEQ ID NO: 548 L comprising a polypeptide, (xlvi) V of SEQ ID NO: 556 H Polypeptide and V of SEQ ID NO: 558 L comprising a polypeptide, (xlvii) V of SEQ ID NO: 566 H Polypeptide and V of SEQ ID NO: 568 L comprising a polypeptide, (xlviii) V of SEQ ID NO: 576 H Polypeptide and V of SEQ ID NO: 578 L comprising a polypeptide, (xlix) V of SEQ ID NO: 586 H Polypeptide and V of SEQ ID NO: 588 L comprising a polypeptide, (l) V of SEQ ID NO: 596 H Polypeptide and V of SEQ ID NO: 598 L comprising a polypeptide, (li) V of SEQ ID NO: 606 H Polypeptide and V of SEQ ID NO: 608 L comprising a polypeptide, (lii) V of SEQ ID NO: 616 H Polypeptide and V of SEQ ID NO: 618 L comprising a polypeptide, (liii) V of SEQ ID NO: 626 H Polypeptide and V of SEQ ID NO: 628 L comprising a polypeptide, (liv) V of SEQ ID NO: 636 H Polypeptide and V of SEQ ID NO: 638 L comprising a polypeptide, (lv) V of SEQ ID NO: 646 H Polypeptide and V of SEQ ID NO: 648 L comprising a polypeptide, (lvi) V of SEQ ID NO: 656 H Polypeptide and V of SEQ ID NO: 658 L comprising a polypeptide, (lvii) V of SEQ ID NO: 666 H Polypeptide and V of SEQ ID NO: 668 L comprising a polypeptide, (lviii) V of SEQ ID NO: 676 H Polypeptide and V of SEQ ID NO: 678 L comprising a polypeptide, (lix) V of SEQ ID NO: 686 H Polypeptide and V of SEQ ID NO: 688 L comprising a polypeptide, (lx) V of SEQ ID NO: 696 HPolypeptide and V of SEQ ID NO: 698 L comprising a polypeptide, (lxi) V of SEQ ID NO: 706 H Polypeptide and V of SEQ ID NO: 708 L comprising a polypeptide, (lxii) V of SEQ ID NO: 716 H Polypeptide and V of SEQ ID NO: 718 L comprising a polypeptide, (lxiii) V of SEQ ID NO: 726 H Polypeptide and V of SEQ ID NO: 728 L comprising a polypeptide, (lxiv) V of SEQ ID NO: 736 H Polypeptide and V of SEQ ID NO: 738 L comprising a polypeptide, (lxv) V of SEQ ID NO: 746 H Polypeptide and V of SEQ ID NO: 748 L comprising a polypeptide, (lxvi) V of SEQ ID NO: 756 H Polypeptide and V of SEQ ID NO: 758 L comprising a polypeptide, (lxvii) V of SEQ ID NO: 766 H Polypeptide and V of SEQ ID NO: 768 L comprising a polypeptide, (lxviii) V of SEQ ID NO: 776 H Polypeptide and V of SEQ ID NO: 778 L comprising a polypeptide, (lxix) V of SEQ ID NO: 786 H Polypeptide and V of SEQ ID NO: 788 L comprising a polypeptide, (lxx) V of SEQ ID NO: 796 H Polypeptide and V of SEQ ID NO: 798 L comprising a polypeptide, (lxxi) V of SEQ ID NO: 806 H Polypeptide and V of SEQ ID NO: 808 L comprising a polypeptide, (lxxii) V of SEQ ID NO: 816 HPolypeptide and V of SEQ ID NO: 818 L It is an object of the present invention to provide a VISTA agonist, comprising a polypeptide, as described in any of the foregoing.

[0038] It is a specific object of the present invention to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, optionally comprising a human IgG2 constant domain, wherein at least one cysteine ​​residue has been deleted or changed to another amino acid.

[0039] The following immunosuppressive effects are observed: (i) decreased immune response, (ii) decreased T cell activation, (iii) decreased cytotoxic T cell activity, (iv) decreased natural killer (NK) cell activity, (v) decreased T cell activity, (vi) decreased proinflammatory cytokine secretion, (vii) decreased IL-2 secretion, (viii) decreased interferon-γ production, (ix) decreased Th1 response, (x) decreased Th2 response, (xi) increased number and / or activity of regulatory T cells, and (xii) increased regulatory cell activity and / or bone marrow-derived cytokines. (xiii) an increase in regulatory cell activity and / or an increase in activity of one or more of myeloid-derived suppressor cells (MDSC), iMC, mesenchymal stromal cells, and TIE2-expressing monocytes; (xiii) an increase in M2 macrophages; (xiv) an increase in M2 macrophage activity; (xv) an increase in N2 neutrophils; (xvi) an increase in N2 neutrophil activity; (xvii) an increase in inhibition of T cell activation; (xviii) an increase in inhibition of CTL activation; (xi) (xxiii) reduced antigen-specific memory responses; (xxiv) inhibition of cell apoptosis or lysis; (xxv) reduced cytotoxic or cytostatic effects on cells; (xxvi) reduced direct cell killing; (xxvii) reduced Thl7 activity; and / or (xxviii) reduced complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity; or a combination of at least one of them, provided that the anti-VISTA antibody or antigen-binding fragment may induce the opposite effect of one or more of (i) to (xxviii), and optionally, to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, for use in treating autoimmunity, allergy, inflammation, transplantation, or sepsis.

[0040] It is a specific object of the present invention to provide a pharmaceutical or diagnostic composition comprising an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above.

[0041] It is a specific object of the present invention to provide a therapeutic and / or diagnostic method or use of a composition comprising at least one antagonistic antibody or antibody fragment according to any of the preceding claims for use in diagnosis or therapy, the method or use comprising administering to a subject in need of treatment and / or diagnosis, such as for example cancer or an infectious disorder, at least one dose or a therapeutically or diagnostically effective amount of a composition comprising at least one at least one antagonistic antibody or antibody fragment according to any of the preceding claims, and optionally the cancer is a hematological cancer or is a solid tumor, e.g., one surrounded by a tumor stroma comprising myeloid cells, T cells, or a combination of myeloid and T cells, or a cancer selected from leukemia, lymphoma, myelodysplastic syndrome or myeloma, lung cancer, or a combination thereof, or a leukemia, including acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myelogenous) leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, or adult T-cell leukemia.

[0042] It is a specific object of the present invention to provide a therapeutic and / or diagnostic method or the use of a composition containing at least one agonistic antibody or antibody fragment according to any of the preceding claims for use in diagnosis or therapy, which method or use comprises administering to a subject in need of treatment and / or diagnosis at least one dose, or a therapeutically or diagnostically effective amount, of at least one composition comprising at least one agonistic antibody or antibody fragment according to any of the preceding claims or a composition containing according to any of the preceding.

[0043] The following immunosuppressive effects are observed: (i) decreased immune response, (ii) decreased T cell activation, (iii) decreased cytotoxic T cell activity, (iv) decreased natural killer (NK) cell activity, (v) decreased T cell activity, (vi) decreased proinflammatory cytokine secretion, (vii) decreased IL-2 secretion, (viii) decreased interferon-γ production, (ix) decreased Th1 response, (x) decreased Th2 response, (xi) increased number and / or activity of regulatory T cells, and (xii) increased regulatory cell activity and / or bone marrow-derived cytokines. (xiii) an increase in regulatory cell activity and / or an increase in activity of one or more of myeloid-derived suppressor cells (MDSC), iMC, mesenchymal stromal cells, and TIE2-expressing monocytes; (xiii) an increase in M2 macrophages; (xiv) an increase in M2 macrophage activity; (xv) an increase in N2 neutrophils; (xvi) an increase in N2 neutrophil activity; (xvii) an increase in inhibition of T cell activation; (xviii) an increase in inhibition of CTL activation; (xi) (xxiii) reduced antigen-specific memory responses; (xxiv) inhibition of cell apoptosis or lysis; (xxv) reduced cytotoxic or cytostatic effects on cells; (xxvi) reduced direct cell killing; (xxvii) reduced Thl7 activity; and / or (xxviii) reduced complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity; or a combination of at least one of them, provided that the anti-VISTA antibody or antigen-binding fragment may induce the opposite effect of one or more of (i) to (xxviii), and optionally, to provide an agonistic antibody or antibody fragment thereof comprising an antigen-binding region that specifically binds to human VISTA as described above, for use in treating autoimmunity, allergy, inflammation, transplantation, or sepsis.

[0044] It is a specific object of the present invention to provide a method for use in the treatment or prevention of allergy, autoimmunity, transplantation, gene therapy, inflammation, cancer, GVHD, or sepsis, or for the treatment or prevention of inflammation, autoimmunity, or allergic side effects associated with any of the foregoing in a human subject, or the use of any agonistic antibody or antibody fragment as described in any of the foregoing.

[0045] An anti-VISTA antibody or antigen-binding fragment or composition, or method or use, as described in any preceding paragraph, further comprising another immunomodulatory antibody or fusion protein selected from an immunoinhibitory antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and / or an agonistic antibody or fusion protein targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS.

[0046] A method or use according to any preceding claim, comprising assaying for VISTA protein in cells or body fluids of the individual before, simultaneously with and / or after treatment.

[0047] A method or use according to any preceding claim, comprising assaying VISTA levels on hematopoietic cells.

[0048] The method or use according to any of the preceding claims, comprising assaying VISTA levels for hematopoietic cells selected from myeloid lineage cells and / or any one or more of lymphocytes, monocytes, or neutrophils, T cells, B cells, natural killer (NK) cells, or natural killer T (NKT) cells.

[0049] Any of the preceding methods or uses, wherein the agonist anti-human VISTA antibody or fragment comprises the same CDRs as an antibody selected from VSTB49 to VSTB116 and an optionally mutated human IgG2 Fc region, or wherein the IgG2 constant region or Fc region retains the ability to bind native FcR and / or to bind CD32A.

[0050] Any of the preceding antibodies, compositions, methods, or uses, wherein the anti-human VISTA antibody or fragment comprises an affinity or KD for human VISTA of 50 M or less as determined by surface plasmon resonance at 37°C.

[0051] The antibody, composition, method, or use described in any of the preceding claims, wherein the anti-human VISTA antibody or fragment comprises an affinity or KD for human VISTA of 1 nM or less as determined by surface plasmon resonance at 37°C.

[0052] It is a specific object of the present invention to provide isolated antagonistic and agonistic anti-human VISTA antibodies, as well as agonistic antibody fragments, comprising an antigen-binding region that specifically binds to human VISTA, wherein the antibody or antibody fragment comprises a variable heavy sequence and a variable light sequence having the CDR polypeptides of any one of the anti-human VISTA antibodies having the sequences shown in Figure 4, with the proviso that, if the antibody or fragment comprises an antagonistic anti-human VISTA antibody or antibody fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0053] It is a specific object of the present invention to provide isolated antagonistic and agonistic anti-human VISTA antibodies, as well as agonistic antibody fragments, comprising an antigen-binding region that specifically binds to human VISTA, wherein the antibody or antibody fragment comprises a variable heavy sequence and a variable light sequence having CDR polypeptides of an anti-human VISTA antibody selected from VSTB49-VSTB116, with the proviso that when the antibody or fragment comprises an antagonistic anti-human VISTA antibody or anti-human VISTA antibody fragment, the anti-human VISTA antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0054] It is another specific object of the present invention to provide isolated antagonistic and agonistic antibodies and antibody fragments comprising the CDRs of an anti-human VISTA antibody selected from VSTB49 to VSTB116, which comprise variable heavy and / or variable light polypeptides having at least 90%, 95%, or 96-99% sequence identity to the variable heavy and variable light polypeptide sequences of VSTB49 to VSTB116, with the proviso that, when the antibody or fragment comprises an antagonistic anti-human VISTA antibody or fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0055] It is another specific object of the present invention to provide isolated antagonistic and agonistic antibodies or antibody fragments comprising variable heavy and / or variable light polypeptides that are identical to the variable heavy and variable light polypeptide sequences of VSTB49 to VSTB116, with the proviso that when the antibody or fragment comprises an antagonistic anti-human VISTA antibody or fragment, the antibody or antibody fragment does not comprise the same CDR as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0056] It is another specific object of the present invention to provide an isolated agonistic or agonistic chimeric, human, humanized, multispecific (e.g., bispecific) anti-human VISTA antibody or antibody fragment comprising an antigen-binding region that specifically binds to human VISTA, wherein the agonistic antibody or antibody fragment comprises a variable heavy and variable light sequence having CDR polypeptides as any one of the anti-human VISTA antibodies comprising the CDRs and variable heavy and variable light polypeptides disclosed in Figure 4, with the proviso that if the antibody or fragment comprises an antagonistic anti-human VISTA antibody or antibody fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

[0057] It is another specific object of the present invention to provide novel immunosuppressants, i.e., anti-human VISTA antibodies and antibody fragments, e.g., those containing a human IgG2 constant domain or IgG2 Fc region, optionally with the FcR binding ability of the human IgG2 constant domain or IgG2 Fc region maintained or enhanced compared to wild-type human IgG2 constant domain or IgG2 Fc region, that stimulate, induce, or mimic the immune effects of human VISTA, e.g., the inhibitory effects of human VISTA on T cell activity, differentiation, and proliferation, and the expression of pro-inflammatory cytokines.

[0058] It is another specific object of the present invention to provide novel antagonists, i.e., novel anti-human VISTA antibodies and antibody fragments that antagonize or block the immune effects of human VISTA, particularly the inhibitory effects of human VISTA on T cell activity, differentiation, and proliferation, and on the expression of pro-inflammatory cytokines.

[0059] Enhance or mimic the suppressive effects of VISTA on T cell immunity, i.e., CD4 + or CD8 + T cell proliferation, CD4 + or CD8 + It is another specific object of the present invention to provide novel immunosuppressive antibodies and antibody fragments that inhibit the inhibitory effect of VISTA on T cell activation and the production of immune cytokines, particularly pro-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-17, TNF-α, and / or GM-CSF (granulocyte-macrophage colony-stimulating factor), as well as the promoting effect of VISTA on the expression of chemokines or chemotactic factors such as KC (keratinocyte chemoattractant) or MIP-2 (macrophage inflammatory protein 2).

[0060] Blocking or reducing the suppressive effect of VISTA on T cell immunity, i.e., CD4 + or CD8 + T cell proliferation, CD4 + or CD8+ It is another specific object of the present invention to provide novel antibodies and antibody fragments that enhance the inhibitory effect of VISTA on T cell activation and the production of proinflammatory immune cytokines, particularly proinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17, TNF-α, and / or GM-CSF (granulocyte-macrophage colony-stimulating factor), and the stimulatory effect of VISTA on the expression of chemokines or chemotactic factors such as KC (keratinocyte chemoattractant) or MIP-2 (macrophage inflammatory protein 2).

[0061] It is another specific object of the present invention to provide novel immunosuppressive or agonistic anti-human VISTA antibodies and antibody fragments that are specific for a particular epitope or that compete with a particular anti-human VISTA antibody for binding to human VISTA.

[0062] The suppressive effect of VISTA on immunity, e.g., the suppressive effect of VISTA on T cell immunity, i.e., CD4 + or CD8 + T cell proliferation, CD4 + or CD8 + It is another specific object of the present invention to provide novel immunosuppressive anti-human VISTA antibodies or agonistic anti-human VISTA antibodies and antibody fragments that are specific for a particular epitope or that compete with a particular anti-human VISTA antibody for binding to human VISTA, which stimulate (enhance, induce, or mimic) the inhibitory effects of T cell activation and / or suppress the promoting effect of VISTA on the production of proinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17, TNF-α, and / or GM-CSF (granulocyte-macrophage colony-stimulating factor), and the expression of chemokines or chemotactic factors such as KC (keratinocyte chemoattractant) or MIP-2 (macrophage inflammatory protein 2).

[0063] The present invention also relates to immunosuppressants, particularly those in which the prevention or inhibition or reduction of an immune response is therapeutically desirable, and more particularly to T cell immunity, or more particularly to CD4 +or CD8 + The present invention also relates to the specific use of these agonistic anti-human VISTA antibodies and antibody fragments as prophylactic or therapeutic agents in the treatment of conditions in which the prevention or inhibition or reduction of mediated T cell immunity is therapeutically beneficial, such as autoimmune, inflammatory, allergic disorders, sepsis, GVHD, and / or in the treatment of transplant or cell therapy recipients, e.g., CAR-T recipients, or in the reduction of inflammatory side effects of some conditions, such as cancer.

[0064] The present invention also relates to immunosuppressants, particularly those for which enhanced immunity is desired, such as T cell immunity or CD4 + or CD8 + The present invention also relates to the use of novel antagonistic anti-human VISTA antibodies and antibody fragments as prophylactic or therapeutic agents in the treatment of conditions in which mediated T cell immunity is therapeutically beneficial, such as cancer and infectious diseases.

[0065] It is another specific object of the present invention to provide agonist or antagonist anti-human VISTA antibodies according to the present invention that are attached to a detectable label, linker, or therapeutic moiety.

[0066] It is another specific object of the present invention to provide a diagnostic or pharmaceutical composition comprising a diagnostically or therapeutically effective amount of an agonist or antagonist anti-human VISTA antibody according to the present invention, e.g., one that contains the same CDRs as any of the antibodies having the sequences shown in Figure 4, suitable for use in human therapy, such as compositions that can be administered intravenously, subcutaneously, or intramuscularly.

[0067] It is another specific object of the present invention to provide diagnostic or therapeutic methods that use the agonist antibodies of the present invention in combination with another immune agonist, for example a PD-1 or PD-L1 agonist, e.g., the PD-1 or PD-L1 agonist is selected from an anti-PD-1 antibody or antibody fragment, an anti-PD-L1 antibody or antibody fragment, a PD-L1 polypeptide or fragment thereof, which may be monovalent or multimeric, a PD-1 polypeptide or fragment thereof, which may be monovalent or multimeric, or a complex or fusion protein comprising any of the foregoing.

[0068] It is another specific object of the present invention to provide diagnostic or therapeutic methods that use the antagonist antibodies of the present invention in conjunction with another immune antagonist, for example a PD-1 or PD-L1 antagonist, e.g., the PD-1 or PD-L1 agonist is selected from an antagonist anti-PD-1 antibody or antibody fragment, or an antagonist anti-PD-L1 antibody or antibody fragment.

[0069] It is another specific object of the present invention to provide a method of contacting immune cells, e.g., human immune cells, in vitro or in vivo with an antagonistic or agonistic antibody according to the present invention, e.g., the contacted cells are injected into a human subject, such as a subject with cancer or an infectious disease, or a subject with an inflammatory, allergic, or autoimmune condition. [Brief explanation of the drawings]

[0070] [Figure 1A-D]Figure 1 shows in vitro and in vivo screening assays that can be used to identify inhibitory VISTA mAbs. A) Purified T cells were plated on anti-CD3 in the presence of the indicated mAb for 72 hours. Proliferation was measured by H3 incorporation. B) Purified DO11.10 T cells were stimulated with ISQ-pulsed APCs in the presence of the indicated antibodies for 6 days. Proliferation was measured using CTV dilution dye. C) GVHD was induced by transferring C57BL / 6 cells into irradiated BALB / c recipients. Mice were injected IP with 200 μg of antibody on days 0, 2, and 4 post-transfer, and survival was analyzed. D) Mice were treated with 10 mpk of the indicated antibody 3 hours before administration of ConA (15 mpk), and plasma IL-2 was analyzed by Luminex at 6 dpi. [Figure 2A-F] We demonstrate that the agonist VISTA antibody is immunosuppressive in multiple autoimmune disease models. A) NZB / W F1 mice were treated with either 8G8 or Ham Ig (200 μg) 3X / week starting at week 25 and continuing until the end of the experiment. "X" indicates the time when all control-treated mice were sacrificed. B) Mice were treated with 200 μg of antibody 3 hours before administration of 15 mg / kg (mpk) ConA, and survival was followed for 80 hours. C) Mice were treated sequentially with collagen II mAb followed by LPS, and arthritis was measured by measuring paw swelling. 8G8 and Ham Ig (200 μg) were administered 3X daily. D) Mice were treated with imiquimod daily in the ears. On day 14, 8G8 or Ham Ig (200 μg) was administered daily, and ear thickness was measured with a caliper. E, F) Imiquimod was applied daily to the backs of mice. On day 9, mice were euthanized and the skin was sectioned and stained for CD3 expression by IHC. [Figure 3] Figure 1 shows VISTA expression in WT and hV-KI mice. CD4+ T cells, CD8+ T cells, Tregs (CD4+FoxP3+), and monocytes (CD11b+, Ly6C+, Ly6G-) were isolated from lymph nodes of WT and VISTA KI mice and stained with αVISTA antibody against mouse or human protein, respectively. [Figure 4]Contains sequences of different anti-human VISTA antibodies, including INX800, INX801, and INX900 to INX919. [Figure 5] 1 shows the effect of exemplary anti-human VISTA antibodies, INX800 and INX801, in a ConA hepatitis model evaluating the effect of anti-human VISTA antibodies on the expression of different cytokines, chemokines, and chemoattractants. [Figure 6] FIG. 1 shows the effects of exemplary anti-human VISTA antibodies, INX800 and INX801, in an in vivo graft-versus-host disease (GVHD) animal model. [Figure 7] 1 shows the effect of exemplary agonistic anti-human VISTA antibodies, namely INX800 or INX801, on CD3-driven T cell immune responses. [Figure 8] 1 shows the effect of exemplary agonistic anti-human VISTA antibodies, namely INX800 or INX801, on the numbers of specific T cell populations or total T cell numbers. [Figure 9] The effects of exemplary anti-human VISTA antibodies in a ConA assay and on the expression of select pro-inflammatory cytokines and inflammatory markers, namely IL-2, gamma interferon, and IL-12p70, are compared. [Figures 10A-10C] Different IgG2 isoforms are shown. (A) Disulfide shuffling results in isoforms A and B, along with an A / B transition (Figure from Zhang, A et al., 2015). (B) Isoforms are distinguishable by RP-HPLC. (C) Observed RP-HPLC chromatogram of INX901. [Figure 11] Chemical enrichment of IgG2A or B isoforms is shown. (Black line, top) Chromatogram shows the predominant left-most peak defining the B form. (Red line, bottom) Chromatogram shows the predominant right-hand peak defining the A form. [Figure 12]Comparing INX901 Fc silent variants with respect to disulfide shuffling. (Top) INX901 on an IgG2 backbone displays the expected mixture of A, A / B, and B isoforms. (Middle) INX901 Si on a silent IgG1 backbone exists as a single isoform. (Bottom) INX901 HSi has an IgG1 silent Fc region with the CH1 / hinge from IgG2, allowing disulfide shuffling equivalent to native IgG2. [Figure 13] The biochemically altered INX901 forms were still able to reduce cytokine production in MLRs. Supernatants from two separate MLRs were analyzed for cytokine production at 72 hours by Luminex analysis. INX901 parent, strain A, and strain B all reduced TNFα and IL-2 production in a dose-dependent manner. [Figure 14] The genetically locked INX901 form was still able to reduce cytokine production in MLRs, whereas the Fc-silent variants were unable to. Supernatants from each MLR were analyzed for cytokine production at 72 hours by Luminex analysis. The INX901 parent, A-locked, and B-locked forms all reduced TNFα and IL-2 production in a dose-dependent manner. The Si and HSi variants, which contain mutations that silence the Fc domain, did not consistently suppress cytokine production. [Figure 15] The genetically locked INX908 form was still able to reduce cytokine production in MLRs, whereas the Fc-silent variants were unable to. Supernatants from each MLR were analyzed for cytokine production at 72 hours by Luminex analysis. The INX908 parent, A-locked, and B-locked forms all reduced TNFα and IL-2 production in a dose-dependent manner. The Si and HSi variants, which contain mutations that silence the Fc domain, did not consistently suppress cytokine production. [Figure 16]The Pepscan® technology used to identify linear and discontinuous epitopes bound by agonist anti-human VISTA antibodies is outlined below. [Figure 17] This shows that the agonist anti-human VISTA antibodies bind to the same core sequence. [Figure 18] 1 summarizes the epitope analysis of different anti-human VISTA antibodies according to the present invention. [Figure 19] The epitope bound by the agonistic anti-human VISTA antibody is shown and the key residues involved in binding are further identified. DETAILED DESCRIPTION OF THE INVENTION

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the present invention and testing of the present invention, suitable methods and materials are described herein. The materials, methods, and examples are merely illustrative and are not intended to be limiting. The names used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for treating patients.

[0072] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0073] As used herein, "activating receptor" broadly refers to an immune cell receptor that binds to an antigen, a complex antigen (e.g., in the context of an MHC molecule), an Ig fusion protein, a ligand, or an antibody. Activating receptors include, but are not limited to, T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors. For example, T cell receptors are present on T cells and associated with CD3 molecules. T cell receptors are stimulated by antigens in the context of MHC molecules (as well as by polyclonal T cell activation reagents). TCR-mediated T cell activation results in numerous changes, such as protein phosphorylation, membrane lipid changes, ion flux, cyclic nucleotide changes, RNA transcription changes, protein synthesis changes, and cell volume changes. For example, T cell receptors are present on T cells and associated with CD3 molecules. T cell receptors are stimulated by antigens in the context of MHC molecules (as well as by polyclonal T cell activation reagents). T cell activation via the TCR results in numerous changes, including protein phosphorylation, membrane lipid changes, ion fluxes, cyclic nucleotide alterations, changes in RNA transcription, changes in protein synthesis, and changes in cell volume.

[0074] As used herein, "adjuvant" refers to an agent used to stimulate the immune system and increase response to a vaccine without having any specific antigenic effect itself.

[0075] As used herein, "agonist" refers to a molecule, typically an antibody or fusion protein, that enhances or mimics the immune effect of a particular molecule. Generally in this application, this refers to anti-human VISTA agonist antibodies and antibody fragments that enhance or mimic the immune effect of human VISTA, particularly the inhibitory effect of VISTA on T cell immunity (CD4+ and / or CD8+ T cell immunity), the expression of pro-inflammatory cytokines, and the expression of certain chemokines and chemotactic factors.

[0076] As used herein, "useful in the diagnosis" or "useful in the detection" of a disease means that the expression level of a particular marker polypeptide or expressed RNA is detected, alone or in conjunction with one or more other markers, to assess whether a subject has cells characteristic of a particular disease state or the development of a particular disease state, or an immune dysfunction such as immunosuppression characterized by expression of VISTA, or aberrant immune upregulation characterized by cells with reduced VISTA levels, such as during an autoimmune, inflammatory, or allergic response, for example, in individuals with chronic and non-chronic diseases.

[0077] As used herein, "allergic disease" broadly refers to diseases associated with allergic reactions. More specifically, "allergic disease" is defined as a disease in which an allergen is identified and there is a strong correlation between exposure to the allergen and the onset of pathological changes, and the pathological changes have been proven to have an immunological mechanism. In this specification, immunological mechanism means that white blood cells exhibit an immune response to allergen stimulation.

[0078] As used herein, "amino acid" broadly refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, carbon bonded to the amino group), and R groups (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0079] As used herein, "anergy" or "tolerance," or "long-term antigen-specific T cell suppression" or "prolonged immunosuppression," broadly refer to refraction to activating receptor-mediated stimulation. Refraction is generally antigen-specific and persists even after cessation of exposure to the tolerizing antigen. For example, anergy (as opposed to unresponsiveness) in T cells is characterized by a lack of cytokine production, such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, reexposure to the same antigen (even in the presence of costimulatory molecules) renders the cells unable to produce cytokines and therefore unable to proliferate. However, anergic T cells can mount a response to unrelated antigens and can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or proliferation assays using indicator cell lines. Alternatively, a reporter gene construct can be used. For example, anergic T cells cannot initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5'IL-2 gene enhancer or by multimers of API sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134). Modulation of costimulatory signals leads to the modulation of the effector function of immune cells.

[0080] As used herein, "antagonist" refers to a molecule, typically an antibody or fusion protein, that blocks or reduces the immune effect of a particular molecule. Generally, in this application, this refers to anti-human VISTA antagonist antibodies and antibody fragments that block or reduce the immune effects of human VISTA, particularly the inhibitory effects of VISTA on T cell immunity (CD4+ and / or CD8+ T cell immunity), the expression of pro-inflammatory cytokines, and the expression of certain chemokines and chemotactic factors.

[0081] As used herein, "antibody" broadly refers to an "antigen-binding portion" of an antibody (also used interchangeably with "antibody portion," "antigen-binding fragment," and "antibody fragment"), as well as to a whole antibody molecule. As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody (e.g., VISTA or a particular portion thereof) that retain the ability to specifically bind to an antigen. As referred to herein, the term "antibody" includes whole polyclonal and monoclonal antibodies, as well as any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof, and bispecific and multispecific antibodies, e.g., those that bind to multiple antigens or multiple antigen epitopes. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain is composed of at least one heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C, C, D, E, E, F, H, I, L ... H1、 Each light chain is composed of at least one light chain variable region (referred to herein as V L The light chain constant region is composed of one domain, CL-. H and V LThe regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. More generally, the term "antibody" is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits and recognizes an epitope, where one or more noncovalent interactions stabilize the complex between the molecular structure and the epitope. The prototypic antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other birds, etc., are considered "antibodies."

[0082] The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Non-limiting examples of antigen-binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (a) V L , V H , C L , and C H1 (b) a Fab fragment, which is a monovalent fragment consisting of a V domain; (b) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (c) a V H and C H1 (d) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (e) a V H (f) isolated complementarity-determining regions (CDRs) of the dAb fragment (Ward, et al. (1989) Nature 341:544-546), and (g) isolated complementarity-determining regions (CDRs). L and V HThe two domains are encoded by separate genes, but they are L Area and V H The domains can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain (known as single-chain Fv (scFv)) that pairs to form a monovalent molecule. See, e.g., Bird, et al. (1988) Science 242:423-426, Huston, et al. (1988) Proc Natl. Acad. Sci. USA 85:5879-5883, and Osbourn, et al. (1998) Nat. Biotechnol. 16:778. Single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Any V of a particular scFv can be produced as a single protein chain (known as single-chain Fv (scFv)). See, e.g., Bird, et al. (1988) Science 242:423-426, Huston, et al. (1988) Proc Natl. Acad. Sci. USA 85:5879-5883, and Osbourn, et al. (1998) Nat. Biotechnol. 16:778. Single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. H and V L The sequences can be ligated to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG molecules or other isotypes. L can also be used to generate Fab, Fv, or other fragments of immunoglobulins, using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are fragments of V H and V LA bispecific antibody is a bivalent antibody in which the domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and creating two antigen-binding sites. See, for example, Holliger, et al. (1993) Proc Natl. Acad. Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123. Furthermore, an antibody or antigen-binding portion thereof (antigen-binding fragment, antibody fragment, antibody portion) can be part of a larger immunoadhesion molecule formed by covalent or noncovalent bonding of the antibody or antibody portion to one or more other proteins or peptides. Examples of immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules. Kipriyanov, et al. (1994) Mol. Immunol. 31:1047-1058. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein. Antibodies can be polyclonal, monoclonal, xenoantibodies, alloantibodies, syngeneic antibodies, or modified forms thereof, such as humanized, chimeric, bispecific, or multispecific antibodies.

[0083] The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen" and refer to an immunoglobulin or fragment thereof that specifically binds to an antigen.

[0084] As used herein, "antigen" broadly refers to a molecule or portion of a molecule capable of being bound by an antibody that can further induce an animal to produce an antibody capable of binding to the antigen's epitope. An antigen can have one epitope or two or more epitopes. The specific reaction referred to herein indicates that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with many other antibodies that may be elicited by other antigens. In the case of a desired enhanced immune response against a specific antigen of interest, exemplary antigens include, but are not limited to, infectious disease antigens that can elicit a protective immune response.

[0085] As used herein, "antigen-presenting cells" refers broadly to professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0086] As used herein, " antisense nucleic acid molecule " refers broadly to the nucleotide sequence that is complementary to the mRNA sequence or complementary to the " sense " nucleic acid that encodes a protein that is complementary to the coding strand of a gene (for example, complementary to the coding strand of a double-stranded cDNA molecule).Therefore, antisense nucleic acid molecules can hydrogen bond to sense nucleic acid molecules.

[0087] As used herein, "apoptosis" refers broadly to programmed cell death, which can be characterized using techniques known in the art. Apoptotic cell death can be characterized by cell shrinkage, membrane microspore formation, and chromatin condensation, resulting in cell fragmentation. Cells undergoing apoptosis also exhibit a characteristic pattern of internucleosomal DNA breaks.

[0088] As used herein, "autoimmunity" or "autoimmune disease or condition" refers broadly to and includes diseases or disorders arising from, directed against, or resulting from an individual's own tissues or their co-isolation or manifestation. As used herein, autoimmune conditions include inflammatory or allergic conditions, e.g., chronic diseases characterized by a host immune response to self-antigens potentially associated with tissue destruction, such as rheumatoid arthritis.

[0089] As used herein, "B cell receptor" (BCR) broadly refers to the complex between membrane Ig (mIg) and other transmembrane polypeptides (e.g., IgA and Ig) found on B cells. The signaling function of mIg is triggered by crosslinking of the receptor molecule by oligomeric or multimeric antigens. B cells can also be activated by anti-immunoglobulin antibodies. BCR activation results in a number of changes in B cells, including tyrosine phosphorylation.

[0090] As used herein, "cancer" broadly refers to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division resulting in malignant growth or tumors (e.g., uncontrolled cell growth). As used herein, the term "cancer" or "cancerous" should be understood to encompass any neoplastic disease (whether invasive, non-invasive, or metastatic) characterized by abnormal and uncontrolled cell division resulting in malignant growth or tumors, the non-limiting examples of which are described herein. This includes any physiological condition in a mammal typically characterized by uncontrolled cell growth. Examples of cancers are illustrated in the Examples. Additional cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, carcinoma), and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma and Waldenstrom's hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; multiple myeloma and post-transplant lymphoproliferative disorder (PTLD). Other cancers suitable for treatment by the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.More specific examples of such cancers include colorectal cancer, bladder cancer, ovarian cancer, melanoma, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, and hepatic cancer. carcinoma), and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma and Waldenstrom's hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of colorectal cancer, breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In an exemplary embodiment, the cancer is early or advanced (including metastatic) bladder, ovarian, or melanoma. In another embodiment, the cancer is colorectal cancer. Cancerous conditions suitable for treatment according to the present invention include cancers that express or do not express VISTA, and further include non-metastatic or non-invasive, as well as invasive or metastatic cancers, where expression of VISTA by immune, stromal, or diseased cells suppresses anti-tumor and anti-invasive immune responses. The method of the present invention is particularly suitable for treating angiogenic tumors.Cancers according to the present invention include cancers that express or do not express VISTA, and further include non-metastatic or non-invasive, as well as invasive or metastatic cancers, where expression of VISTA by immune, stromal, or disease cells suppresses anti-tumor and anti-invasive immune responses, as well as those characterized by angiogenic tumors.

[0091] As used herein, "chimeric antibody" refers broadly to an antibody molecule in which the constant region or a portion thereof has been altered, substituted, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, the variable region, or a portion thereof, has been altered, substituted, or exchanged with a variable region having a different or altered antigen specificity.

[0092] As used herein, "coding region" broadly refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, while the term "non-coding region" refers to the regions of a nucleotide sequence that are not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0093] As used herein, the term "conservatively modified variants" applies to both amino acid and nucleic acid sequences and broadly refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. A "silent variation" is one type of conservatively modified nucleic acid variation. All nucleic acid sequences herein that encode polypeptides also account for all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule.

[0094] As used herein, "complementarity-determining region," "hypervariable region," or "CDR" broadly refers to one or more hypervariable or complementarity-determining regions (CDRs) found in the variable regions of the light or heavy chains of an antibody. See Kabat, et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. These terms include the hypervariable regions defined by Kabat, et al. (1983) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, or the hypervariable loops of the three-dimensional structure of an antibody. Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. The CDRs in each chain are held in close proximity by framework regions, and CDRs from the other chain contribute to the formation of the antigen-binding site. Within the CDRs, there are select amino acids designated as selectivity-determining regions (SDRs), which represent important contact residues used by the CDRs in antibody-antigen interactions. (Kashmiri Methods 36:25-34(2005)).

[0095] As used herein, " control amount " refers broadly to any amount or range of amounts of marker that can be compared with the test amount of marker.For example, the control amount of marker can be the amount of marker in patients with specific disease or condition, or in individuals who do not have such disease or condition.The control amount can be either absolute amount (for example, microgram / ml) or relative amount (for example, relative intensity of signal).

[0096] As used herein, the term "costimulatory receptor" refers broadly to a receptor that transmits a costimulatory signal to an immune cell, such as CD28 or ICOS. As used herein, the term "inhibitory receptor" includes a receptor that transmits a negative signal to an immune cell, such as a T cell or NK cell.

[0097] As used herein, "costimulation" broadly refers to the ability of a costimulatory molecule to provide a second, non-activating receptor-mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion (e.g., in a T cell that has received a T cell-receptor-mediated signal). Immune cells that have received a cell receptor-mediated signal (e.g., via an activating receptor) may be referred to herein as "activated immune cells." With respect to T cells, the transmission of the costimulatory signal to the T cell involves a signal transduction pathway that is not inhibited by cyclosaporin A. In addition, the costimulatory signal can induce cytokine secretion (e.g., IL-2 and / or IL-10) in T cells and / or prevent the induction of antigen unresponsiveness, anergy, or cell death in T cells.

[0098] As used herein, "costimulatory polypeptide" or "costimulatory molecule" refers to a polypeptide that regulates T cell responses upon interaction with a cell surface molecule on a T cell.

[0099] As used herein, "costimulatory signaling" refers to the signaling activity resulting from the interaction between costimulatory polypeptides on antigen-presenting cells and their receptors on T cells during an antigen-specific T cell response. While not wishing to be limited by a single hypothesis, antigen-specific T cell responses are believed to be mediated by two signals: 1) the association of the T cell receptor (TCR) with an antigenic peptide presented in the context of the MHC (signal 1), and 2) a second, antigen-dependent signal (signal 2) delivered by contact between a different costimulatory receptor / ligand pair. While not wishing to be limited by a single hypothesis, this "second signal" is critical in determining the type of T cell response (activation versus inhibition), as well as the strength and duration of that response, and is controlled by both positive and negative signals from costimulatory molecules, such as the B7 family of proteins.

[0100] As used herein, "B7" polypeptide refers to members of the B7 family of proteins that costimulate T cells, including, but not limited to, B7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3, and biologically active fragments and / or variants thereof. Exemplary biologically active fragments include the extracellular domain or a fragment of the extracellular domain that costimulates T cells.

[0101] As used herein, "cytoplasmic domain" refers broadly to the portion of a protein that spans the cytoplasm of a cell.

[0102] As used herein, "diagnosis" broadly refers to identifying the presence or nature of a pathological condition. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the proportion of diseased individuals who have a positive test result (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not affected and have a negative test result in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who have a positive test result. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0103] As used herein, "diagnosing" or "diagnostic" broadly refers to classifying a disease or condition and / or determining the likelihood that an individual has a disease state (e.g., based on the absence or presence of VISTA expression, and / or increased or decreased immune expression, and / or putative disease cells), determining disease severity, monitoring disease progression, predicting disease outcome and / or likelihood of recovery. The term "detecting" may optionally encompass any of the foregoing. Diagnosis of disease according to the present invention may, in some embodiments, be affected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, the determined level being correlated with disease predisposition, or the presence or absence of disease. It should be noted that a "biological sample obtained from a subject" may optionally include a sample that has not been physically removed from the subject.

[0104] As used herein, "effective amount" broadly refers to the amount of a compound, antibody, antigen, or cell that, when administered to a patient to treat a disease, is sufficient to effect such treatment of the disease. An effective amount is a prophylactically effective amount and / or a preventatively effective amount. An effective amount may be an amount effective to prevent the onset of signs / symptoms, reduce the severity of the onset of signs / symptoms, eliminate the onset of signs / symptoms, delay the development of the onset of signs / symptoms, prevent the development of the onset of signs / symptoms, and / or prevent the onset of signs / symptoms. The "effective amount" may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of the present invention.

[0105] As used herein, "extracellular domain" or "ECD" refers broadly to the portion of a protein that spans the surface of a cell.

[0106] As used herein, "expression vector" refers broadly to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the present invention in any cell, including prokaryote, yeast, fungus, plant, insect, or mammalian cells, in vitro or in vivo, constitutively or inducibly. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or that integrate into the host cell genome. Expression systems may or may not have the ability to autonomously replicate, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes that contain only the minimum elements necessary for transcription of the recombinant nucleic acid.

[0107] As used herein, "family" refers broadly to the polypeptides and nucleic acid molecules of the present invention and is intended to mean two or more polypeptides or nucleic acid molecules that share a common structural domain or motif and have sufficient amino acid or nucleotide sequence homology as defined herein. Family members may be natural or non-naturally occurring and may be from either the same or different species. For example, a family may contain a first polypeptide of human origin and other distinct polypeptides of human origin, or alternatively, may contain homologs of non-human origin (e.g., simian polypeptides). Family members may also share common functional characteristics.

[0108] As used herein, "Fc receptor" (FcR) broadly refers to the cell surface receptor for the Fc portion of immunoglobulin molecules (Ig). Fc receptors are found on many cells involved in the immune response. Among the human FcRs identified to date are those that recognize IgG (designated FcyR), IgE (FceRI), IgA (FcaR), and polymerized IgM / A (FcεμR). FcRs are found on the following cell types: FceRI (mast cells), FceRII (many leukocytes), FcaR (neutrophils), and FcμR (glandular epithelium, hepatocytes) (Hogg Immunol. Today 9:185-86 (1988)). The extensively studied FcyRs are central to cellular immune defense and are involved in stimulating the release of inflammatory mediators and hydrolases involved in the pathogenesis of autoimmune diseases. (Unkeless, Annu. Rev. Immunol. 6:251-87 (1988)). Macrophage / monocyte, polymorphonuclear leukocyte, and natural killer (NK) cell FcyRs provide an important link between effector cells and Ig-secreting lymphocytes because they confer an element of specific recognition mediated by IgG. Human leukocytes possess at least three different types of FcyR for IgG: hFcγRI (CD64) (found on monocytes / macrophages), hFcyRIIA or hFcyRIIB (CD32 or CD32A) (found on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and FcγRIIIIA (CD16A) or FcγRIIIIB (CD16B) (found on NK cells, neutrophils, eosinophils, and macrophages).

[0109] As used herein, "framework region" or "FR" refers broadly to one or more of the framework regions in the light and heavy chain variable regions of an antibody. See Kabat, et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987). These terms include those amino acid sequence regions interposed between the CDRs in the light and heavy chain variable regions of an antibody.

[0110] As used herein, "heterologous" refers broadly to a portion of a nucleic acid, indicating that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids having two or more sequences from unrelated genes (e.g., a promoter from one source and a coding region from another source) arranged to create a new functional nucleic acid are typically produced by recombinant means. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0111] As used herein, "high affinity" refers to an affinity of at least 10 with respect to a target antigen or receptor. -6 M, more preferably 10 -7 M, and even more preferably at least 10 -8 M, and even more preferably at least 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 "High affinity" for an IgG antibody or fusion protein herein refers broadly to an antibody or fusion protein having a KD of at least 10 M with respect to a target antigen or receptor. -6 M or less, preferably 10 -7 M or less, preferably 10 -8 M or less, preferably 10 -9 M or less, and even more preferably 10 -10 M, 10 -11 M or 10 -12 With respect to antibodies specifically, "high affinity" binding may differ for different antibody isotypes. For example, "high affinity" binding for an IgM isotype is considered to be 10 -7 M or less, preferably 10 -8 K below M D It refers to an antibody having the following structure:

[0112] As used herein, "homology" broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Homology can be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison using default parameters, for example, using the BlastP software of the National Center of Biotechnology Information (NCBI). The term "sequence identity" can be used interchangeably with "homology."

[0113] As used herein, "host cell" broadly refers to a cell into which a nucleic acid molecule of the present invention, such as a recombinant expression vector of the present invention, is introduced. Host cells can be prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa, or HEK-293, including cultured cells, explants, and in vivo cells. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, the progeny may not actually be identical to the parent cell but still fall within the scope of the term as used herein.

[0114] "Human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. This includes fully human monoclonal antibodies, as well as conjugates and variants thereof, for example, which are linked to an effector agent, such as a therapeutic or diagnostic agent.

[0115] As used herein, "humanized antibody" broadly refers to antibodies produced by non-human cells with variable and constant regions that have been altered to resemble antibodies produced by human cells. For example, amino acids in a non-human antibody are altered to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the present invention may contain amino acid residues, for example, in CDRs, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0116] As used herein, "hybridization" refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands through the formation of hydrogen bonds between complementary nucleotides when the strands are positioned antiparallel to one another.

[0117] As used herein, "IgV domain" and "IgC domain" broadly refer to Ig superfamily member domains. These domains correspond to structural units with distinct folding patterns, called Ig folds. The Ig fold consists of a sandwich of two β-sheets, each consisting of antiparallel β-strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, domains. The IgC domains of Ig, TCR, and MHC molecules share the same type of sequence pattern, referred to as the C-I set within the Ig superfamily. Other IgC domains are in other sets. IgV domains also share a sequence pattern, referred to as the V-set domains. IgV domains are longer than C-domains and form an additional pair of β-strands.

[0118] As used herein, " immune cell " refers broadly to the cells of hematopoietic origin and play a role in immune response. Immune cells include, but are not limited to, lymphocytes such as B cells and T cells, natural killer cells, dendritic cells, and bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0119] As used herein, "immunoassay" refers broadly to an assay that uses an antibody to specifically bind to an antigen. Immunoassays can be characterized by the use of the specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen.

[0120] As used herein, "immune-related disease (or disorder or condition)" should be understood to encompass any disease disorder or condition selected from the group including, but not limited to, autoimmune diseases, inflammatory disorders, and immune disorders associated with transplant rejection of a graft, such as acute and chronic organ transplants, allogeneic stem cell transplants, autologous stem cell transplants, bone marrow transplant rejection, and graft-versus-host disease.

[0121] As used herein, "immune response" broadly refers to T cell-mediated and / or B cell-mediated immune responses affected by modulation of T cell costimulation. Exemplary immune responses include B cell responses (e.g., antibody production), T cell responses (e.g., cytokine production and cytotoxicity), and activation of cytokine-responsive cells, such as macrophages. As used herein, the term "downregulation" with respect to an immune response includes a decrease in any one or more immune responses, while the term "upregulation" with respect to an immune response includes an increase in any one or more immune responses. It is understood that upregulation of one type of immune response can lead to a corresponding downregulation of another type of immune response. For example, upregulation of the production of a particular cytokine (e.g., IL-10) can lead to downregulation of a cellular immune response.

[0122] As used herein, an "immune," "immunological," or "immune" response refers to the acquisition of a humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response directed against a peptide in a recipient patient. Such a response may be an active response induced by administration of an immunogen, or a passive response induced by administration of antibodies or primed T cells. Without wishing to be limited by a single hypothesis, a cellular immune response is elicited by presentation of a polypeptide epitope in the context of class II or class I MHC molecules, resulting in the expression of antigen-specific CD4 + T helper cells and / or CD8 + Activates cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating antibodies and T cells separately from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0123] An "immunogenic agent" or "immunogen" is a moiety that is capable of inducing an immunological response against itself upon administration to a mammal, optionally with an adjuvant.

[0124] As used interchangeably herein, the terms "inflammatory disorder," "inflammatory condition," and / or "inflammation" refer broadly to chronic or acute inflammatory diseases, specifically including inflammatory autoimmune diseases and inflammatory allergic conditions. These conditions include examples of inflammatory disorders characterized by a dysregulated immune response to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammatory disorders underlie a wide variety of human diseases. Non-immune diseases with aetiological etiology in the inflammatory process include cancer, atherosclerosis, and ischemic heart disease. Examples of inflammation-related disorders include chronic prostate cancer, glomerulonephritis, hypersensitivity reactions, pelvic inflammatory disease, reperfusion injury, sarcoidosis, vasculitis, interstitial cystitis, hypocomplementemic urticarial vasculitis, pericarditis, myositis, antisynthetase syndrome, scleritis, macrophage activation syndrome, Behçet's syndrome, PAPA syndrome, Blau syndrome, gout, adult and juvenile Still's disease, cryropyrin-associated periodic fever syndrome, and Muckle-Wells syndrome. , familial cold-induced autoinflammatory syndrome, neonatal-onset multisystem inflammatory disease, familial Mediterranean fever, chronic infantile neurological, cutaneous, and articular syndrome, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, TNF receptor-associated periodic syndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

[0125] As used herein, "inhibitory signal" broadly refers to a signal transmitted through an inhibitory receptor molecule on an immune cell. The signal can antagonize the signal through an activating receptor (e.g., through a TCR, CD3, BCR, or Fc molecule) and result in the production of second messengers, proliferation, or inhibition of effector function in immune cells, such as a decrease in phagocytosis, antibody production, or cytotoxicity, or a decrease in immune cells producing mediators (e.g., cytokines (e.g., IL-2) and / or mediators of allergic responses), or the acquisition of anergy.

[0126] As used herein, "isolated" broadly refers to material that has been removed from its original environment in which it naturally occurs, and thus includes "recombinant" polypeptides that have been altered by the hand of man from their natural environment. Isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained within a host cell, or any material that has been removed from its original environment and thus altered by the hand of man (e.g., an "isolated antibody"). For example, as used herein, "isolated" or "purified" broadly refers to a protein, DNA, antibody, RNA, or biologically active portion thereof that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological material is derived, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. As used herein, the term "isolated" refers to a compound of interest (e.g., a polynucleotide or polypeptide) that has been separated from its natural environment, such as by concentrating a peptide to a concentration not found in nature, in an environment different from that in which the compound naturally occurs. "Isolated" includes compounds that are in a sample that are substantially enriched for the compound of interest and / or from which the compound of interest is partially or substantially purified.

[0127] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of antibodies that specifically bind to antigens other than VISTA, and that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds VISTA). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0128] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0129] As used herein, "K-assoc" or "Ka" broadly refers to the association rate of a particular antibody-antigen interaction, while as used herein, the terms "Kdiss" or "Kd" refer to the dissociation rate of a particular antibody-antigen interaction.

[0130] As used herein, "K D The term "dissociation constant" refers to the ratio of Kd to Ka (i.e., Kd / Ka), expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art, such as plasmon resonance (BIAcore®), ELISA, and KINEXA. D A preferred method for determining K is using surface plasmon resonance, preferably using a biosensor system such as a BIAcore® system, or by ELISA. Typically, these methods are accomplished at 25°C or 37°C. Antibodies for therapeutic use generally have a K of 50 nM or less, or more typically 1 nM or less, as determined by surface plasmon resonance at 25°C or 37°C. D It has.

[0131] As used herein, a "label" or a "detectable moiety" refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0132] As used herein, " low stringency conditions ", " medium stringency conditions ", " high stringency conditions " or " very high stringency conditions " refer broadly to the conditions for nucleic acid hybridization and washing.The guidance for carrying out hybridization reaction can be found in Ausubel, et al., Short Protocols in Molecular Biology (5th Ed.) John Wiley & Sons, NY (2002). Exemplary specific hybridization conditions include, but are not limited to, (1) low stringency hybridization conditions of 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at at least 50°C (the temperature of the washes can be increased up to 55°C for low stringency conditions); (2) medium stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions of 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC and 1% SDS at 65°C.

[0133] As used herein, "mammal" broadly refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by hairy skin and, in females, mammary glands that produce milk to nourish their young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, tinklings, cows, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals include, but are not limited to, bovine, ovine, porcine, primate, and rodent species. Mammals also include any and all species listed in the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington, D.C.

[0134] A "multispecific antibody" refers to an antibody having two or more antigen-binding regions, including bispecific antibodies, which can bind to different antigens or different epitopes of the same antigen.

[0135] As used herein, a "naturally-occurring nucleic acid molecule" refers broadly to RNA or DNA having a nucleotide sequence that occurs in nature (eg, encodes a natural protein).

[0136] As used herein, "nucleic acid" or "nucleic acid sequence" broadly refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single-stranded or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides containing known analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0137] As used herein, "operably linked" refers broadly to when two DNA fragments are joined together so that the amino acid sequences encoded by the two DNA fragments are in frame.

[0138] As used herein, "paratope" broadly refers to the portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of an antibody). The paratope can be a small region (e.g., 15-22 amino acids) of the Fv region of an antibody and can include portions of the heavy and light chains of the antibody. See Goldsby, et al., Antigens (Chapter 3) Immunology (5th Ed). New York: W.H. Freeman and Company, pages 57-75.

[0139] "Patient" or "subject" or "recipient," "individual," or "treated individual" are used interchangeably herein and refer broadly to any animal in need of treatment to either alleviate a disease state, or to relieve a disease state, or to prevent the occurrence or recurrence of a disease state. Also, as used herein, "patient" refers broadly to any animal that has a history, susceptibility, symptom, and sign risk factors, has been previously diagnosed, is at risk for a disease, or is a member of a patient population for a disease. A patient may be a human or a clinical patient, such as a companion animal, domesticated animal, farm animal, exotic animal, or zoo animal.

[0140] "Polypeptide," "peptide," and "protein" are used interchangeably and refer broadly to polymers of amino acid residues of any length, regardless of modification (e.g., phosphorylation or glycosylation). The terms apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" expressly include glycoproteins as well as non-glycoproteins.

[0141] As used herein, "promoter" broadly refers to an array of nucleic acid sequences that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter optionally also includes distal enhancer or repressor elements, which may be located several thousand base pairs from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental control.

[0142] As used herein, "prophylactically effective amount" refers broadly to the amount of a compound that, when administered to a patient for the prevention of a disease or prevention of recurrence of a disease, is sufficient to provide such prevention for the disease or recurrence. A prophylactically effective amount may be an amount effective to prevent the occurrence of signs and / or symptoms. The "prophylactically effective amount" may vary depending on the disease being treated and its severity, as well as the patient's age, weight, medical history, predisposition to the condition, and pre-existing conditions.

[0143] "Prophylactic vaccine" and / or "prophylactic vaccination" refer to a vaccine used to prevent a disease or symptoms associated with a disease, such as cancer or an infectious condition.

[0144] As used herein, "prevention" broadly refers to a course of therapy in which signs and / or symptoms are absent from, in remission of, or previously present in a patient. Prevention includes preventing disease from occurring after treatment of the disease in a patient. Furthermore, prevention includes treating patients who may develop the disease, particularly those who are susceptible to the disease (e.g., members of a patent population, those with risk factors, or those at risk of developing the disease).

[0145] As used herein, "recombinant," in reference to a product, e.g., a cell, or a nucleic acid, protein, or vector, refers broadly to and indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0146] As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), thus allowing for the V H and V L The amino acid sequences of the regions are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0147] As used herein, "signal sequence" or "signal peptide" broadly refers to a peptide containing about 15 or more amino acids that occurs at the N-terminus of a secreted or membrane-bound polypeptide and contains a large number of hydrophobic amino acid residues. For example, a signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, and even more preferably about 19 amino acid residues, with at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, or phenylalanine). A "signal sequence," also referred to in the art as a "signal peptide," serves to direct a polypeptide containing such a sequence to the lipid bilayer and is cleaved upon secretion.

[0148] As used herein, the terms "specifically (or selectively) bind" or "specifically (or selectively) immunoreactive with" or "specifically interact with or bind" to an antibody refer broadly to a protein or peptide (or other epitope) and, in some embodiments, to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other organisms. For example, under specified immunoassay conditions, a specified antibody binds to a specific protein with at least two-fold greater than background (non-specific signal) and does not substantially bind in significant amounts to other proteins present in the sample. Typically, a specific or selective reaction is at least two-fold greater than background signal or noise, more typically about 10-100 times greater than background.

[0149] As used herein, "specifically hybridizable" and "complementary" broadly refer to a nucleic acid that can form hydrogen bond(s) with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional type. The binding free energy of a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid, such as RNAi activity, to proceed. The determination of the binding free energy of a nucleic acid molecule is well known in the art. (See, for example, Turner, et al. CSH Symp. Quant. Biol. LII: 123-33 (1987); Frier, et al. PNAS 83: 9373-77 1986); Turner, et al. J. Am. Chem. Soc. 109: 3783-85 (1987)). Percent complementarity refers to the proportion of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., at least about 5, 6, 7, 8, 9, 10 out of 10 are at least about 50%, 60%, 70%, 80%, 90%, and 100% (inclusive) complementary). "Fully complementary" or 100% complementarity broadly refers to all of the contiguous residues of a nucleic acid sequence that hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0150] " Substantial complementarity " refers to a polynucleotide strand that exhibits at least about 90% complementarity, excluding regions of the polynucleotide strand, such as overhangs, that are selected to be non-complementary.Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of oligomeric compounds to non-target sequences under the conditions where specific binding is desired, i.e., in the case of in vivo assays or therapeutic treatments, under physiological conditions, or in the case of in vitro assays, under the conditions where the assay is performed.Non-target sequences can typically differ by at least 5 nucleotides.

[0151] As used herein, a "sign" of disease refers broadly to any abnormality indicative of disease that can be determined upon examination of a patient, which is an objective indicator of disease, as opposed to a symptom, which is a subjective indicator of disease.

[0152] As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semi-solid structure to which another material can be attached, including, but not limited to, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), as well as engineered and porous materials.

[0153] As used herein, VISTA "soluble ectodomain (ECD)" or "ectodomain" or "soluble VISTA protein(s) / molecule(s)" refers to a non-cell surface-bound VISTA molecule or any portion thereof, including, but not limited to, a VISTA fusion protein or a VISTA ECD-Ig fusion protein, or fragments and derivatives thereof, in which the VISTA extracellular domain or a fragment thereof is fused to an immunoglobulin (Ig) portion to confer solubility to the fusion molecule; a protein having the VISTA extracellular domain fused or conjugated to a biologically active protein or portion of a chemically active protein, such as the papillomavirus E7 gene product, melanoma-associated antigen p97, or HIV envelope protein, or fragments and derivatives thereof; a hybrid (chimeric) fusion protein, such as VISTA-Ig, or fragments and derivatives thereof. Such fusion proteins are described in more detail below.

[0154] As used herein, "soluble VISTA protein(s) / molecule(s)" includes VISTA molecules having the transmembrane domain removed to confer protein solubility, or fragments and derivatives thereof; fragments, portions, or derivatives thereof, and also soluble VISTA mutant molecules. Soluble VISTA molecules used in methods according to at least some embodiments of the present invention may or may not include a signal (leader) peptide sequence.

[0155] In the context of therapy or diagnosis herein, a "subject" or "patient" or "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals, and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles, i.e., any suitable subject to be treated by the present invention, including, but not limited to, avian and mammalian subjects, preferably mammals. Any mammalian subject in need of treatment by the present invention is suitable. Human subjects of both genders and at all stages of development (i.e., neonates, infants, juveniles, adolescents, and adults) can be treated by the present invention. The present invention may also be performed on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cows, goats, sheep, and horses for veterinary purposes and for drug screening and drug development purposes. "Subject" is used interchangeably with "individual" and "patient."

[0156] As used herein, "substantially free of chemical precursors or other chemicals" refers broadly to preparations of VISTA protein in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein. In one embodiment, the term "substantially free of chemical precursors or other chemicals" includes preparations of VISTA protein having less than about 30% (by dry weight) chemical precursors or non-VISTA chemicals, more preferably less than about 20% chemical precursors or non-VISTA chemicals, even more preferably less than about 10% chemical precursors or non-VISTA chemicals, and most preferably less than about 5% chemical precursors or non-VISTA chemicals.

[0157] As used herein, a "symptom" of disease refers broadly to any morbid sign or deviation from normal in structure, function, or sensation experienced by a patient and that is indicative of disease.

[0158] As used herein, "T cells" refers broadly to CD4+ T cells and CD8+ T cells. The term T cells also includes both T helper type 1 T cells and T helper type 2 T cells.

[0159] As used herein, "therapy," "therapeutic," "treating," or "treatment" broadly refers to treating a disease, halting or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease, causing regression of a disease or its clinical symptoms. Therapy includes preventing, treating, relieving, reducing, alleviating, and / or causing relief from a disease, disease signs and / or symptoms. Therapy includes the alleviation of signs and / or symptoms in patients with ongoing disease signs and / or symptoms (e.g., inflammation, pain). Therapy also encompasses "prevention." For purposes of therapy, the term "reduction" broadly refers to a clinically significant decrease in signs and / or symptoms. Therapy includes treatment of recurrent or recurrent signs and / or symptoms (e.g., inflammation, pain). Therapy includes, but is not limited to, the elimination of ever-present signs and / or symptoms, as well as the reduction of existing signs and / or symptoms, and the elimination of existing signs and / or symptoms. Therapy includes the treatment of chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms (eg, inflammation, pain).

[0160] As used herein, "Treg cells" (sometimes also referred to as suppressor T cells or inducible Treg cells or iTregs) refer to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and can suppress autoimmune disease. + CD4 + CD25 + Regulatory T cells (Tregs) are important in maintaining peripheral tolerance under normal conditions.

[0161] As used herein, "transmembrane domain" broadly refers to an amino acid sequence of about 15 amino acid residues in length that spans the plasma membrane. More preferably, the transmembrane domain contains at least about 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. The transmembrane domain is rich in hydrophobic residues and typically has an α-helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids in the transmembrane domain, such as leucine, isoleucine, tyrosine, or tryptophan, are hydrophobic. Transmembrane domains are described, for example, in Zagotta, et al., Annu. Rev. Neurosci. 19:235-263 (1996).

[0162] As used herein, "transgenic animal" refers broadly to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more of the animal's cells contain a "transgene." The term "transgene" refers to exogenous DNA that is integrated into the genome of a cell from which the transgenic animal develops and remains in the genome of the mature animal, e.g., directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.

[0163] As used herein, "unresponsiveness" refers broadly to the refraction of immune cells to stimulation, e.g., via activating receptors or cytokines. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen.

[0164] As used herein, "variable region" or "VR" refers broadly to the domains within each pair of antibody light and heavy chains that are directly involved in binding the antibody to an antigen. Each heavy chain contains a variable domain (V H ), followed by several constant domains. Each light chain has a variable domain (V L ), and a constant domain at its other end, where the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0165] As used herein, the term "vector" broadly refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. Techniques and procedures are generally performed by conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook, et al. Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring Harbor Laboratory Press (2001). Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may follow manufacturer's specifications or be performed as commonly accomplished in the art or as described herein.

[0166] Having defined certain terms and phrases used in this application, the anti-VISTA antibodies and antigen-binding antibody fragments encompassed by the present invention and methods for making and using same are further described below.

[0167] The present invention relates to antibodies and antibody fragments comprising an antigen-binding region that binds to IgV domain suppressor of T cell activation (VISTA). VISTA is a checkpoint regulator that negatively suppresses immune responses. See Wang et al., "VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses," J. Exp. Med., 208(3)577-92 (2011). This protein is expressed on normal human neutrophils, monocytes, and T cell subsets. In addition, cynomolgus monkey cells express VISTA in a pattern similar to normal human cells. VISTA is also expressed in peripheral blood cells, for example, from cancer patients.

[0168] Binding of an antagonist anti-VISTA antibody or antibody fragment according to the invention to VISTA antagonizes at least one of the immune effects of VISTA, thereby suppressing the suppressive effects of VISTA on immunity, e.g., T cell immunity and / or cytokine expression. In contrast, binding of an agonist anti-VISTA antibody or antibody fragment according to the invention to VISTA stimulates, induces, or mimics at least one of the immune effects of VISTA, thereby promoting at least one of the suppressive effects of VISTA on immunity, e.g., suppression of T cell immunity or suppression of expression of certain pro-inflammatory cytokines, or its promoting effect on expression of certain chemotactic factors and chemokines.

[0169] Such antibody fragments include, for example, Fab and F(ab') 2、and scFv antibody fragments. These antibodies or antibody fragments may contain antibody constant regions, or fragments or variants thereof. Such antibodies and antibody fragments include those that bind to VISTA proteins expressed on hematopoietic cells and other cells, such as myeloid cells and / or lymphocytes, monocytes, neutrophils, T cells, natural killer (NK) cells, natural killer T (NKT) cells, tumor cells, and / or tumor microenvironment (TME) cells. The tumor microenvironment is the cellular environment of a tumor. It includes surrounding immune cells, fibroblasts, blood vessels, other cells, signaling molecules, and the extracellular matrix.

[0170] Antibodies that block or inhibit the effects of VISTA can be used to enhance human immune responses, particularly immune responses to malignancies and infections. By using control molecules that stimulate VISTA, such as soluble VISTA, for example, VISTA-Ig and target agonist anti-human VISTA antibodies and fragments, can be used to suppress unwanted human immune responses, such as autoimmunity, allergies, GVHD, sepsis, or unwanted inflammatory immune responses.

[0171] The target application provides novel antagonistic and agonistic anti-human VISTA antibodies, including those containing the same CDRs as any of the anti-human VISTA antibodies having the sequences shown in Figure 4. Although several antagonistic anti-human VISTA antibodies have been reported in the literature prior to the present invention, no agonistic anti-human VISTA antibodies or antibody fragments have been reported.

[0172] As disclosed in the experimental examples below, the inventors first produced two chimeric anti-human VISTA antibodies derived from a mouse anti-human VISTA antibody (1E8 having the sequence of Figure 4) containing an unmodified IgG2 human constant region or an IgG2 constant region, respectively, in which the cysteine ​​residue at position 127 of the kappa chain was changed to a serine residue. As shown in the Examples and Figures referenced herein, both antibodies were found to stimulate or mimic the immune suppressive effects of VISTA based on at least (i) their ability to reduce the expression of certain pro-inflammatory cytokines, such as IL-2, IL-4, IL-6, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-α), as well as the reduction in the expression of certain chemokines or chemotactic factors, such as KC (keratinocyte-derived chemokine) or MIP-2 (macrophage inflammatory protein-2); (ii) their ability to suppress T cell activity in GVHD models; and (iii) their ability to suppress CD3-driven T cell responses.

[0173] In addition, following the isolation of these two agonistic antibodies, additional 10 chimeric agonistic anti-human VISTA antibodies containing human IgG2 constant regions or Fc regions were obtained using similar methods. These antibodies were derived from antibodies designated herein as GG8, VSTB95 (INX903), VSTB103 (INX904), VSTB53 (INX905), VSTB92 (INX908), VSTB50 (INX900), VSTB56 (INX901), VSTB63 (INX902), VSTB54 (INX906), and VSTB66 (INX907) (having the sequences of Figure 4).

[0174] In particular, these chimeric anti-human VISTA antibodies have the variable sequences shown in Figure 4 and a human IgG2 constant region. As reported in Summary Tables 1 and 2 (see below), these anti-human VISTA antibodies, when assessed by the use of antibody binning, were found to bind to two distinct epitope groups, designated Group 1 and Group 2. As depicted in Figure 4, the epitopes corresponding to Group 2 include residues of two different peptides present in human VISTA, namely, NLTLLDSGL and VQTGKDAPSNC.

[0175] As shown in Tables 1 and 2 (see below), these 12 different anti-human VISTA antibodies were found to be immunosuppressive in at least one model of immunosuppression, and many in several models of immunosuppression. In particular, INX905, INX908, INX901, INX902, and INX906 were shown to be immunosuppressive in two different assay formats. While all of these antibodies are immunosuppressive and appear to induce, enhance, or stimulate the immunosuppressive effects of VISTA, INX901, INX902, and INX906, as well as INX908, appear to be the most immunosuppressive.

[0176] Additionally, other chimeric anti-human VISTA antibodies comprising human IgG2 constant domains containing the variable sequences of other anti-VISTA antibodies shown in Figure 4 will be screened for their immunosuppressive properties and their ability to stimulate or mimic the immunosuppressive and other effects of human VISTA. Based on the results obtained to date, this screening should identify other agonistic anti-human VISTA antibodies, particularly those that bind to the same epitope. In addition, agonistic anti-human VISTA antibodies according to the present invention have been shown to be effective (immunosuppressive) in a number of autoimmune and inflammatory animal disease models, including those of arthritis, lupus, or SLE, GVHD, inflammatory bowel disease (IBD) or colitis, chronic and acute infectious disease, or hepatotoxicity and psoriasis. Based thereon, the subject anti-human VISTA agonistic antibodies should be well suited for use in the therapeutic and prophylactic treatment of autoimmune, allergic, and inflammatory conditions.

[0177] As described, chimeric IgG2 anti-human VISTA antibodies having the sequences shown in Figure 4 have been shown to be immunosuppressive in different models of immunosuppression. These antibodies further elicit these immunosuppressive effects in a specific immunomodulatory manner, rather than by causing depletion of specific types of T cells or by depleting T cells in general.

[0178] As further shown in the Examples, chimeric IgG2 agonistic anti-human VISTA antibodies containing mutations in the hinge region induce substantially the same suppressive effect on immunity; i.e., mutations within the IgG2 constant region do not appear to induce enhanced suppression under the experimental conditions tested. Rather, both the IgG2A and IgG2B forms, as well as mixtures thereof, induced the same immunosuppressive effect. Additionally, based on the experiments disclosed in the Examples, it appears that FcγR binding may contribute to the agonistic properties of the subject anti-human VISTA antibodies. In particular, it was found that the inclusion of a silent IgG2 constant region eliminated the immunosuppressive properties of the tested agonist antibodies. Based on these results, it is hypothesized that one or more FcγRs influence the agonistic properties of these antibodies, and in particular, it is hypothesized that FcγRIIA (CD32 or CD32A) or FcγRIIB (CD32B) binding may be involved in the agonistic properties of the subject agonist antibodies.

[0179] It is expected that using these same methods, other agonistic anti-human VISTA IgG2 antibodies will be obtained, such as others derived from the anti-human VISTA antibody having the sequence shown in Figure 4. As mentioned, 12 agonistic anti-human VISTA antibodies have been obtained to date, including those having the sequence contained in Figure 4. Based on these results, it is expected that other agonistic anti-human VISTA antibodies may be generated and shown to be immunosuppressive. It is also expected that other agonistic anti-human VISTA antibodies may be generated that bind to the same or overlapping epitopes and / or compete with any of the antibodies containing the sequence shown in Figure 4. In exemplary embodiments, these antibodies bind to epitopes corresponding to Group 1 or Group 2 antibodies, or compete for binding to human VISTA with such antibodies.

[0180] Methods for identifying the specific epitope(s) bound by an antibody are known in the art. In the Examples, Applicant discloses the elucidation of the epitopes bound by several anti-VISTA antibodies according to the present invention. Thus, in exemplary embodiments, agonistic anti-human VISTA antibodies according to the present invention comprise an IgG2 constant region or fragment thereof in form A, form B, or a mixture of the foregoing. In exemplary embodiments, these antibodies bind to one or more FcγRs, e.g., they bind to the same FcγRs as intact or wild-type human IgG2 Fc regions. In other exemplary embodiments, the antibodies bind to CD32 (CD32A and / or CD32B). This can be achieved by using wild-type or modified IgG2 constant regions that bind to CD32 (CD32A and / or CD32B). Furthermore, agonistic antibodies can be modified to incorporate another polypeptide, such as another Fc polypeptide or antigen-binding region, that binds to an FcγR, such as CD32A and / or CD32B.

[0181] The IgG2 Fc region or constant region contained in the agonist anti-human VISTA antibodies of the invention can optionally be modified, for example, to alter effector function, e.g., to alter FcR binding, FcRN binding, complement fixation, glycosylation, etc. In particular, the IgG2 Fc region or constant region contained in the agonist anti-human VISTA antibodies of the invention can optionally be modified by converting the cysteine ​​at position 27, or further optionally by converting another cysteine ​​residue or other residues to another amino acid, such as serine, for example, in the hinge region. Other possible Fc modifications are disclosed below.

[0182] These VISTA agonist antibodies may be used to treat or prevent disease states or to treat, reduce, or ameliorate pathological effects associated therewith, such as inflammation, in the treatment or prevention of conditions in which suppression of T cell immunity or increased expression of proinflammatory cytokines and / or chemokines and chemotactic factors is therapeutically or prophylactically beneficial. These conditions include, among others, autoimmunity, allergy, inflammatory disorders, sepsis, GVHD, and for the inhibition of unwanted T cell immune responses against transplanted cells, tissues, or organs, such as CAR-T cells or gene therapy constructs or cell-containing transplanted cells, tissues, or organs.

[0183] As noted, exemplary conditions that may be treated therapeutically or prophylactically using agonistic anti-human VISTA antibodies according to the present invention include autoimmune conditions, allergic conditions, inflammatory conditions, GVHD, transplantation, and sepsis. Also, as noted, agonistic anti-human VISTA antibodies according to the present invention have been shown to be therapeutically effective and immunosuppressive in a number of animal disease models, including arthritis, inflammatory bowel disease (IBD), lupus, GVHD, chronic acute infection / hepatotoxicity, and psoriasis disease models. Thus, the antibodies of the present invention should be well suited for use in treating conditions in which suppression of immunity, particularly T cell immunity, is therapeutically desirable. A. Uses of Agonistic or Antagonistic Anti-Human VISTA Antibodies and Fragments in Therapy and Diagnostics

[0184] Compositions containing agonists according to the present invention inhibit T cell immunity, which can be used to treat therapeutically desirable conditions, such as autoimmune, allergic, or inflammatory conditions. These compositions contain an amount of an agonist antibody or antibody fragment according to the present invention effective to suppress T cell activation or proliferation or cytokine expression or other effects of VISTA in a subject in need thereof. Such autoimmune, inflammatory, and allergic conditions include, for example, arthritic conditions such as rheumatoid arthritis, psoriatic arthritis, psoriasis, scleroderma, multiple sclerosis, lupus, IBD, ITP, diabetes, GVHD, sarcoidosis, allergic asthma, hepatitis associated with hepatotoxicity, and for inhibiting unwanted T cell immune responses against transplanted cells, tissues, or organs, such as CAR-T cells or gene therapy constructs or cell-containing transplants.

[0185] Specific conditions in which the antibodies of the invention may be used alone or in conjunction with other therapeutic agents, particularly other immunosuppressive molecules, include: Acquired immune deficiency syndrome (AIDS), acquired splenic atrophy, acute anterior uveitis, acute disseminated encephalomyelitis (ADEM), acute gouty arthritis, acute necrotizing hemorrhagic leukoencephalitis, acute or chronic sinusitis, acute suppurative meningitis (or other central nervous system inflammatory disorders), acute severe inflammation, Addison's disease, adrenalitis, adult-onset diabetes mellitus (type II diabetes), adult-onset idiopathic hypoparathyroidism (AOIH), agammaglobulinemia, agranulocytosis, vasculitis (including vasculitis, optionally large vasculitis, optionally polymyalgia rheumatica, and giant cell (Takayasu) arteritis), allergic conditions, allergic contact dermatitis, allergic dermatitis, allergic granulomatous vasculitis, allergic hypersensitivity disorder, allergies allergic neuritis, allergic reaction, alopecia areata, alopecia totalis, Alport syndrome, alveolitis, optionally allergic alveolitis or fibrosing alveolitis, Alzheimer's disease, amyloidosis, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), eosinophil-related disorders, optionally eosinophilia, anaphylaxis, ankylosing spondylitis, vascular ectasia, antibody-mediated nephritis, anti-GBM / anti-TBM nephritis, antigen-antibody complex-mediated disease, anti-glomerular basement membrane disease, antiphospholipid syndrome, antiphospholipid syndrome (APS), aphthous stomatitis, aplastic anemia, cardiac arrhythmia, arteriosclerosis, arteriosclerotic disorders, arthritis, optionally rheumatoid arthritis such as acute arthritis or chronic rheumatoid arthritis, progressive chronic arthritis, osteoarthritis, ascariasis, aspergilloma, eosinophilic granuloma, aspergillosis, aspermiogenese, asthma, optionally asthma bronchiale, bronchial asthma, or autoimmune asthma, ataxia telangiectasia, ataxic sclerosis, atherosclerosis, autism, autoimmune angioedema, autoimmune aplastic anemia, autoimmune atrophic gastritis, autoimmune diabetes, autoimmune diseases of the testes and ovaries including autoimmune orchitis and oophoritis, autoimmune disorders associated with collagen diseases, autoimmune autonomic neuropathy, autoimmune ear diseases, optionally autoimmune inner ear disease (AGED), autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, autoimmune enteropathy syndrome, autoimmune hypogonadism, autoimmune hearing loss, autoimmune Autoimmune hemolysis, autoimmune hepatitis, autoimmune hepatological diseases, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune neutropenia, autoimmune pancreatitis, autoimmune polyendocrinopathy, autoimmune polyglandular syndrome type 1, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, autoimmune-mediated gastrointestinal diseases, axonal and neuronal neuropathy, Baro's disease, Behçet's disease, benign familial and ischemia-reperfusion injury, benign lymphocytic vasculitis, Berger's disease (IgA nephropathy), Bird fancier's disease, blindness, Beck's disease, bronchiolitis obliterans (non-transplant) vs. NSIP, bronchitis, bronchopulmonary aspergillosis, Bruton's disease, bullous pemphigoid, Kaplan's syndrome, cardiomyopathy, cardiovascular ischemia, Castleman's syndrome, celiac disease, celiac sprue (gluten enteropathy), cerebellar degeneration, diseases with cerebral ischemia and angiogenesis, Chagas' disease, channelopathies, any epilepsy, channelopathies of the CNS, chorioretinitis, choroiditis, autoimmune blood disorders, chronic active hepatitis or autoimmune chronic active hepatitis, chronic contact dermatitis, chronic eosinophilic pneumonia, chronic fatigue syndrome, chronic hepatitis, chronic hypersensitivity pneumonitis, chronic hypersensitivity interstitial pneumonia, chronic chronic inflammatory arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic refractory inflammation, chronic mucocutaneous candidiasis, chronic neuropathy, any IgM polyneuropathy or IgM-mediated neuropathy, chronic obstructive airway disease, chronic pulmonary inflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO), chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, CNS inflammatory disease, CNS vasculitis, celiac disease, Cogan's syndrome, cold agglutinin disease, colitis polyposa, ulcerative colitis, ulcerative colitisulcerosa), colitis such as collagen colitis, conditions with T cell infiltration and chronic inflammatory response, congenital heart block, congenital rubella infection, Coombs positive anemia, coronary artery disease, Coxsackie myocarditis, CREST syndrome (calcification, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia, Cushing's syndrome, cyclitis, optionally chronic cyclitis, iridocyclitis, or Fuchs' cyclitis, cystic fibrosis, cytokine-induced toxicity, hearing loss, osteoarthritis, demyelinating diseases, optionally autoimmune demyelinating diseases, demyelinating neuropathy, dengue fever, dermatitis herpetiformis and atopic dermatitis, dermatitis (contact dermatitis, cutaneous myositis, including skin diseases with an acute inflammatory component, Devic's disease (neuromyelitis optica), diabetic aortopathy, diabetic neuropathy, diabetic retinopathy, Diamond-Blackfan anemia, diffuse interstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus, diseases with leukocyte emigration, Dressler's syndrome, Dupuytren's contracture, echovirus infections, eczema including allergic or atopic eczema, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, encephalomyelitis, optionally allergic encephalomyelitis or encephalomyelitis allergy, and experimental allergic encephalomyelitis (EAE), intra-arterial hyperplasia hyperplasia, endocarditis, endocrine ophthalmopathy, endometriosis, endomyocardial fibrosis, endophthalmia phacoanaphylactica, endophthalmitis, allergic enterocolitis, eosinophilia-myalgia syndrome, eosinophilic fasciitis, epidemic keratoconjunctivitis, epidermolysis bullosa acquisita (EBA), episclerosis, episcleritis, Epstein-Barr virus infection, persistent erythema elevatum et diutinum, erythema multiforme, erythema nodosum leprosum, erythema nodosum, erythroblastosis fetalis, esophageal hypoperistalsis, essential mixed cryoglobulinemia, cribriform granulomatosis, Evan's syndrome, experimental allergic encephalomyelitis (EAE), factor VIII deficiency, farmer's lung, rheumatic fever (Febris rheumatica, Felty's syndrome, fibromyalgia, fibrosing alveolitis, filariasis, focal segmental glomerulosclerosis (FSGS), food poisoning, frontal gastric atrophyGlomerulonephritis (GN) with and without nephrotic syndrome, such as atrophy, giant cell arthritis (chronoarthritis), giant cell hepatitis, giant cell polymyalgia, glomerulonephritides, chronic or acute glomerulonephritis (e.g., primary GN), Goodpasture's syndrome, gouty arthritis, Granulocyte transfusion-associated syndrome, granulomatous diseases including lymphomatoid granulomatosis, granulomatosis with polyangiitis (GPA), granulomatous uveitis, Graves' disease, Guillain-Barré syndrome, guttate psoriasis, paroxysmal hemoglobinuria paroxysmatica, Hermann-Rich disease, Hashimoto's disease, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemochromatosis, hemolytic anemia or immune-mediated hemolytic anemia (including autoimmune hemolytic anemia (AIHA) and hemolytic anemia), hemophilia A, Henoch-Schönlein purpura, herpes gestationis, human immunodeficiency virus (HIV) infection, hyperalgesia, hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathic diabetes insipidus, idiopathic facial nerve palsy, idiopathic hypothyroidism, idiopathic IgA nephropathy IgA nephropathy, idiopathic membranous GN or idiopathic membranous nephropathy, idiopathic nephritic syndrome, idiopathic pulmonary fibrosis, idiopathic sprue, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated disorders, optionally anaphylaxis and allergic or atopic rhinitis, IgG4-related sclerosing disease, focal ileitis, immune complex nephritis, immune responses with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, immune-mediated GN, immunoregulatory lipoproteins (adult or acute respiratory distress syndrome) syndrome (ARDS), inclusion body myositis, infectious arthritis, infertility due to antisperm antibodies, inflammation of all or part of the uvea, inflammatory bowel disease (IBD), inflammatory hyperproliferative dermatoses, inflammatory myopathies, insulin-dependent diabetes mellitus (type 1), insulitis, interstitial cystitis, interstitial lung disease, interstitial pulmonary fibrosis, iritis, ischemia-reperfusion injury, arthritis, juvenile arthritis, juvenile dermatomyositis, juvenile diabetes, juvenile-onset (type 1) diabetes mellitus (including pediatric insulin-dependent diabetes mellitus (IDDM)), juvenile-onset (juvenile-onset) related conditions. Rheumatoid arthritis, Kawasaki syndrome, keratoconjunctivitis sicca, kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy, leukopenia, leukocyte adhesion deficiency, leukocytoclastic vasculitis, leukopenia, lichen planus, scleroatrophic leukemia, lignified conjunctivitis, linear immunoglobulin A (IgA) skin, linear immunoglobulin A disease (LAD), Löffler's syndrome, lupoid hepatitis, lupus (including nephritis, encephalitis, childhood, non-renal, extrarenal, discoid, and alopecia), systemic lupus erythematosus (SLE), disseminated lupus erythematosusdisseminatus), Lyme arthritis, Lyme disease, lymphoid interstitial pneumonia, malaria, male and female autoimmune infertility, moderate vasculitis of the maxilla (including Kawasaki disease and polyarteritis nodosa), membranous or membranous proliferative GN (MPGN), (including types I and II, as well as rapidly progressive GN, membranous GN (membranous nephropathy)), Meniere's disease, meningitis, microscopic colitis, microscopic polyangiitis, migraine, minimal change nephropathy, mixed connective tissue disease (MCTD), infectious mononucleosis, Mooren's ulcer, Muscher-Habermann disease, multifocal motor neuropathy, multiple endocrine disorders, sepsis, multiple organ injury syndromes such as those following trauma or hemorrhage, multiple organ injury syndromes, multiple sclerosis (MS) such as spino-ocular MS, multiple sclerosis, mumps , muscle diseases, myasthenia gravis such as thymoma-associated myasthenia gravis, myasthenia gravis, myocarditis, myositis, narcolepsy, necrotizing enterocolitis, and transmural colitis, as well as autoimmune inflammatory bowel disease, necrotizing, cutaneous, or hypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephropathy, nephrotic syndrome, neurological diseases, neuromyelitis optica (Devic), neuromyelitis optica, neuromyotonia, neutropenia, non-cancerous lymphocytosis, non-granulomatous uveitis, non-malignant thymoma, ocular and orbital inflammatory disorders, ocular cicatricial pemphigoid, oophoritis, sympathetic ophthalmia, opsoclonus-myoclonus syndrome (OMS), opsoclonus or opsoclonus-myoclonus syndrome (OMS), and secondary neuropathy, optic neuritis, granulomatous orchitisgranulomatosa), osteoarthritis, relapsing rheumatoid arthritis, pancreatitis, pancytopenia, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paraneoplastic syndromes (including neurological paraneoplastic syndromes), optionally Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, parasitic diseases (e.g. leishmaniasis), paroxysmal nocturnal hemoglobin PNH, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonnage-Turner syndrome, parvovirus infection, pemphigoid including bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris), erythematous pemphigus, pemphigus foliaceus, pemphigus mucomembranous pemphigoid, pemphigus vulgaris, peptic ulcer, periodic paralysis, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia anemia) (anemia perniciosa), pernicious anemia, phacoantigenic uveitis, pulmonary cirrhosis, POEMS syndrome, polyarteritis nodosa, types I, II, and III polyarthritis chronica, polychondritis (e.g., refractory or relapsing polychondritis), polyendocrine autoimmune disease, polyendocrine deficiency, polyglandular syndrome, optionally autoimmune polyglandular syndrome (or polyendocrine dyscrasia syndrome), Polymyalgia rheumatica, polymyositis, polymyositis / dermatomyositis, polyneuropathy, polyradiculitis acuta, postcardiotomy syndrome, posterior uveitis or autoimmune uveitis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, poststreptococcal nephritis, post-vaccination syndrome, presenile dementia, primary biliary cirrhosis, primary hypothyroidism, primary idiopathic myxedema, primary lymphocytosis (including monoclonal B-cell lymphocytosis), optionally benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS, primary myxedema, primary progressive MS (PPMS), and Relapsing-remitting MS (RRMS), primary sclerosing cholangitis, progestational dermatitis, progressive systemic sclerosis, proliferative arthritis, psoriasis (plaque psoriasis, psoriasis, psoriatic arthritis, etc.), pulmonary alveolar proteinosis, pulmonary infiltrate eosinophilia, pure red cell anemia or aplasia (PRCA), pure red cell aplasia, suppurative or non-suppurative sinusitis, pustular psoriasis and psoriasis of the nails, pyelitis, pyoderma gangrenosum, Quervain thyroiditis, Raynaud's phenomenon, reactive arthritis, recurrent miscarriage, decreased blood pressure response, reflex sympathetic dystrophy, refractory sprue, leukocytosis Reiter's syndrome, relapsing polychondritis, reperfusion injury of myocardium or other tissue, reperfusion injury, respiratory distress syndrome, restless legs syndrome, retinal autoimmunity, retroperitoneal fibrosis, Raynaud's syndrome, rheumatic disease, rheumatic fever, rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virus infection, Sumpter's syndrome, sarcoidosis, schistosomiasis, Schmidt's syndrome, SCID and Epstein-Barr virus-associated disease, scleritis, sclerodactyly, scleroderma, optionally Systemic scleroderma, sclerosing cholangitis, disseminated sclerosis, sclerosis (including systemic sclerosis), sensorineural hearing loss, seronegative spondyloarthritis, Sheehan's syndrome, Shulman's syndrome, silicosis, Sjögren's syndrome, sperm and testicular autoimmunity, sphenoid sinusitis, Stevens-Johnson syndrome, stiff-man (or stiff-person) syndrome, subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, sudden hearing loss, Susac's syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic lupus erythematosusSystemic lupus erythematosus (SLE) or systemic lupus erythematodes, cutaneous SLE, systemic necrotizing vasculitis, ANCA-associated vasculitis, any Churg-Strauss vasculitis or syndrome (CSS), tabes dorsalis, Takayasu's arteritis, telangiectasia, temporal arteritis / giant cell arteritis, thromboangiitis obliterans, thrombocytopenia (including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune-mediated thrombocytopenia, e.g., idiopathic thrombocytopenic purpura (ITP) (including chronic or acute ITP), thrombocytopenic purpura (TTP)), thyrotoxicosis, tissue injury, Tolosa-Hunt syndrome, toxic epidermal necrolysis , toxic shock syndrome, transfusion reactions, transient hypogammaglobulinemia of infancy, transverse myelitis, transverse myelitis, tropical pulmonary eosinophilia, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), urticaria, optionally chronic allergic urticaria and chronic idiopathic urticaria (including chronic autoimmune urticaria), uveitis, anterior uveitis, retino-uveitis, valvulitis, vascular insufficiency, vasculitis, spondyloarthritis, vesicular dermatosis, vitiligo, Wegener's granulomatosis (granulomatosis with polyangiitis (GPA)), Wiskott-Aldrich syndrome, or X-linked hyper-IgM syndrome.

[0186] In contrast, compositions containing VISTA antagonist antibodies according to the present invention promote T cell immunity, which can be used to treat conditions for which it is therapeutically valuable, such as cancer and infectious conditions, including viral, bacterial, yeast, fungal, protozoal, and parasitic infections. These compositions contain an amount of an antagonist according to the present invention effective to promote T cell activation or proliferation or cytokine expression or other effects of VISTA in a subject in need thereof. Such cancer conditions include, for example, blood cancers and solid tumors, such as leukemia, lymphoma, myelodysplastic syndrome, myeloma, lung cancer, and other cancers identified herein.

[0187] It should be understood that the disease states identified herein are exemplary and not exhaustive.

[0188] The subject agonists and antagonists may be combined with other therapeutic agents, which may be administered at the same or different times and in the same or different compositions. For example, the subject agonists may be administered in a therapeutic regimen that includes administration of a PD-1 or PD-L1 agonist, CTLA4-Ig, a cytokine, a cytokine agonist or antagonist, or another receptor agonist or antagonist. Downregulation of immune responses

[0189] Upregulation or enhancement of the inhibitory function of a VISTA polypeptide may be used to downregulate an immune response. Downregulation may take the form of inhibiting or blocking an immune response already underway, or may involve preventing the induction of an immune response. The function of activated immune cells may be inhibited by downregulating immune cell responses, by inducing specific anergy in immune cells, or both. For example, a VISTA agonist antibody may bind to a VISTA polypeptide expressed on various immune cells, thereby downregulating the immune response. This agonist antibody may be monospecific or multispecific, e.g., it may include a bispecific antibody such as a BiTE. For example, such an antibody may contain a VISTA antigen-binding portion and another antigen-binding portion, e.g., which targets a cell surface receptor on an immune cell, e.g., a T cell, B cell, or myeloid cell. In addition to containing a VISTA antigen-binding portion, such an antibody may also contain a binding portion that binds to a B cell antigen receptor, a T cell antigen receptor, or an Fc or other receptor, in order to target the molecule to a specific cell population. This choice of second antigen for the bispecific antibody provides flexibility in the selection of the cell population to be targeted. VISTA agonist antibodies that promote or mimic VISTA activity may enhance the interaction of VISTA with its natural binding partner. As disclosed herein, other human VISTA activating or agonist antibodies may be identified by their ability to inhibit T cell activity or proliferation, and / or based on their immunosuppressive effects in vitro or in inflammatory, allergic, or autoimmune disease models.

[0190] Several art-recognized readouts of cell activation can be used, for example, to measure cell proliferation or effector function (e.g., antibody production, cytokine production, phagocytosis) in the presence of an activating agent. The ability of a test antibody to stimulate or promote the action of human VISTA, thereby blocking this activation, can be readily determined by measuring the ability of the agent to affect a decrease in the measured proliferation or effector function. Thus, the ability of a test antibody to be immunosuppressive and block immune activation can be determined by measuring cytokine production and / or proliferation at different concentrations of antigen.

[0191] Co-administration of a VISTA agonist antibody according to the present invention with an antigen can induce tolerance to a specific antigen. For example, tolerance to a specific polypeptide immune response to an allergen or foreign polypeptide to which an immune response is undesirable can be induced. For example, patients receiving factor VIII frequently produce antibodies against this coagulation factor. Co-administration of a VISTA agonist antibody according to the present invention, which stimulates or mimics VISTA activity or the interaction with its natural binding partner, with recombinant factor VIII can suppress this undesirable immune response.

[0192] The VISTA agonist antibodies of the present invention can be used in combination with another agent that blocks the activity of a costimulatory receptor on an immune cell or stimulates the activity of another immunosuppressive receptor or ligand expressed on an immune cell to downregulate the immune response. Exemplary molecules include antagonistic antibodies targeting one or more of PD-1, PDL-1 agonists, soluble forms of CTLA-4, anti-B7-1 antibodies, anti-B7-2 antibodies, LAG-3, TIM-3, BTLA, B7-H4, B7H3, etc., and / or agonistic antibodies targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS, or combinations thereof. These moieties may be incorporated into a single composition or compound, for example, a bispecific antibody containing a VISTA agonist antibody according to the present invention and further comprising another immunoagonist antibody, or may comprise a fusion polypeptide containing a VISTA agonist antibody according to the present invention fused to another immunosuppressive polypeptide or other active agent. Alternatively, these moieties may be administered as separate or distinct entities (simultaneously or sequentially) in the same or different compositions to downregulate an immune cell-mediated immune response in a subject.

[0193] Examples of specific immune inhibitory molecules that can be combined with a VISTA agonist antibody according to the present invention include antibodies that block costimulatory signals (e.g., against CD28 or ICOS), antibodies that activate inhibitory signals via CTLA4, and / or antibodies against other immune cell markers (e.g., against CD40, CD40 ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-1-Fc), and immunosuppressants (e.g., rapamycin, cyclosporin A, or FK506).

[0194] In further embodiments, bispecific antibodies containing VISTA agonist antibodies according to the present invention are useful for targeting specific cell populations, for example, using markers found only on certain cell types, such as B lymphocytes, monocytes, dendritic cells, or Langerhans cells. Downregulating immune responses by activating VISTA activity or VISTA-immune cell interactions (thus stimulating the negative signaling function of VISTA) is useful in downregulating immune responses, for example, in the case of tissue, skin, and organ transplants, in graft-versus-host disease (GVHD) or allergies, or in autoimmune and inflammatory diseases such as systemic lupus erythematosus, IBD, RA, psoriasis, and multiple sclerosis. For example, blocking immune cell function results in reduced tissue destruction in tissue transplants. Typically, in tissue transplants, rejection of the graft is initiated through its recognition as foreign by immune cells, followed by an immune response that destroys the graft. Administration of a molecule that enhances the activity of VISTA on immune cells or the interaction of VISTA with its natural binding partner(s) prior to or at the time of transplantation, alone or in combination with another down-regulator, can inhibit the generation of costimulatory signals. Furthermore, promotion of VISTA activity can be sufficient to anergize immune cells, thereby inducing tolerance in a subject.

[0195] In some diseases or some subjects, it may be necessary to block the costimulatory function of other molecules to achieve sufficient immunosuppression or tolerance.For example, it may be desirable to block the function of B7-1 and B7-2 (separately or together in a single composition) by administering soluble forms of a combination of peptides that have the activity of each of these antigens or block antibodies against these antigens before or at the time of transplantation.Alternatively, it may be desirable to promote the inhibitory activity of VISTA and further inhibit the costimulatory activity of B7-1 and / or B7-2.

[0196] The subject anti-human VISTA agonist antibodies are particularly useful for treating autoimmune diseases. Many autoimmune disorders result from the inappropriate activation of immune cells that are reactive against self-tissue and promote the production of cytokines and autoantibodies involved in disease pathology. Preventing the activation of autoreactive immune cells can reduce or eliminate disease symptoms. Administration of a subject anti-human VISTA agonist antibody that promotes VISTA activity or VISTA's interaction with its natural binding partner(s) can induce antigen-specific tolerance of autoreactive immune cells, which can result in long-term disease alleviation. Furthermore, co-administration of an agent that blocks immune cell costimulation by disrupting the receptor-ligand interaction of B7 molecules with costimulatory receptors can be useful in inhibiting immune cell activation to prevent the production of autoantibodies or cytokines that may be involved in the disease process.

[0197] Downregulation of immune responses through stimulation of VISTA activity or VISTA's interaction with its natural binding partner(s) using a subject anti-human VISTA agonist antibody may also be useful in treating autoimmune attacks of autologous tissue. Thus, conditions caused by or exacerbated by autoimmune attack (e.g., heart disease, myocardial infarction, or atherosclerosis) may be restored or improved by increasing VISTA activity or the binding of VISTA to its natural binding partner. Thus, it is within the scope of the present invention to regulate conditions exacerbated by autoimmune attack, such as autoimmune disorders (as well as conditions such as heart disease, myocardial infarction, and atherosclerosis) by using a subject anti-human VISTA agonist antibody to stimulate VISTA activity or the interaction of VISTA with its counterreceptor.

[0198] As mentioned above, the efficacy of agonist anti-human VISTA antibodies according to the present invention for preventing or alleviating autoimmune and inflammatory disorders can be determined using several well-characterized animal models of human autoimmune and inflammatory diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis. See Paul, ed., Fundamental Immunology, Raven Press, New York, 1989, pages 840-856.

[0199] Inhibition of immune cell activation is further therapeutically useful in the treatment of allergies and allergic reactions, for example, by inhibiting IgE production. A subject anti-human VISTA agonist antibody that promotes or mimics VISTA activity or the interaction of VISTA with its natural binding partner(s) can be administered to an allergic subject to inhibit immune cell-mediated allergic responses in the subject. Stimulation of VISTA activity or its interaction with its natural binding partner(s) can involve exposure to an allergen along with the appropriate MHC molecule. Allergic responses can be systemic or local in nature, depending on the route of entry of the allergen and the pattern of IgE deposition on mast cells or basophils. Thus, immune cell-mediated allergic responses can be inhibited locally or systemically by administration of a subject anti-human VISTA agonist antibody. Selection of anti-VISTA antibodies that bind to the same epitope

[0200] In certain embodiments, agonistic anti-VISTA antibodies according to the present invention possess desirable functional properties, such as immune stimulation and modulation of related functions. As shown in Figure 4 and disclosed in the Examples, the epitope specificity of several anti-human VISTA agonist antibodies according to the present invention has been elucidated. Because some antibodies shown to bind to the same epitope have been found to be immunosuppressive, other VISTA agonist antibodies that bind to the same or overlapping epitopes may be identified, i.e., they are predicted to interact with one or more of the amino acid residues of the human VISTA polypeptide that bind to the exemplary VISTA agonist antibodies. Other antibodies with the same epitope specificity and / or the ability to cross-compete with the desired antibody for binding to the VISTA antigen may be selected. For example, the epitope specificity of a desired antibody may be determined using a library of overlapping peptides comprising the entire VISTA polypeptide, e.g., 15-mer or overlapping peptide libraries comprising portions containing the desired epitope of VISTA and antibodies that bind to the same peptide or one or more residues thereof within the library are determined to bind to the same linear or conformational epitope. In the examples, epitope specificity was determined using the Pepscan® method, which can be used to identify linear and conformational epitopes.

[0201] Modification of agonist antibodies according to the present invention Additionally, or as an alternative to modifications made within the framework or CDR regions, antibodies according to at least some embodiments of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Further, antibodies according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), or modified to alter its glycosylation, also to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0202] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0203] In another embodiment, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has toxic Staphylococcal protein A (SpA) binding relative to the native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0204] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, and T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, the antibody can be modified in the CHI or CL region to contain salvage receptor binding epitopes obtained from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0205] In yet other embodiments, the Fc region is altered by replacing at least one amino acid with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0206] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0207] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO 94 / 29351 by Bodmer et al.

[0208] In yet another example, the Fc region is modified to increase the affinity of the antibody for an Fγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293 , 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication No. WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn have been mapped, and mutants with improved binding have been described (see Shields, R. Lett. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcyRIII. Furthermore, the following combination mutants have been shown to improve FcyRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A. Furthermore, mutations such as M252Y / S254T / T256E or M428L / N434S improve binding to FcRn and increase antibody circulating half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0209] In yet another embodiment, antibodies can be modified to prevent in vivo Fab arm exchange. Specifically, this process involves the exchange of IgG4 half molecules (one heavy chain plus one light chain) with other IgG4 antibodies, effectively resulting in a functionally monovalent b-specific antibody. Mutations to the hinge region and constant domain of the heavy chain can prevent this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).

[0210] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0211] Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies according to at least some embodiments of the present invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (a(1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 cell lines were created by targeted distribution of the FUT8 gene in CHO / DG44 cells using two exchange vectors (see U.S. Patent Application Publication No. 2004 / 0110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α1,6 linkage-related enzymes. Hanai et al. also describe cell lines, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662), that have low or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of an antibody.PCT Publication No. WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, RR et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication No. WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., P(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increased bisecting GlcNac structure, resulting in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme, for example, the fucosidase α-L-fucosidase, which removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14:5516-23).

[0212] Another modification of the antibodies herein contemplated by the present invention is pegylation or the addition of other water-soluble moieties, typically polymers, for example, to enhance half-life. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(Ci-Cio)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an unglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to antibodies according to at least some embodiments of the present invention. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0213] Antibody Engineering Methods In certain embodiments, V H and V L Agonist anti-VISTA antibodies according to the invention having the sequences V H and / or V LThe CDR regions of a VISTA antibody or variants thereof can be recombinantly combined with known framework regions and / or other CDRs to create additional engineered anti-VISTA antibodies according to at least some embodiments of the present invention. Other types of modifications include those described in the previous section. The starting material for the engineering method is the V CDR region of the V CDR provided herein. H and / or V L In order to create an engineered antibody, one or more of the V sequences provided herein, or one or more CDR regions thereof, may be used. H It is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VL and / or VL sequences, or one or more CDR regions thereof. Rather, the information contained in the sequence is used as starting material to create "second generation" sequences derived from the original sequence, and the "second generation" sequences are then prepared and expressed as proteins.

[0214] Standard molecular biology techniques can be used to prepare and express the altered antibody sequences. Preferably, the anti-VISTA antibodies encoded by the altered antibody sequences each retain one, some, or all of the functional properties of the anti-VISTA antibodies produced by the methods provided herein and having the sequences, including the ability to bind to specific K D These include binding to a VISTA antigen with a level below that of the VISTA antigen, and / or modulating an immune response, and / or selectively binding to a desired target cell, e.g., expressing a VISTA antigen.

[0215] The functional properties of the altered antibodies can be assessed using standard assays available in the art and / or described herein. In certain embodiments of antibody engineering methods according to at least some embodiments of the present invention, mutations can be introduced randomly or selectively along with all or part of the anti-VISTA antibody coding sequence, and the resulting modified anti-VISTA antibodies can be screened for binding activity and / or other desired functional properties.

[0216] Mutation methods have been described in the art.For example, PCT Publication No. WO02 / 092780 by Short describes the method for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof.Alternatively, PCT Publication No. WO03 / 074679 by Lazar et al. describes the method of using computational screening methods to optimize the physiochemical properties of antibody.

[0217] Nucleic acid molecules encoding antibodies The present invention further provides nucleic acids encoding anti-VISTA antibodies, or fragments or conjugates thereof, according to the present invention. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids according to at least some embodiments of the present invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0218] Nucleic acids according to at least some embodiments of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies made by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding the antibodies can be recovered from the library.

[0219] V H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L - or V H -the coding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As defined above, "operably linked" means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments are in frame.

[0220] V H The isolated DNA encoding the region is V HThe VH-encoding DNA can be converted into a full-length heavy chain gene by operably linking it to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1, IgG2, or IgG4 constant region. For Fab fragment heavy chain genes, the VH-encoding DNA is linked to the heavy chain C H1 It may be operably linked to another DNA molecule encoding only the constant region.

[0221] V L The isolated DNA encoding the region is V L The coding DNA for the light chain constant region C L The light chain constant region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking it to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa (κ) or lambda (λ) constant region, but is most preferably a κ constant region.

[0222] To generate the scFv gene,H - and V L The coding DNA fragment is V H and V L The V fragment is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, so that the sequence can be expressed as a contiguous single-chain protein. L and V H The domains are joined by flexible linkers (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci., USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0223] Production of anti-VISTA monoclonal antibodies Anti-VISTA monoclonal antibodies (mAbs) and antigen-binding fragments according to the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B lymphocytes.

[0224] The preferred animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Chimeric or humanized antibodies of the present invention can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (see, e.g., U.S. Pat. No. 5,225,539 to Winter, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0225] According to at least some embodiments of the present invention, the antibody is a human monoclonal antibody. Such human monoclonal antibodies directed against VISTA can be generated using transgenic or transchromosomic mice that have parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb Mouse™ and KM Mouse™, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb Mouse™ (Medarex Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy chain μ and γ and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ, γ, and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474):856-859). Thus, the mice exhibit reduced mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG κ monoclonals (Lonberg, N. et al. (1994) (supra); reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546).The preparation and use of HuMab Mouse® and the genomic modifications performed by such mice are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591, and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the entire contents of which are specifically incorporated herein by reference in their entireties. Further, U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all to Lonberg and Kay, U.S. Patent No. 5,545,807 to Surani, all to Lonberg and See PCT Publication Nos. WO92 / 03918, WO93 / 12227, WO94 / 25585, WO97 / 13852, WO98 / 24884, and WO99 / 45962 to Kay, and PCT Publication No. WO01 / 14424 to Korman et al.

[0226] In another embodiment, human antibodies according to at least some embodiments of the invention can be produced using mice carrying human immunoglobulin sequences on a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM Mice™," are described in detail in PCT Publication No. WO 02 / 43478 to Ishida.

[0227] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and may be used to produce anti-VISTA antibodies according to at least some embodiments of the present invention. For example, an alternative transgenic system called the Xenomouse (Abgenix, Inc.) may be used; such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0228] Additionally, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and may be used to produce anti-VISTA antibodies according to at least some embodiments of the present invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," may be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA 97:722-727. Additionally, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and may be used to produce anti-VISTA antibodies according to at least some embodiments of the present invention.

[0229] Human monoclonal antibodies according to at least some embodiments of the present invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies have been established in the art. See, for example, U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner, 5,427,908 and 5,580,717 to Dower et al., 5,969,108 and 6,172,197 to McCafferty et al., and 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0230] Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al. Immunization of human Ig mice

[0231] In some embodiments, human Ig mice can be used to produce human anti-VISTA antibodies according to the present invention, for example, by immunizing such mice with a purified or enriched preparation of VISTA antigen and / or recombinant VISTA, or a VISTA fusion protein, as described in Lonberg, N. et al. (1994) Nature 368(6474):856-859, Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, and PCT Publication Nos. WO 98 / 24884 and WO 01 / 14424. Preferably, mice will be 6-16 weeks old at the time of the first injection. For example, human Ig mice can be immunized intraperitoneally with a purified or recombinant preparation of VISTA antigen (at a dose ranging from 0.5 to 500 μg).

[0232] Generally, transgenic mice respond to an initial intraperitoneal (IP) immunization with antigen in complete Freund's adjuvant, followed by IP immunizations every two weeks with antigen in incomplete Freund's adjuvant (for a maximum of six total immunizations). However, adjuvants other than Freund's have also been shown to be effective. In addition, whole cells in the absence of adjuvant have been shown to be highly immunogenic. The immune response can be monitored over the course of the immunization protocol with plasma samples obtained by retroorbital bleeds. Plasma is screened by ELISA (described below), and mice with sufficient titers of anti-VISTA human immunoglobulin are used for fusions. Mice can be boosted intravenously three days before sacrifice and spleen removal. It is expected that two to three fusions for each immunization will need to be performed. 6 to 24 mice are typically immunized for each antigen. Both HCo7 and HCol2 strains are commonly used. Additionally, both HCo7 and HCol2 transgenes can be bred together into a single mouse with two different human heavy chain transgenes (HCo7 / HCo12). Alternatively, or in addition, the KM Mouse™ strain can be used. In an exemplary embodiment, these mice are engineered to selectively produce human IgG2 antibodies. Generation of hybridomas producing human monoclonal antibodies

[0233] In certain embodiments, hybridomas producing human monoclonal anti-VISTA antibodies according to the present invention can be generated using spleen cells and / or lymph node cells from immunized mice, isolated, and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused with 50% PEG to one-sixth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL1580). Cells are plated at approximately 2x105 cells in flat-bottom microtiter plates and then incubated for 2 weeks in selection medium containing 20% ​​fetal clonal serum, 18% "653" conditioned medium, 5% IGEN, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and IX HAT (Sigma; HAT is added 24 hours after fusion). After approximately 2 weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, usually after 10-14 days, medium can be observed. The antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0234] To purify human monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0235] Generation of transfectomas producing human monoclonal antibodies In certain embodiments, anti-VISTA antibodies according to the present invention can be produced in host cell transfectomas, for example, using a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein are H The segment is C in the vector H operatively connected to the V L The segment is C in the vector LThese segments can be used to create full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding the heavy and light chain constant regions of the desired isotype so that they are operably linked to the segments. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0236] Characterization of antibody binding to antigen In certain embodiments, the binding specificity of agonistic anti-VISTA antibodies according to the present invention is determined by known antibody binding assay techniques, such as ELISA. In an exemplary ELISA, microtiter plates are coated with purified antigen, herein VISTA, at 0.25 μg / ml in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS / Tween and then incubated with a secondary reagent conjugated to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent) for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405-650. Preferably, mice achieving the highest titers are used for fusions.

[0237] The ELISA assay described above can also be used to screen for hybridomas that show positive reactivity with the VISTA immunogen. Hybridomas that bind with high avidity to VISTA are subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells (by ELISA) can be selected to generate a 5-10 vial cell bank stored at -140°C and for antibody purification.

[0238] To purify anti-VISTA antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80°C.

[0239] To determine whether selected anti-VISTA monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using VISTA-coated ELISA plates, as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

[0240] To determine the isotype of purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype, such as IgG2. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated with 1 μg / ml anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate is reacted with up to 1 μg / ml of test monoclonal antibody or purified isotype control for 1-2 hours at ambient temperature. The wells can then be reacted with either human IgG1- or human IgM-specific alkaline phosphatase-conjugated probes. The plate is developed and analyzed as described above.

[0241] Each anti-VISTA human IgG can be further tested for reactivity with the VISTA antigen by Western blot. Briefly, the VISTA antigen is prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

[0242] In another aspect, the invention features antibody-drug conjugates (ADCs) composed of an antibody (or antibody fragment, often cytotoxic), such as a single-chain variable fragment (scFv), linked to a payload drug. The antibody binds the ADC to target cancer cells. In many cases, the ADC is then internalized by the cell and the drug is released intracellularly. Because of the targeting, side effects are fewer and provide a broader therapeutic window. The hydrophilic linker (e.g., PEG4Mal) helps prevent the drug from being effluxed from resistant cancer cells by MDR (multidrug resistance) transporters.

[0243] In another aspect, the invention features an immunoconjugate comprising an anti-VISTA antibody, or fragment thereof, conjugated to a therapeutic agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such complexes are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0244] Other examples of therapeutic cytotoxins that may be conjugated to antibodies according to at least some embodiments of the invention include duocarmycins, calicheamicins, maytansines, and auristatins, and their derivatives. One example of a calicheamicin antibody conjugate is commercially available (Mylotarg™ Wyeth).

[0245] Cytotoxins can be conjugated to antibodies according to at least some embodiments of the present invention using linker technology available in the art.Examples of the types of linkers used for conjugating cytotoxins to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers.For example, linkers can be selected that are susceptible to cleavage by the low pH in lysosomal compartments, or proteases, such as cathepsins (e.g., cathepsins B, C, D), that are preferentially expressed in tumor tissues. For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, PA et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, TM (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0246] The antibodies of the present invention can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art. Radioimmunoconjugates, including Zevalin® (BiogenlDEC) and Bexxar® (Corixa Pharmaceuticals), are commercially available, and similar methods can be used to prepare radioimmunoconjugates using antibodies according to at least some embodiments of the present invention.

[0247] Agonist anti-human VISTA antibodies and conjugates according to at least some embodiments of the present invention can be used to modify a given biological response, and the drug moiety should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other growth factors.

[0248] Techniques for conjugating such therapeutic moieties to antibodies are well known and include, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use See, "Of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0249] bispecific molecules Multispecific anti-VISTA agonist antibodies are also encompassed in accordance with at least some embodiments of the present invention. Multispecific antibodies are monoclonal antibodies with binding specificities at at least two different sites. In another aspect, the present invention features bispecific molecules comprising an anti-VISTA antibody, or fragment thereof, in accordance with at least some embodiments of the present invention. Antibodies, or antigen-binding portions thereof, in accordance with at least some embodiments of the present invention can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand of a receptor), to generate bispecific molecules that bind to at least two different binding sites or target molecules. Antibodies in accordance with at least some embodiments of the present invention can, in fact, be derivatized or linked to two or more other functional molecules to generate multispecific molecules that bind to three or more different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create bispecific molecules in accordance with at least some embodiments of the present invention, an antibody can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to generate a bispecific molecule. In certain embodiments, one of the binding specificities of the bispecific antibody is for VISTA, and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of VISTA. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express VISTA. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0250] Bispecific antibodies, according to at least some embodiments of the invention, are antibodies that can simultaneously bind to two targets that are of different structures. Bispecific antibodies (bsAbs) and bispecific antibody fragments (bsFabs), according to at least some embodiments of the invention, have at least one arm that specifically binds to a B-cell antigen or epitope and at least one other arm that specifically binds to a targetable conjugate.

[0251] In at least some embodiments, the present invention also encompasses fusion antibody proteins, which are recombinantly produced antigen-binding molecules in which two or more different single-chain antibody or antibody fragment segments with the same or different specificities are linked. A variety of bispecific fusion antibody proteins can be produced using molecular engineering. In one form, the bispecific fusion antibody protein is monovalent, consisting, for example, of a sentinel having a single binding site for one antigen and a Fab fragment having a single binding site for a second antigen. In another form, the bispecific fusion antibody protein is bivalent, consisting, for example, of an IgG having two binding sites for one antigen and two scFvs having two binding sites for a second antigen.

[0252] The present invention further encompasses engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies" (see, e.g., US 2006 / 0025576A1) and "dual-acting FAb" or "DAB" antibodies (see, e.g., US 2008 / 0069820) that comprise antigen-binding sites that bind VISTA and another distinct antigen. Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for VISTA and a second binding specificity for a second target epitope. According to at least some embodiments of the present invention, the second target epitope is an Fc receptor, e.g., human FcyRI (CD64) or a human FcαR receptor (CD89). Accordingly, the present invention includes bispecific molecules capable of binding both to FcyR-, FcαR-, or FcsR-expressing effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)), and to target cells expressing VISTA, respectively. These bispecific molecules target VISTA-expressing cells to effector cells and trigger Fc receptor-mediated effector cell activities such as phagocytosis of VISTA-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion generation.

[0253] According to at least some embodiments of the invention in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the anti-Fc binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) moiety, e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell.

[0254] An "anti-enhancement factor moiety" can be an antibody, functional antibody fragment, or ligand that binds to a given molecule, e.g., an antigen or receptor, thereby enhancing the effect of a binding determinant on an Fc receptor or target cell antigen. The "anti-enhancement factor moiety" can bind to an Fc receptor or target cell antigen. Alternatively, the anti-enhancement factor moiety can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor moiety can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an increased immune response against the target cell).

[0255] According to at least some embodiments of the invention, the bispecific molecule comprises as binding specificity at least one antibody, or antibody fragment thereof, comprising, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv. The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof (such as an Fv or single-chain construct), as described in U.S. Patent No. 4,946,778 to Ladner et al., the contents of which are expressly incorporated by reference.

[0256] In one embodiment, the binding specificity for an Fcγ receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight gamma chain genes located on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms, divided into three Fcγ receptor classes: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). In one preferred embodiment, the Fcγ receptor is human high-affinity FcyRI. Human FcyRI is a 72-kDa molecule that exhibits high affinity for monomeric IgG. The production and characterization of certain preferred anti-Fcγ monoclonal antibodies are described by Fanger et al. in PCT Publication No. WO 88 / 00052 and U.S. Pat. No. 4,954,617, the teachings of which are incorporated herein by reference in their entireties. These antibodies bind to an epitope of FcγRI, FcγRII, or FcγRIII at a site distinct from the Fcγ binding site of the receptor, and therefore, their binding is not substantially blocked by physiological levels of IgG. Known anti-FcγRI antibodies include mAb22, mAb32, mAb44, mAb62, and mAb197. A hybridoma producing mAb32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. In another embodiment, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody are described in Graziano, RF et al. (1995) J. Immunol. 155(10):4996-5002 and PCT Publication No. WO94 / 10332. The H22 antibody-producing cell line has been deposited at the American Type Culture Collection under the designation HA022CLI and has accession number CRL 11177.

[0257] In yet another embodiment, the binding specificity of the Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., the Fc-α receptor (FcαRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of the α-gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced ​​transmembrane isoforms ranging from 55 to 10 kDa. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophils, and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI binds both IgA1 and IgA2 with moderate affinity (approximately 5×10 -7 M -1 ), which increases upon exposure to cytokines such as G-CSF or GM-CSF (Morton, HC et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62, and A77, which bind to FcαRI outside the IgA ligand-binding domain, have been described (Monteiro, RC et al. (1992) J. Immunol. 148:1764).

[0258] Although human monoclonal antibodies are preferred, other antibodies that can be used in bispecific molecules according to at least some embodiments of the present invention are murine, chimeric, and humanized monoclonal antibodies. The bispecific molecules of the present invention can be prepared by conjugating component binding specificities, such as anti-FcR and anti-VISTA binding specificities, using methods known in the art. For example, the binding specificities of each bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyl-dithiopropionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139:2367-2375). Preferred conjugation agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.). When the binding moieties are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, before conjugation.

[0259] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAbXmAb, mAbXFab, FabXF(ab')2, or ligandXFab fusion protein. Bispecific molecules according to at least some embodiments of the present invention can be single-chain molecules containing one single-chain antibody and a binding determinant, or single-chain bispecific molecules containing two binding determinants. Bispecific molecules can contain at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858.

[0260] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); controlled Fab arm exchange (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110(13):5145-50 (2013)); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al. Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and single-chain Fv (sFv) dimers (e.g., Gruber et al., J. Immunol., 152:5368 (1994)), as well as by preparing triabodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0261] Binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of interest specifically by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

[0262] Uses of antagonist antibodies and pharmaceutical compositions containing them cancer immunotherapy Unlike tumor-targeted therapy, which aims to inhibit molecular pathways important for tumor growth and development and / or deplete tumor cells, cancer immunotherapy aims to stimulate the patient's own immune system to eliminate cancer cells and provide long-lasting tumor destruction.In cancer immunotherapy, various approaches can be used, among which are therapeutic cancer vaccines that induce tumor-specific T cell responses, and immunostimulatory antibodies (i.e., inhibitory receptors = immune checkpoint antagonists) that eliminate immunosuppressive pathways.

[0263] Clinical responses from targeted therapy or conventional anti-cancer therapy tend to be transient, as cancer cells develop resistance, resulting in tumor recurrence. However, the clinical use of cancer immunotherapy in the past few years has shown that this type of therapy can have durable clinical responses, demonstrating a dramatic impact on long-term survival. However, although the response is long-term, only a small number of patients respond (as opposed to conventional therapy or targeted therapy, in which a large number of patients respond, but the response is transient).

[0264] By the time tumors are clinically detected, they have already evaded the immune defense system by acquiring immune resistance and suppression properties and creating an immunosuppressive tumor microenvironment through various mechanisms and various immune cells. Therefore, it is becoming increasingly clear that a combination of therapies is required for clinical efficacy in cancer immunotherapy.

[0265] Combination approaches are necessary and anticipated to increase the number of patients who benefit from immunotherapy, expand the number and types of responsive cancers, and broaden the potential cancer indications for checkpoint agents well beyond the initial indications currently demonstrating the efficacy of immune checkpoint blockade as monotherapy. Combining immunomodulatory approaches is meant to maximize outcomes and overcome the resistance mechanisms of most tumors to single approaches. Thus, tumors traditionally considered non-immunogenic may become immunogenic and respond to immunotherapy, but with the co-administration of pre-immunogenic therapies designed to increase the patient's anti-tumor immune response. Potential priming agents are detailed below.

[0266] The scientific basis underlying the dramatically increased efficacy of combination therapy asserts that immune checkpoint blockade as a monotherapy induces tumor regression only when there is a pre-existing strong anti-tumor immune response that is "released" when the pathway is blocked. According to at least some embodiments of the present invention, VISTA-specific antibodies, antibody fragments, conjugates, and compositions comprising them are used in combination therapy and in cancer immunotherapy for the treatment of all types of cancer.

[0267] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, and in this example relates to the treatment of inflammatory side effects of cancer therapy. However, as described below, the use of antibodies and pharmaceutical compositions is also provided for the treatment of infectious diseases, sepsis, and / or autoimmune conditions, and / or for inhibiting unwanted immune activation following gene therapy. Those in need of treatment include those already suffering from cancer as well as those in whom cancer is to be prevented. Thus, mammals treated herein may have been diagnosed with cancer or may be predisposed or susceptible to cancer. As used herein, the term "treating" refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, inhibiting, halting the deleterious effects of, or stabilizing the discernible symptoms of the cancerous disease, disorder, or condition. Managing the cancer is also included. By "manage" is meant a decrease in the severity of the disease, a decrease in the frequency of disease episodes, a decrease in the duration of such episodes, a decrease in the severity of such episodes, a slowing / reducing of the growth or proliferation of cancer cells, a slowing of the progression of at least one symptom, or an amelioration of at least one measurable physical parameter. For example, an immunostimulatory anti-VISTA antibody would treat cancer or infectious disease by promoting T cell or NK or cytokine immunity against target cells, e.g., cancer, infected cells, or pathogen cells, thereby depleting cells involved in the disease state. An agonistic anti-VISTA antibody would decrease T cell or NK activity and / or secretion of pro-inflammatory cytokines involved in the disease pathology of certain immune disorders, such as autoimmune, inflammatory, or allergic conditions, thereby treating or ameliorating the disease pathology and tissue destruction that may be associated with such conditions (e.g., joint destruction associated with rheumatoid arthritis conditions).

[0268] For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic animals, and farm animals, as well as zoo animals, sport animals, or pet animals, such as dogs, horses, cats, and cows. Preferably, the mammal is a human. Preferably, the mammal is a human diagnosed with one of the above-mentioned diseases, disorders, or conditions, or alternatively, is predisposed to at least one type of cancer.

[0269] A "therapeutically effective amount" refers to an amount of an agent according to the present invention that is effective to treat a disease or disorder in a mammal. The therapeutic agents of the present invention can be provided to a subject alone or as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier.

[0270] The anti-VISTA antibodies, fragments thereof, conjugates thereof, and / or pharmaceutical compositions comprising same described herein, according to at least some embodiments of the present invention, may also be administered in combination with other enhancing agents and / or other therapies. According to at least some embodiments, the anti-VISTA antibodies may be used in combination with any of the therapies known in the art of standard of care for cancer treatment (e.g., which can be found at http: / / www.cancer.gov / cancertopics).

[0271] For example, the combination therapy may include an anti-VISTA antibody, fragment thereof, conjugate, and / or pharmaceutical composition in the same combination with at least one other therapeutic or immunomodulatory agent, other compound or immunotherapeutic agent, or immune stimulatory strategy described herein.

[0272] Antagonistic VISTA antibodies include anti-CTLA4 mAbs such as ipilimumab and tremelimumab; anti-PD-1 mAbs such as nivolumab (BMS-936558 / MDX-1106 / ONO-4538), AMP224, CT-011, and MK-3475, and anti-PDL-1 antagonists such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3, anti-TIM-3, anti-BTLA, anti-B7-H4, and anti-B7-H3 mAbs such as IMP-321; and anti-CD40 mAbs such as CP-870,893, lucatumumab, and dacetuzumab. It may be used in combination with agonistic antibodies targeting immune checkpoints, including mAbs; anti-CD137 mAbs such as BMS-663513, urelumab, and PF-05082566; anti-OX40 mAbs such as anti-OX40; anti-GITR mAbs such as TRX518; anti-CD27 mAbs such as CDX-1127; and anti-ICOS mAbs.

[0273] Cytokines are molecular messengers that enable immune system cells to communicate with each other to generate coordinated, robust, yet self-limited responses against target antigens. Cytokine-based therapies embody direct attempts to stimulate a patient's own immune system to reject cancer. The growing interest over the past two decades in harnessing the immune system to eradicate cancer has been accompanied by significant efforts to characterize cytokines and harness their vast signaling networks to develop cancer treatments. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site, enhancing tumor cell recognition by cytotoxic effector cells. Numerous animal tumor model studies have demonstrated that cytokines have broad antitumor activity, leading to several cytokine-based approaches to cancer therapy (Lee and Margolin 2011, Cancers 3(4):3856-93). Many cytokines are in preclinical or clinical development as agents to enhance anti-tumor immune responses for cancer immunotherapy, including IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, and IL-21, IL-23, IL-27, GM-CSF, IFNα (interferon alpha), IFNβ, and IFNγ, among others.

[0274] The antagonist anti-VISTA antibodies and pharmaceutical compositions containing them may also be administered in conjunction with other compounds or immunotherapies. For example, the combination therapy may include a compound of the present invention in combination with at least one other therapeutic or immunomodulatory agent or immune stimulatory strategy, including, but not limited to, tumor vaccines, adoptive T cell therapy, Treg depletion, chemotherapeutic agents such as antibodies (e.g., bevacizumab, erbitux), peptides, peptibodies, small molecules, cytotoxic and cytostatic agents (e.g., paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, carboplatin), immune modifiers such as interferons and interleukins, immunostimulatory antibodies, growth hormones or other cytokines, folic acid, vitamins, minerals, aromatase inhibitors, RNAi, histone deacetylase inhibitors, proteasome inhibitors, etc.

[0275] According to at least some embodiments, immune cells, preferably T cells, may be contacted in vivo or ex vivo with a therapeutic agent of interest to modulate an immune response. The T cells contacted with the therapeutic agent can be any cell that expresses a T cell receptor, including α / β and γ / δ T cell receptors. T cells include all cells that express CD3, including T cell subsets that also express CD4 and CDS. T cells include naive and memory cells, as well as effector cells such as CTLs. T cells also include cells such as Th1, Tel, Th2, Th2, Th3, Thl7, Th22, Treg, and Trl cells. T cells also include NKT cells and similar distinct classes of T cell lineages.

[0276] Use of agonistic anti-VISTA antibodies and pharmaceutical compositions containing them for the treatment of autoimmune diseases According to at least some embodiments, the anti-VISTA antibodies described herein, fragments thereof, conjugates thereof, or medicaments containing them, which function as VISTA stimulating therapeutic agents, may be used to treat immune system-related diseases.

[0277] Optionally, the immune system related condition includes for blocking or promoting immune stimulation mediated by immune related conditions, autoimmune diseases listed herein, transplant rejection, and graft-versus-host disease, and / or VISTA immune related diseases listed herein, and / or for immunotherapy (promoting or inhibiting immune stimulation).

[0278] Optionally, the immune condition is selected from an autoimmune disease, transplant rejection, an inflammatory disease, an allergic condition, or graft-versus-host disease. Optionally, the treatment is combined with another moiety useful in treating the immune-related condition.

[0279] Thus, treatment of multiple sclerosis using an agent according to at least some embodiments of the invention can be combined with any known therapeutic agent or method for treating multiple sclerosis, for example, as optionally described herein.

[0280] Thus, treatment of rheumatoid arthritis or other arthritic conditions using a subject agonist antibody can be combined with any known therapeutic agent or method for treating rheumatoid arthritis, e.g., optionally as described herein.

[0281] Thus, treatment of IBD using a subject agonist antibody can be combined with any known therapeutic agent or method for treating IBD, eg, as optionally described herein.

[0282] Thus, treatment of psoriasis using a subject agonist antibody can be combined with any known therapeutic agent or method for treating psoriasis, eg, as optionally described herein.

[0283] Thus, treatment of type 1 diabetes using a subject agonist antibody can be combined with any known therapeutic agent or method for treating type 1 diabetes, for example, as optionally described herein.

[0284] Thus, treatment of uveitis using a subject agonist antibody can be combined with any known therapeutic agent or method for treating uveitis, for example, as optionally described herein.

[0285] Thus, treatment of psoriasis using a subject agonist antibody can be combined with any known therapeutic agent or method for treating psoriasis, eg, as optionally described herein.

[0286] Thus, treatment of Sjogren's syndrome using a subject agonist antibody can be combined with any known therapeutic agent or method for treating Sjogren's syndrome, for example, as optionally described herein.

[0287] Thus, treatment of systemic lupus erythematosus using a subject agonist antibody can be combined with any known therapeutic agent or method for treating systemic lupus erythematosus, for example, as optionally described herein.

[0288] Thus, treatment of GVHD using a subject agonist antibody can be combined with any known therapeutic agent or method for treating GVHD, eg, as optionally described herein.

[0289] Thus, treatment of chronic or acute infection and / or associated hepatotoxicity, e.g., hepatitis, using a subject agonist antibody can be combined with any known therapeutic agent or method for treating chronic or acute infection and / or associated hepatotoxicity, e.g., as optionally described herein.

[0290] In the above-described therapy, preferably, a subject having one of the aforementioned or other autoimmune or inflammatory conditions is administered an immune inhibitory anti-VISTA antibody disclosed herein, or an antigen-binding fragment according to the invention, which antibody mimics or stimulates at least one VISTA-mediated effect on immunity, e.g., suppressing cytotoxic T cell or NK activity, and / or proinflammatory cytokine production involved in disease pathology, thereby preventing or ameliorating disease symptoms, potentially resulting in long-term disease remission, e.g., due to induction of Tregs, which induce T cell tolerance or long-term immunosuppression.

[0291] The therapeutic agents enumerated herein and / or pharmaceutical compositions comprising same, according to at least some embodiments of the invention, can be administered as the sole active ingredient or together with other drugs in immunomodulatory regimens, or together with other anti-inflammatory agents, for example, to treat or prevent acute or chronic rejection of allo- or xenografts, or inflammatory or autoimmune disorders, or to induce tolerance.

[0292] Use of agonistic anti-VISTA antibodies and pharmaceutical compositions containing them for the treatment of sepsis According to at least some embodiments, the VISTA antibodies, fragments thereof, conjugates, and / or pharmaceutical compositions described herein can be used to treat sepsis, a potentially life-threatening complicated infection. Sepsis represents a complex clinical syndrome that develops when the initial host response to infection is inappropriately amplified, dysregulated, and harmful to the host. The initial hyperinflammatory phase in sepsis (the "cytokine storm") is followed by a state of immunosuppression (Hotchkiss et al. 2013 Lancet Infect. Dis. 13:260-268). The later stage of this immune disorder, also referred to as "immune paralysis," manifests when the primary infection is not cleared, reactivation of viruses such as HSV and cytomegalovirus, and the development of new secondary infections, often by organisms that are not particularly virulent in immunocompetent patients. The majority of current sepsis patients ultimately survive these initial bouts of hyperinflammation only in the intensive care unit, with sepsis-induced multiorgan dysfunction over the following days to weeks. Sepsis-induced immunosuppression is increasingly recognized as the most significant immune dysfunction in these vulnerable patients. Poor pathogen clearance after primary infection and / or susceptibility to secondary infections contribute to the high morbidity and mortality associated with sepsis.

[0293] In accordance with at least some embodiments of the present invention, there is provided the use of therapeutic agents and / or pharmaceutical compositions comprising same, as well as combinations of known therapeutic agents, as enumerated herein, effective in treating sepsis.

[0294] In accordance with at least some embodiments of the present invention, there is provided the use of the therapeutic agents listed herein and / or pharmaceutical compositions comprising same, which can be combined with standard or emerging treatments for sepsis, therapies that block the cytokine storm in the early hyperinflammatory phase of sepsis, and / or therapies that have immunostimulatory effects to overcome the sepsis-induced immunosuppressive phase.

[0295] Some of the standard treatment combinations for sepsis recommended by the "International Guidelines for Management of Severe Sepsis and Septic Shock" (Dellinger et al 2013 Intensive Care Med 39:165-228) are listed below. 1. Broad-spectrum antibiotics active against all possible pathogens (bacterial and / or fungal - treatment begins when sepsis is diagnosed, but no specific pathogen is identified) - e.g., cefotaxime (Claforan®), ticarcillin and clavulanate (Timentin®), piperacillin and tazobactam (Zosyn®), imipenem and cilastatin (Primaxin®), meropenem (Merrem®), clindamycin (Cleocin), metronidazole (Flagyl®), ceftriaxone (Rocephin®), ciprofloxacin (Cipro®), cefepime (Maxipime®), levofloxacin (Levaquin®), vancomycin, or any combination of the drugs listed. 2. Vasopressors: e.g. norepinephrine, dopamine, epinephrine, vasopressin 3. Steroids: e.g., hydrocortisone, dexamethasone, or fludrocortisone; Intravenous or otherwise inotropic therapy: e.g., dobutamine for septic patients with myocardial dysfunction. 4. Recombinant human activated protein C (rhAPC) such as drotrecogin alfa (activated) (DrotAA). 5. Beta-blockers also reduce local and systemic inflammation. 6. Metabolic interventions such as pyruvate, succinate, or high-dose insulin replacement. Use of anti-VISTA antibodies and pharmaceutical compositions containing them to reduce unwanted immune activation following gene or cell therapy or transplantation

[0296] As used herein, the term "gene therapy" encompasses any type of gene therapy, vector-mediated gene therapy, gene transfer, viral-mediated gene transfer, and further encompasses certain cell therapies, such as CAR T-cell therapy and CAR NK-cell therapy. According to at least some embodiments of the present invention, the agonistic VISTA antibodies, fragments thereof, conjugates, and / or pharmaceutical compositions described herein that target VISTA and have inhibitory activity on the immune response may be used as therapeutic agents to reduce undesired immune activation following gene or cell therapy used to treat various genetic diseases. Without wishing to be limited by a single hypothesis, such antibodies have VISTA-like inhibitory activity on the immune response and / or optionally enhance VISTA immunoinhibitory activity by inhibiting pathogenic T cells and / or NK cells.

[0297] Many gene therapy products for the treatment of genetic diseases are currently undergoing clinical trials. Recent studies have described therapeutic success for several genetic diseases using gene therapy vectors. Gene therapy strategies are characterized by three key elements: the gene to be transferred, the target tissue into which the gene is introduced, and the vector (gene delivery vehicle) used to facilitate entry of the gene into the target tissue. The majority of gene therapy clinical trials utilize viral vectors as highly efficient delivery vehicles, including retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, pseudotyped viruses, and herpes simplex viruses. However, interactions between the human immune system and all components of gene therapy vectors appear to represent one of the major limitations to sustained therapeutic efficacy. Human studies have demonstrated a high potential for host immune responses to viral vectors. Such immune responses to the virus or transgene product itself, resulting in the formation of neutralizing antibodies and / or the destruction of transduced cells by cytotoxic cells, can greatly hinder therapeutic efficacy (Seregin and Amalfitano 2010 Viruses 2:2013; Mingozzi and High 2013 Blood 122:23; Masat et al 2013 Discov Med. 15:379). Therefore, developing strategies to circumvent immune responses and facilitate long-term expression of transgenic therapeutic proteins is one of the major challenges for successful clinical gene therapy.

[0298] Factors that influence the immune response to transgenic proteins encoded by viral vectors include the route of administration, vector dose, transgenic protein immunogenicity, host inflammatory status, and capsid serotype. These factors are thought to influence immunogenicity by triggering innate immunity, cytokine production, APC maturation, antigen presentation, and ultimately, the priming of naive T lymphocytes to functional effector antigens (Mingozzi and High 2013 Blood 122:23). Therefore, the idea of ​​attenuating immune activation by disrupting these very mechanisms seems logical, with the goal of inducing short-term immunosuppression, avoiding early immune priming following vector administration, and promoting long-term tolerance.

[0299] Immunomodulatory treatment by targeting two nonredundant checkpoints in the immune response at the time of vector delivery has been tested in animal models as a strategy to inhibit unwanted immune activation after gene therapy, especially after multiple injections. Studies of vector-mediated immune responses to adenoviral vectors instilled into the lungs of mice or monkeys showed that transient treatment with anti-CD40L antibodies suppressed the adenovirus-induced immune response, allowing the animals to be subsequently re-challenged with adenoviral vectors. Short treatment with this antibody resulted in long-term effects on immune function and long-term inhibition of adenovirus-specific humoral responses, well beyond the point at which the antibody effect was no longer significant, indicating the therapeutic potential of blocking this costimulatory pathway as an immunomodulatory regimen to enable the administration of gene transfer vectors (Scaria et al. 1997 Gene Ther. 4:611; Chirmule et al. 2000 J. Virol. 74:3345). Another study showed that coadministration of CTLA4-Ig and anti-CD40L Ab before and after primary vector administration reduced immune responses to the vector, prolonged long-term adenovirus-mediated gene expression, and enabled secondary adenovirus-mediated gene transfer even after the immunosuppressive effects of these agents were no longer present, suggesting that it may be possible to obtain persistent and secondary adenovirus-mediated gene transfer with transient immunosuppressive therapy (Kay et al. 1997 Proc. Natl. Acad. Sci. USA 94:4686). In another study, similar administration of CTLA4-Ig and anti-CD40L Ab prevented the formation of neutralizing Abs against the vector and enabled gene transfer expression, provided the treatment was administered during each gene transfer injection (Lorain et al. 2008 Molecular Therapy 16:541). Furthermore, administration of CTLA4-Ig to mice, even a single dose, resulted in suppression of immune responses at early time points and prolonged transgene expression (Adriouch et al 2011 Front. Microbiol. 2:199). However, CTLA4-Ig alone was not sufficient to permanently wipe out the immune response against the transgene product.Combination therapy targeting two immune checkpoints with CTLA4-Ig and PD-L1 or PDL-2 resulted in synergistic improvement of transgene tolerance at later time points, likely by targeting two non-redundant mechanisms of immune regulation, resulting in long-term transgene persistence and expression (Adriouch et al 2011 Front. Microbiol. 2:199).

[0300] In accordance with at least some embodiments of the present invention, the subject agonists may be used to overcome limitations in immune responses to gene therapy and to reduce undesired immune activation following gene therapy alone or with other active agents. Current approaches include excluding patients with antibodies to the delivery vector, administering high vector doses, using empty capsids to adsorb anti-vector antibodies to allow subsequent vector transduction, and repeated plasmapheresis cycles to adsorb immunoglobulins and reduce anti-vector antibody titers.

[0301] Novel approaches attempting to overcome these limitations can be divided into two broad categories: selective modification of the Ad vector itself and preventative host immunomodulation (Seregin and Amalfitano 2010 Viruses 2:2013). The first category includes several innovative strategies, including (1) Ad-capsid display of specific inhibitors or ligands, (2) covalent modification of the entire Ad vector capsid, (3) use of tissue-specific promoters and local administration routes, (4) use of genomically modified Ad, and (5) development of chimeric or alternative serotype Ad.

[0302] The second category of methods involves the use of immunosuppressants or specific compounds to block important immune pathways known to be induced by viral vectors. Immunosuppressants have been tested in preclinical studies and have shown efficacy in preventing or eradicating immune responses to the transfer vector and transgene product. These include general immunosuppressants such as cyclosporine A; cyclophosphamide; FK506; steroids such as glucocorticoids or dexamethasone; TLR9 blockade such as the TLR9 antagonist oligonucleotide ODN-2088; TNF-α blockade with TNF-α antibodies or TNFR-Ig antibodies; and Erk and other signaling inhibitors such as U0126. In the clinical setting, glucocorticoid administration has been successfully used to blunt T cell responses directed against the viral capsid during hepatic gene transfer of an adenovirus-associated virus (AAV) vector expressing a human factor IX transgene to patients with severe hemophilia B (Nathwani et al 2011 N. Engl. J. Med. 365:2357).

[0303] In contrast to previous approaches utilizing drugs that tend to "blanket" and nonspecifically immunosuppress the host, more selective immunosuppressive approaches are being developed. These include the use of agents that provide blockade of positive costimulatory interactions, such as those between CD40 and CD154, ICOS and ICOSL, CD28 and CD80 or CD86 (including CTLA4-Ig), NKG2D and NKG2D ligands, LFA-1 and ICAM, LFA-3 and CD2, 4-1BB and 4-1BBL, OX40 and OX40L, and GITR and GITRL, as well as agents that stimulate negative costimulatory receptors, such as CTLA-4, PD-1, BTLA, LAG-3, TIM-1, TEVI-3, and KIR, as well as B7-H4 and B7-H3 receptors. Some of these have been utilized in preclinical or clinical transplantation studies (Pilat et al. 2011 Sem. Immunol. 23:293).

[0304] In the above-mentioned gene or cell therapy or in the treatment of transplantation indications, preferably, a subject having or receiving cell or gene therapy, or transplanted tissue or organ, is administered an immune inhibitory anti-VISTA antibody disclosed herein, or an antigen-binding fragment according to the invention, which antibody enhances, stimulates, or mimics at least one VISTA-mediated effect on immunity, such as its inhibitory effect on cytotoxic T cell or NK activity, and / or its inhibitory effect on the production of proinflammatory cytokines, or its stimulatory effect on Tregs, thereby preventing or reducing host immune responses against the cells or genes used in the therapy, or unwanted immune responses against the transplanted cells, organ, or tissue. Preferably, the treatment induces prolonged immune tolerance to the transplanted or injected cells, tissue, or organ. In some cases, for example, in the case of transplant cells, tissues, or organs containing immune cells, the immunoinhibitory anti-VISTA antibodies or antigen-binding fragments disclosed herein may be contacted with the cells, tissues, or organs prior to infusion or transplantation, and / or potentially with the immune cells of the transplant recipient to tolerize the immune cells and prevent unwanted or GVHD immune responses.

[0305] Pharmaceutical Composition In another aspect, the invention provides compositions, e.g., pharmaceutical compositions, containing an anti-human VISTA antibody according to the invention and, optionally, one or a combination of another immunosuppressant or other active agent. Accordingly, pharmaceutical compositions are featured that include a therapeutically effective amount of an anti-human VISTA antibody according to at least some embodiments of the invention. In particular, the invention features pharmaceutical compositions that include a therapeutically effective [immunosuppressive] amount of at least one agonist anti-human VISTA antibody or antibody fragment according to the invention.

[0306] Pharmaceutical compositions according to at least some embodiments of the present invention (i.e., in the case of the VISTA antagonist antibodies disclosed herein) can be used for the treatment of cancer (whether non-metastatic, invasive, or metastatic), and / or liver toxicity associated with immune-related disorders, autoimmune, allergic, GVHD, inflammatory, or infectious disorders, and / or sepsis (i.e., in the case of the VISTA agonist antibodies disclosed herein). "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Thus, mammals treated herein may have been diagnosed with the disorder or may be predisposed or susceptible to the disorder. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals such as dogs, horses, cats, and cows. Preferably, the mammal is a human.

[0307] The term "therapeutically effective amount" refers to an amount of an agent according to the present invention that is effective to treat a disease or disorder in a mammal. Therapeutics of the present invention can be provided to a subject alone or as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier. In many cases, agonist or antagonist anti-VISTA antibodies according to the present invention will be used in combination with other immunotherapeutics or other therapeutic agents useful in treating a particular condition.

[0308] A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0309] Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and optionally additives such as detergents and solubilizing agents (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Non-aqueous solvents or vehicles may also be used, as detailed below.

[0310] Examples of suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Depending on the route of administration, active compounds that specifically bind to any one of the VISTA proteins or bispecific molecules, i.e., monoclonal or polyclonal antibodies, and antigen-binding fragments containing same, and conjugates and / or alternative scaffolds, may be coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound. Pharmaceutical compounds according to at least some embodiments of the present invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrogen bromide, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and non-toxic organic amines such as N,N-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0311] Pharmaceutical compositions according to at least some embodiments of the present invention may include a pharmaceutically acceptable antioxidant, examples of which include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite, (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and p-tocopherol, and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0312] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0313] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Except where any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical composition according to at least some embodiments of the present invention is contemplated.Auxiliary active compounds can also be incorporated into the composition.

[0314] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients listed above, followed by sterilization microfiltration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0315] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients, and then optionally sterilized microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from the above-listed ingredients.For the preparation of sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization) to produce a powder of active ingredient plus any additional desired ingredients from the previously sterilized filtered solution.

[0316] The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for therapeutic agents according to at least some embodiments of the present invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, intestinal, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal, and sublingual), intravesical, intravitreal, intraperitoneal, intravaginal, brain delivery (e.g., intraventricular, intracerebral, and convection-enhanced diffusion), CNS delivery (e.g., intrathecal, perispinal, and intraspinal), or parenteral (subcutaneous, intramuscular, intravenous, and intradermal), transmucosal (e.g., sublingual), administration via injection or implant, or other parenteral administration, e.g., by injection or infusion, or other delivery routes and / or forms known in the art. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In specific embodiments, proteins, therapeutic agents, or pharmaceutical compositions according to at least some embodiments of the invention may be administered intraperitoneally or intravenously.

[0317] Alternatively, VISTA specific antibodies according to the present invention may be administered parenterally, for example by topical, epidermal, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0318] Active compound can be prepared with carrier that protects compound from rapid release, for example, sustained release preparations, including implant, transdermal patch and microencapsulated delivery system.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid can be used.Many methods for preparing such preparations are patented and generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0319] Therapeutic compositions can be administered using medical devices known in the art. For example, in preferred embodiments, therapeutic compositions according to at least some embodiments of the present invention can be administered using needle-based hypodermic injection devices, such as those described in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful in the present invention include U.S. Pat. No. 4,487,603 (disclosing an implantable microinfusion pump for dispensing drugs at a controlled rate), U.S. Pat. No. 4,486,194 (disclosing a therapeutic device for administering drugs through the skin), U.S. Pat. No. 4,447,233 (disclosing a drug infusion pump for delivering drugs at a precise infusion rate), U.S. Pat. No. 4,447,224 (disclosing a variable flow rate implantable infusion device for continuous drug delivery), U.S. Pat. No. 4,439,196 (disclosing an osmotic drug delivery system with multi-chamber compartments), and U.S. Pat. No. 4,475,196 (disclosing an osmotic drug delivery system). These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0320] In certain embodiments, anti-VISTA antibodies can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can enhance targeted drug delivery by containing one or more moieties that are selectively transported to specific cells or organs (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (e.g., U.S. Patent No. 5,416,016 to Low et al.), mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134), p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 357:140; Lett. 346:123, JJ Killion and IJ Fidler (1994) Immunomethods 4:273.

[0321] In yet another embodiment, the immunoconjugates of the present invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins, immunosuppressants) to cells bearing VISTA cell surface receptors by linking such compounds to the antibodies disclosed herein. Thus, the present invention also provides methods for localizing ex vivo or in vivo cells expressing VISTA (e.g., with a detectable label such as a radioisotope, fluorescent compound, enzyme, or enzyme co-factor). Alternatively, the immunoconjugates can be used to kill cells bearing VISTA cell surface receptors by targeting cytotoxins or radiotoxins to the VISTA antigen.

[0322] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., soluble polypeptide conjugates containing the ectodomain of the VISTA antigen, antibodies, immunoconjugates, alternative scaffolds, and / or bispecific molecules, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. Pharmaceutical compounds according to at least some embodiments of the present invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxic effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrogen bromide, hydroiodic acid, phosphorous acid, etc., and non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, etc., and non-toxic organic amines, such as N,N-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0323] Pharmaceutical compositions according to at least some embodiments of the present invention may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite); (2) oil-soluble antioxidants (such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and p-tocopherol); and (3) metal chelators (such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid). Examples of suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0324] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0325] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Except where any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions according to at least some embodiments of the present invention is contemplated.Auxiliary active compounds can also be incorporated into the composition.

[0326] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients listed above, followed, if necessary, by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0327] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients as needed, and then sterilizing and microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients that are required from the above-listed ingredients.For the preparation of sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient plus any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0328] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the subject being treated and the specific mode of administration.The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of that composition that produces a therapeutic effect.Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent, when combined with a pharmaceutically acceptable carrier.

[0329] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several discrete doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active compound, together with the necessary pharmaceutically acceptable carrier, calculated to produce the desired therapeutic effect. The specifications for dosage unit forms according to at least some embodiments of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology of formulating such active compounds for the treatment of individual susceptibility.

[0330] For administration of the VISTA antibodies disclosed herein, dosages range from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, of the host's body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1 to 10 mg / kg. Exemplary treatment regimes involve administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every three months, or once per six months. Preferred dosing regimens for the antibodies disclosed herein, according to at least some embodiments of the present invention, include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, wherein the antibodies disclosed herein are given using one of the following dosing schedules: (i) once every four weeks for six doses, then once every three months; (ii) once every three weeks; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight once every three weeks.

[0331] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody is within the ranges indicated herein. The antibodies disclosed herein are typically administered multiple times. The interval between single doses can be, for example, daily, weekly, monthly, every three months, or yearly. The intervals can also be irregular, as indicated by measuring the patient's blood levels of antibody to the target antigen. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 micrograms / ml, and in some methods, about 25-300 micrograms / ml.

[0332] Alternatively, the therapeutic agent can be administered as a sustained-release formulation, in which case frequent administration is not required. The dosage and frequency depend on the half-life of the therapeutic agent in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The half-life of fusion proteins can vary widely. The dosage and frequency of administration can vary depending on whether the treatment is preventative or therapeutic. In preventative applications, relatively low doses are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete recovery from the symptoms of the disease. Thus, patients can be administered a preventative regimen.

[0333] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention or its ester, salt, or amide employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and factors well known in the medical field.

[0334] Having described the invention, the following examples are provided to further illustrate the invention and its inherent advantages. [Example]

[0335] Example 1 : Use of the assay to screen immunosuppressive anti-mouse VISTA Abs We developed various assays to screen for putative agonistic anti-murine VISTA antibodies. As shown in Figure 1, immunosuppressive anti-VISTA mAbs were identified using in vitro and in vivo screening assays. In the experiment shown in Figure 1A, purified T cells were plated on anti-CD3 in the presence of the indicated mAb for 72 hours. Proliferation was measured by H3 incorporation. In the experiment shown in Figure 1B, purified DO11.10 T cells were stimulated with ISQ-pulsed APCs in the presence of the indicated antibody for 6 days. Proliferation was measured using CTV dilution dye. In the experiment shown in Figure 1C, GVHD was induced by transferring C57BL / 6 cells into irradiated BALB / c recipients. Mice were IP injected with 200 μg of antibody on days 0, 2, and 4 after transfer, and survival was analyzed. In the experiment in Figure 1D, mice were treated with 10 mpk of the indicated antibody 3 hours before administration of ConA (15 mpk), and plasma IL-2 was analyzed at 6 by Luminex.

[0336] More specifically, in the first assay, CD4 + T cells were isolated and incubated with Ab1, Ab2, or Ab3 before being added to anti-CD3-coated plates. After 3 days of culture, T cells were pulsed with tritiated thymidine, which was incorporated by proliferating cells. Notably, both Ab1 and Ab2 induced a significant decrease in T cell proliferation rate, while Ab3 had no effect (Figure 1). In a similar assay in which transgenic T cells were instead stimulated with antigen-pulsed APCs, T cell proliferation was measured by proliferation dye dilution. Similar to the anti-CD3 assay, Ab1 suppressed antigen-specific T cell proliferation by approximately 50% (Figure 1B). These data indicate that Ab3 mAb blocks mVISTA function (i.e., enhances immune responses), while Ab1 and Ab3 stimulate mVISTA function and downregulate primary immune responses.

[0337] We also determined whether Ab3 and Ab1 could be distinguished using in vivo animal models, specifically GVHD and ConA hepatitis models. Mice with GVHD treated with a control antibody (Ham Ig) had progressive disease and, as expected, had to be euthanized by 4 weeks after transplantation (Figure 1C). Ab3-treated mice were also prone to GVHD; indeed, most mice died before the control-treated group, indicating that Ab3 may exacerbate the disease. Conversely, none of the Ab1-treated mice showed overt symptoms of GVHD, and nearly all remained healthy for at least 40 days. Specifically, in these experiments, mice with GVHD treated with a control antibody (Ham Ig) had progressive disease and, as expected, had to be euthanized by 4 weeks after transplantation (Figure 1C). Ab3-treated mice were also prone to GVHD; indeed, most mice died before the control-treated group, indicating that Ab3 may exacerbate the disease. Conversely, none of the Ab1-treated mice showed overt symptoms of GVHD, and nearly all remained healthy for at least 40 days.

[0338] In the ConA model, we tested whether each VISTA antibody affected the well-characterized T cell cytokine response to ConA. Notably, Ab1 induced a decrease in plasma cytokine levels of IL-2, but Ab3 did not (Figure 1D). Specifically, in the ConA model, we tested whether each VISTA antibody affected the well-characterized T cell cytokine response to ConA. Notably, Ab1 induced a decrease in plasma cytokine levels of IL-2, but Ab3 did not (Figure 1D).

[0339] Thus, these results demonstrate that both anti-VISTA mAbs (Ab1 and Ab2) are immunosuppressive and can be distinguished from the inflammatory immunosuppressive anti-mouse VISTA antibody (Ab3). As shown in Figure 1, Ab1 is effective (immunosuppressive) in multiple inflammatory models, including GVHD, NZB / W F1 lupus-like glomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagen antibody-induced arthritis (CAIA), and imiquimod-induced psoriasis. In each of these diseases, administration of Ab1 during disease progression significantly reduced pathology and / or mortality. Each listed model has unique requirements for T cells for disease progression. Both GVHD and ConA are driven by Th1 T cell responses.

[0340] Example 2 : Identification of anti-VISTA antibodies that suppress autoimmunity in different autoimmune disease models In the experiments shown in Figures 2A-F, the effects of different anti-mouse VISTA Abs were again compared in different disease models. In the experiment shown in Figure 2A, NZB / W F1 mice were treated with either Ab1 or Ham Ig (200 μg) 3x / week starting at week 25 and continuing until the end of the experiment. "X" indicates the time point at which all mice in the control-treated group were sacrificed. In the experiment shown in Figure 2B, mice were treated with 200 μg of antibody 3 hours before administration of 15 mg / kg (mpk) ConA, and survival was followed for 80 hours. In the experiment shown in Figure 2C, mice were treated sequentially with collagen II mAb followed by LPS, and arthritis was measured by measuring paw swelling. In the experiment shown in Figure 2D, Ab1 and Ham Ig were administered 3x / day (200 μg). Imiquimod was applied daily to the ears of mice. On day 14, Ab1 or Ham-Ig (200 μg) was administered daily, and ear thickness was measured with a caliper. In the same experiment shown in Figure 2E-F, imiquimod was applied daily to the backs of mice. On day 9, mice were euthanized, and the skin was sectioned and stained for CD3 expression by IHC.

[0341] As shown in Figure 2A-F, in each of these experiments, administration of Ab1 during the progression of the specific disease significantly reduced pathology and / or mortality. Each listed model has unique requirements for T cells for disease progression. Both GVHD and ConA are driven by Th1 T cell responses.

[0342] Imiquimod-induced psoriasis is an IL-17 / 23-driven disease in which T cells are recruited to the dermal layer of the skin. Ab1 binds to CD3 + dramatically reduced the number of cells (Fig. 2E and F) but had no effect on splenic T cell populations (data not shown), indicating that this anti-mouse VISTA Ab preferentially suppressed immunity in inflammatory lesions.

[0343] NZB / W F1 lupus is a multifactorial disease with contributions from B cells, T cells, and myeloid cells. In this model, therapeutic administration of Ab1 reduced proteinuria levels, indicating reduced kidney damage. Finally, CAIA does not involve adaptive immunity but is instead driven by macrophages and granulocytes. Suppression by anti-VISTA in this model indicates that antibodies can also affect the myeloid compartment. Thus, the inhibitory VISTA mAb appears to mediate effects on both the T cell and innate immune compartments.

[0344] Thus, as shown in Figures 1 and 2, both monoclonal hamster anti-mouse VISTA Abs, Ab1 and AB2, induced a significant decrease in T cell proliferation rates, whereas Ab3 had no effect (Figure 1). In a similar assay in which transgenic T cells were stimulated with antigen-pulsed APCs, T cell activation was measured by proliferation dye dilution. Similar to the anti-CD3 assay, Ab1 suppressed antigen-specific T cell proliferation by approximately 50% (Figure 1B). These data suggest that Ab1 and Ab2 stimulate VISTA function, thereby downregulating primary immune responses.

[0345] In particular, Ab1, a hamster anti-mouse VISTA antibody, was effective in multiple inflammatory models, including GVHD, NZB / W F1 lupus-like glomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagen antibody-induced arthritis (CAIA), and imiquimod-induced psoriasis (Figures 1 and 2). In each of these diseases, administration of Ab1 during disease progression significantly reduced pathology and / or mortality. Each listed model has unique requirements for T cells for disease progression. Both GVHD and ConA are driven by Th1 T cell responses. As mentioned above, imiquimod-induced psoriasis is an IL-17 / 23-driven disease in which T cells are recruited to the dermal layer of the skin. Therefore, Ab1-mediated suppression in this particular autoimmune model indicates that this antibody may also affect the myeloid compartment. Thus, these immunosuppressive anti-mouse VISTA mAbs appear to mediate effects on both the T cell and innate immune compartments.

[0346] Example 3 : Development of human VISTA knock-in mice for use in screening agonistic anti-human VISTA Abs The previous examples relate to the isolation and characterization of agonistic anti-mouse VISTA Abs. To date, no agonistic anti-human VISTA Abs have been reported in the literature, despite the fact that numerous antagonistic anti-human VISTA antibodies have been identified by the present assignee and other groups. Thus, prior to the present invention, it was unknown whether an agonistic anti-human VISTA antibody would ever be identified.

[0347] Such an antibody would be highly beneficial, as there are currently no approved human therapeutics that exploit the natural function of NCRs to suppress immune responses. While Orencia (CTLA4-Ig) is effective, it acts only by blocking the CD28-B7 interaction and pathway, not by stimulating downregulatory pathways. Engaging this pathway could prove revolutionary in the management of different human autoimmune diseases, as illustrated by the potent immunosuppressive effects of two different agonistic anti-VISTA mAbs shown in the Examples that follow. Furthermore, the immunosuppressive impact of anti-VISTA on both adaptive and innate autoimmune effector mechanisms distinguishes it from many other anti-inflammatory agents.

[0348] In the foregoing, it was assumed that a desirable and necessary reagent for screening agonistic anti-human VISTA Abs would be human VISTA knock-in mice. Human VISTA knock-in mice have ...

Claims

1. An isolated antibody or antibody fragment thereof, comprising an antigen-binding region that specifically binds to human IgV domain suppressor of T cell activation (human VISTA), wherein the antibody or antibody fragment stimulates or enhances one or more of the immune effects of VISTA.

2. The isolated antibody of claim 1 , comprising a human IgG2 constant region or a human IgG2 Fc region.

3. 3. The isolated antibody of claim 2, wherein the human IgG2 constant region or the Fc region of human IgG2 binds to an Fc gamma receptor, including human CD32A.

4. 3. The isolated antibody of claim 2, wherein the IgG2 constant region or IgG2 Fc region comprises native human IgG2 bound to an Fc gamma receptor.

5. 5. The isolated antibody of claim 4, wherein the FcyR comprises one or more of hFcγRI (CD64), FcyRIIA or hFcyRIIB (CD32 or CD32A), and FcγRlllA (CD16A) or FcγRlllB (CD16B).

6. 6. An isolated antibody or antibody fragment according to any one of claims 1 to 5, which competes with or binds to a VISTA epitope that includes or overlaps with an epitope bound by any of the anti-human VISTA antibodies having the sequence of Figure 4.

7. 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, which binds to or interacts with one or more residues of an epitope comprising residues LLDSGLYCCLVVEIRHHHSEHRVH.

8. ７９ EVQTCSERRPIR ９０ , ４８ NVTLTCRLLGPV ６０ , １５３ HHHSEHRVHGAM １６４ , ５２ LTCRLLGPV ６０ , ５６ LLGPVDKGHDVTFYK ７０ , １１３ LAQRHGLESASDHHG １２７ , １５３ HHHSEHRVHGAM １６４ , ９３ TFQDLHLHHGGHQAA １０７ , １４６ CLVVEIRHHSEH158, ５３ TCRLLGPVDKG ６３ , １２３ SDHHG １２７ and / or １５３ HHHSEHRVHGAM １６４ 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, which binds to or interacts with one or more residues of an epitope that comprises one or more residues of.

9. ７９ EVQTCSERRPIR ９０ 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, which binds to or interacts with one or more residues of an epitope that comprises one or more residues of.

10. The agonistic anti-human VISTA antibody or antibody fragment of any one of claims 1 to 9, which promotes or enhances at least one effect of human VISTA on immunity, such as induction of T cell immunity, activation of monocytes, T cell proliferation; induction or suppression of cytokine expression, increased monocyte survival, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing VISTA, and induction of antibody-dependent cellular phagocytosis (ADCP) in cells expressing VISTA.

11. 1. An isolated agonistic or antagonistic anti-human VISTA antibody or antibody fragment thereof, comprising an antigen-binding region which specifically binds to human VISTA, said antibody or antibody fragment comprising a variable heavy sequence and a variable light sequence having the same CDR polypeptide as any one of said anti-human VISTA antibodies having CDRs and variable heavy and variable light peptides as shown in FIG. 4, with the proviso that in the case of an antagonist anti-human VISTA antibody or antibody fragment, said antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

12. 12. The isolated antibody or antibody fragment of claim 11, which comprises the same CDRs as an antibody selected from VSTB49-VSTB116, with the proviso that, in the case of an antagonist anti-human VISTA antibody or fragment, the antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

13. 13. The isolated antibody or antibody fragment of claim 11 or 12, comprising a variable heavy and / or variable light polypeptide having at least 90% sequence identity to a sequence of an anti-human VISTA antibody selected from any one of VSTB49-VSTB116, the variable heavy and variable light polypeptide sequences of which are shown in FIG. 4, with the proviso that in the case of said antibody or fragment comprising an antagonist anti-human VISTA antibody or fragment, said antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

14. 13. The isolated antibody or antibody fragment of claim 11 or 12, comprising a variable heavy and / or variable light polypeptide having at least 95% sequence identity to a sequence of an anti-human VISTA antibody selected from any one of VSTB49-VSTB116, the variable heavy and variable light polypeptide sequences of which are shown in Figure 4, with the proviso that in the case of said antibody or fragment comprising an antagonist anti-human VISTA antibody or antibody fragment, said antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

15. 13. The isolated antibody or antibody fragment of claim 11 or 12, comprising a variable heavy and / or variable light polypeptide having at least 96-99% sequence identity to a sequence of an anti-human VISTA antibody selected from any one of VSTB49-VSTB116, the variable heavy and variable light polypeptide sequences of which are shown in Figure 4, with the proviso that in the case of said antibody or fragment comprising an antagonist anti-human VISTA antibody or antibody fragment, said antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

16. 13. The isolated antibody or antibody fragment of claim 11 or 12, comprising a variable heavy and / or variable light polypeptide that is identical to the sequence of an anti-human VISTA antibody selected from one of VSTB49-VSTB116, the variable heavy and variable light polypeptide sequences of which are shown in Figure 4, with the proviso that in the case of said antibody or fragment comprising an antagonist anti-human VISTA antibody or antibody fragment, said antibody or antibody fragment does not comprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, or VSTB60.

17. 17. The isolated antibody or antibody fragment of any one of claims 1 to 16, which antagonizes or blocks at least one immune effect of human VISTA.

18. 18. The isolated antibody or antibody fragment of any one of claims 1 to 17, which stimulates or blocks at least one immune effect of human VISTA.

19. 19. The isolated antibody or antibody fragment of any one of claims 1 to 18, comprising a human constant domain.

20. 20. The isolated antibody or antibody fragment of any one of claims 1 to 19, comprising a human constant domain selected from IgG1, IgG2, IgG3, and IgG4, optionally modified by deletion, substitution, or addition mutations, or any combination thereof.

21. The antibody fragment is Fab, F(ab') ２ 21. The antibody or antibody fragment of any one of claims 1 to 20, comprising or being an scFv antibody fragment.

22. 22. The antagonistic anti-human VISTA antibody or antibody fragment of any one of claims 1 to 21, which blocks or inhibits at least one of the immune effects of human VISTA selected from the inhibitory effects of human VISTA on T cell immunity, monocyte activation, or T cell proliferation; induction or inhibition of cytokine expression, increasing monocyte survival, inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) of cell-expressed VISTA; and inhibition of antibody-dependent cellular phagocytosis (ADCP) of cell-expressed VISTA.

23. The agonistic anti-human VISTA antibody or antibody fragment of any one of claims 1 to 22, which promotes or enhances at least one of the immune effects of human VISTA, for example selected from the following: inhibitory effects of human VISTA on T cell immunity, monocyte activation, inhibition of T cell proliferation; induction or inhibition of cytokine expression, increased monocyte survival in cells expressing VISTA, inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) and inhibition of antibody-dependent cellular phagocytosis (ADCP) in cells expressing VISTA.

24. 24. The agonist anti-human VISTA antibody or antibody fragment of claim 23, comprising a human IgG2 constant region or a human IgG2 Fc region.

25. The agonist anti-human VISTA antibody or antibody fragment of claim 23 or 24, which promotes or enhances the inhibitory effects of human VISTA on immunity, such as the effects of human VISTA on any one or more of T cell immunity, monocyte activation, T cell proliferation; cytokine expression, monocyte survival, antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing VISTA; and antibody-dependent cellular phagocytosis (ADCP) in cells expressing VISTA.

26. The agonist anti-human VISTA antibody or antibody fragment of any one of claims 23 to 25, which inhibits T cell immunity and / or pro-inflammatory cytokine expression.

27. 27. The agonistic antibody or antibody fragment of any one of claims 1 to 26, which is a human antibody, a humanized antibody, or a chimeric antibody comprising a human Fc region, such as human IgG1, human IgG2, human IgG3, and human IgG4, or a chimera of any of them.

28. 28. The agonist antibody of any one of claims 1 to 27, which is a chimeric agonist antibody, a human agonist antibody, or a humanized agonist antibody.

29. The antibody of any one of claims 1 to 28, comprising a potentially mutated human IgG2 constant domain or a human IgG2 Fc region.

30. and optionally the entire hinge or substantially the entire hinge and CH1 domain, and optionally the entire light chain constant region or substantially the entire light chain constant region of the antibody, comprising a human IgG2 constant domain or fragment thereof, or hIgG1, hIgG3, hIgG4, IgA, IgD, IgE, or IgM, Ｈ１ 30. The agonist antibody of any one of claims 1 to 29, which is substituted with a domain ("H2 region" or "H2 domain").

31. (i) an IgG2 Fc region, in which either or both of the heavy chain cysteine ​​residue at position 127 and the light chain cysteine ​​residue at position 214 (numbering according to Kabat) have been deleted or altered to a different amino acid residue, resulting in increased agonistic properties of the resulting altered antibody compared to an antibody in which those residues are not altered; (ii) the cysteine ​​residue at position 214 of the H2 region of the antibody has been mutated or replaced with another amino acid, and / or 31. The agonistic antibody of any one of claims 1 to 30, wherein one or more of the cysteine ​​residues at positions 127, 232, or 233 have been deleted or substituted with another amino acid, (iii) comprises a human IgG2 constant domain in which at least one cysteine ​​residue has been deleted or changed to another amino acid, and (iv) competes with or binds to the same epitope on human VISTA as VSTB95 (the variable heavy and variable light sequences shown in Figure 4).

32. (i) V of SEQ ID NOs: 100, 101, and 102 Ｈ CDRs and V of SEQ ID NOs: 103, 104, and 105 Ｌ comprising CDRs, (ii) V of SEQ ID NOs: 110, 111, and 112 Ｈ CDRs and V of SEQ ID NOs: 113, 114, and 115 Ｌ comprising CDRs, (iii) V of SEQ ID NOs: 120, 121, and 122 Ｈ CDRs and V of SEQ ID NOs: 123, 124, and 125 Ｌ comprising CDRs, (iv) V of SEQ ID NOs: 130, 131, and 132 Ｈ CDRs and V of SEQ ID NOs: 133, 134, and 135 Ｌ comprising CDRs, (v) V of SEQ ID NOs: 140, 141, and 142 Ｈ CDRs and V of SEQ ID NOs: 143, 144, and 145 Ｌ comprising CDRs, (vi) V of SEQ ID NOs: 150, 151, and 152 Ｈ CDRs and V of SEQ ID NOs: 153, 154, and 155 Ｌ comprising CDRs, (vii) V of SEQ ID NOs: 160, 161, and 162 Ｈ CDRs and V of SEQ ID NOs: 163, 164, and 165 Ｌ comprising CDRs, (viii) V of SEQ ID NOs: 170, 171, and 172 Ｈ CDRs and V of SEQ ID NOs: 173, 174, and 175 Ｌ comprising CDRs, (ix) V of SEQ ID NOs: 180, 181, and 182 Ｈ CDRs and V of SEQ ID NOs: 183, 184, and 185 Ｌ comprising CDRs, (x) V of SEQ ID NOs: 190, 191, and 192 Ｈ CDRs and V of SEQ ID NOs: 193, 194, and 195 Ｌ comprising CDRs, (xi) V of SEQ ID NOs: 200, 201, and 202 Ｈ CDRs and V of SEQ ID NOs: 203, 204, and 205 Ｌ comprising CDRs, (xii) V of SEQ ID NOs: 210, 211, and 212 Ｈ CDRs and V of SEQ ID NOs: 213, 214, and 215 Ｌ comprising CDRs, (xiii) V of SEQ ID NOs: 220, 221, and 222 Ｈ CDRs and V of SEQ ID NOs: 223, 224, and 225 Ｌ comprising CDRs, (xiv) V of SEQ ID NOs: 230, 231, and 232 Ｈ CDRs and V of SEQ ID NOs: 233, 234, and 235 Ｌ comprising CDRs, (xv) V of SEQ ID NOs: 240, 241, and 242 Ｈ CDRs and V of SEQ ID NOs: 243, 244, and 245 Ｌ comprising CDRs, (xvi) V of SEQ ID NOs: 250, 251, and 252 Ｈ CDRs and V of SEQ ID NOs: 253, 254, and 255 Ｌ comprising CDRs, (xvii) the VHCDRs of SEQ ID NOs: 260, 261, and 262, and the VHCDRs of SEQ ID NOs: 263, 264, and 265 Ｌ comprising CDRs, (xviii) V of SEQ ID NOs: 270, 271, and 272 Ｈ CDRs and V of SEQ ID NOs: 273, 274, and 275 Ｌ comprising CDRs, (xix) V of SEQ ID NOs: 280, 281, and 282 Ｈ CDRs and V of SEQ ID NOs: 283, 284, and 285 Ｌ comprising CDRs, (xx) V of SEQ ID NOs: 290, 291, and 292 Ｈ CDRs and V of SEQ ID NOs: 293, 294, and 295 Ｌ comprising CDRs, (xxi) V of SEQ ID NOs: 300, 301, and 302 Ｈ CDRs and V of SEQ ID NOs: 303, 304, and 305 Ｌ comprising CDRs, (xxii) V of SEQ ID NOs: 310, 311, and 312 Ｈ CDRs and V of SEQ ID NOs: 313, 314, and 315 Ｌ comprising CDRs, (xxiii) V of SEQ ID NOs: 320, 321, and 322 Ｈ CDRs and V of SEQ ID NOs: 323, 324, and 325 Ｌ comprising CDRs, (xxiv) V of SEQ ID NOs: 330, 331, and 332 Ｈ CDRs and V of SEQ ID NOs: 333, 334, and 335 Ｌ comprising CDRs, (xxv) V of SEQ ID NOs: 340, 341, and 342 Ｈ CDRs and V of SEQ ID NOs: 343, 344, and 345 Ｌ comprising CDRs, (xxvi) V of SEQ ID NOs: 350, 351, and 352 Ｈ CDRs and V of SEQ ID NOs: 353, 354, and 355 Ｌ comprising CDRs, (xxvii) V of SEQ ID NOs: 360, 361, and 362 Ｈ CDRs and V of SEQ ID NOs: 363, 364, and 365 Ｌ comprising CDRs, (xxviii) V of SEQ ID NOs: 370, 371, and 372 Ｈ CDRs and V of SEQ ID NOs: 373, 374, and 375 Ｌ comprising CDRs, (xxix) V of SEQ ID NOs: 380, 381, and 382 Ｈ CDRs and V of SEQ ID NOs: 383, 384, and 385 Ｌ comprising CDRs, (xxx) V of SEQ ID NOs: 390, 391, and 392 Ｈ CDRs and V of SEQ ID NOs: 393, 394, and 395 Ｌ comprising CDRs, (xxxi) V of SEQ ID NOs: 400, 401, and 402 Ｈ CDRs and V of SEQ ID NOs: 403, 404, and 405 Ｌ comprising CDRs, (xxxii) V of SEQ ID NOs: 410, 411, and 412 Ｈ CDRs and V of SEQ ID NOs: 413, 414, and 415 Ｌ comprising CDRs, (xxxiii) V of SEQ ID NOs: 420, 421, and 422 Ｈ CDRs and V of SEQ ID NOs: 423, 424, and 425 Ｌ comprising CDRs, (xxxiv) V of SEQ ID NOs: 430, 431, and 432 Ｈ CDRs and V of SEQ ID NOs: 433, 434, and 435 Ｌ comprising CDRs, (xxxv) V of SEQ ID NOs: 440, 441, and 442 Ｈ CDRs and V of SEQ ID NOs: 443, 444, and 445 Ｌ comprising CDRs, (xxxvi) V of SEQ ID NOs: 450, 451, and 452 Ｈ CDRs and V of SEQ ID NOs: 453, 454, and 455 Ｌ comprising CDRs, (xxxvii) V of SEQ ID NOs: 460, 461, and 462 Ｈ CDRs and V of SEQ ID NOs: 463, 464, and 465 Ｌ comprising CDRs, (xxxviii) V of SEQ ID NOs: 470, 471, and 472 Ｈ CDRs and V of SEQ ID NOs: 473, 474, and 475 Ｌ comprising CDRs, (xxxix) V of SEQ ID NOs: 480, 481, and 482 Ｈ CDRs and V of SEQ ID NOs: 483, 484, and 485 Ｌ comprising CDRs, (xl) V of SEQ ID NOs: 490, 491, and 492 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 493, 494, and 495, (xli) V of SEQ ID NOs: 500, 501, and 502 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 503, 504, and 505, (xlii) V of SEQ ID NOs: 510, 511, and 512 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 513, 514, and 515, (xliii) V of SEQ ID NOs: 520, 521, and 522 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 523, 524, and 525, (xliv) V of SEQ ID NOs: 530, 531, and 532 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 533, 534, and 535, (xlv) V of SEQ ID NOs: 540, 541, and 542 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 543, 544, and 545, (xlvi) V of SEQ ID NOs: 550, 551, and 552 Ｈ CDRs and the VLCDR polypeptides of SEQ ID NOs: 553, 554, and 555, (xlvii) V of SEQ ID NOs: 560, 561, and 562 Ｈ CDRs and V of SEQ ID NOs: 563, 564, and 565 Ｌ comprising CDRs, (xlviii) V of SEQ ID NOs: 570, 571, and 572 Ｈ CDRs and V of SEQ ID NOs: 573, 574, and 575 Ｌ comprising CDRs, (xlix) V of SEQ ID NOs: 580, 581, and 582 Ｈ CDRs and V of SEQ ID NOs: 583, 584, and 585 Ｌ comprising CDRs, (l) V of SEQ ID NOs: 590, 591, and 592 Ｈ CDRs and V of SEQ ID NOs: 593, 594, and 595 Ｌ comprising CDRs, (li) V of SEQ ID NOs: 600, 601, and 602 Ｈ CDRs and V of SEQ ID NOs: 603, 604, and 605 Ｌ comprising CDRs, (lii) V of SEQ ID NOs: 610, 611, and 612 Ｈ CDRs and V of SEQ ID NOs: 613, 614, and 615 Ｌ comprising CDRs, (liii) V of SEQ ID NOs: 620, 621, and 622 Ｈ CDRs and V of SEQ ID NOs: 623, 624, and 625 Ｌ comprising CDRs, (liv) V of SEQ ID NOs: 630, 631, and 632 Ｈ CDRs and V of SEQ ID NOs: 633, 634, and 635 Ｌ comprising CDRs, (lv) V of SEQ ID NOs: 640, 641, and 642 Ｈ CDRs and V of SEQ ID NOs: 643, 644, and 645 Ｌ comprising CDRs, (lvi) V of SEQ ID NOs: 650, 651, and 652 Ｈ CDRs and V of SEQ ID NOs: 653, 654, and 655 Ｌ comprising CDRs, (lvii) V of SEQ ID NOs: 660, 661, and 662 Ｈ CDRs and V of SEQ ID NOs: 663, 664, and 665 Ｌ comprising CDRs, (lviii) V of SEQ ID NOs: 670, 671, and 672 Ｈ CDRs and V of SEQ ID NOs: 673, 674, and 675 Ｌ comprising CDRs, (lix) V of SEQ ID NOs: 680, 681, and 682 Ｈ CDRs and V of SEQ ID NOs: 683, 684, and 685 Ｌ comprising CDRs, (lx) V of SEQ ID NOs: 690, 691, and 692 Ｈ CDRs and V of SEQ ID NOs: 693, 694, and 695 Ｌ comprising CDRs, (lxi) V of SEQ ID NOs: 700, 701, and 702 Ｈ CDRs and V of SEQ ID NOs: 703, 704, and 705 Ｌ comprising CDRs, (lxii) V of SEQ ID NOs: 710, 711, and 712 Ｈ CDRs and V of SEQ ID NOs: 713, 714, and 715 Ｌ comprising CDRs, (lxiii) V of SEQ ID NOs: 720, 721, and 722 Ｈ CDRs and V of SEQ ID NOs: 723, 724, and 725 Ｌ comprising CDRs, (lxiv) V of SEQ ID NOs: 730, 731, and 732 Ｈ CDRs and V of SEQ ID NOs: 733, 734, and 735 Ｌ comprising CDRs, (lxv) V of SEQ ID NOs: 740, 741, and 742 Ｈ CDRs and V of SEQ ID NOs: 743, 744, and 745 Ｌ comprising CDRs, (lxvi) V of SEQ ID NOs: 750, 751, and 752 Ｈ CDRs and V of SEQ ID NOs: 753, 754, and 755 Ｌ comprising CDRs, (lxvii) V of SEQ ID NOs: 760, 761, and 762 Ｈ CDRs and V of SEQ ID NOs: 763, 764, and 765 Ｌ comprising CDRs, (lxviii) V of SEQ ID NOs: 770, 771, and 772 Ｈ CDRs and V of SEQ ID NOs: 773, 774, and 775 Ｌ comprising CDRs, (lxix) V of SEQ ID NOs: 780, 781, and 782 Ｈ CDRs and V of SEQ ID NOs: 783, 784, and 785 Ｌ comprising CDRs, (lxx) V of SEQ ID NOs: 790, 791, and 792 Ｈ CDRs and V of SEQ ID NOs: 793, 794, and 795 Ｌ comprising CDRs, (lxxi) V of SEQ ID NOs: 800, 801, and 802 Ｈ CDRs and V of SEQ ID NOs: 803, 804, and 805 Ｌ comprising CDRs, (lxxii) V of SEQ ID NOs: 810, 811, and 812 Ｈ CDRs and V of SEQ ID NOs: 813, 814, and 815 Ｌ 32. The antibody or antibody fragment of any one of claims 1 to 31, comprising the CDRs.

33. (i) V of SEQ ID NO: 106 Ｈ Polypeptide and V of SEQ ID NO: 108 Ｌ comprising a polypeptide, (ii) V of SEQ ID NO: 116 Ｈ Polypeptide and V of SEQ ID NO: 118 Ｌ comprising a polypeptide, (iii) V of SEQ ID NO: 126 Ｈ Polypeptide and V of SEQ ID NO: 128 Ｌ comprising a polypeptide, (iv) V of SEQ ID NO: 136 Ｈ Polypeptide and V of SEQ ID NO: 138 Ｌ comprising a polypeptide, (v) V of SEQ ID NO: 146 Ｈ Polypeptide and V of SEQ ID NO: 148 Ｌ comprising a polypeptide, (vi) V of SEQ ID NO: 156 Ｈ Polypeptide and V of SEQ ID NO: 158 Ｌ comprising a polypeptide, (vii) V of SEQ ID NO: 166 Ｈ Polypeptide and V of SEQ ID NO: 168 Ｌ comprising a polypeptide, (viii) V of SEQ ID NO: 176 Ｈ Polypeptide and V of SEQ ID NO: 178 Ｌ comprising a polypeptide, (ix) V of SEQ ID NO: 186 Ｈ Polypeptide and V of SEQ ID NO: 188 Ｌ comprising a polypeptide, (x) V of SEQ ID NO: 196 Ｈ Polypeptide and V of SEQ ID NO: 198 Ｌ comprising a polypeptide, (xi) V of SEQ ID NO: 206 Ｈ Polypeptide and V of SEQ ID NO:208 Ｌ comprising a polypeptide, (xii) V of SEQ ID NO: 216 Ｈ Polypeptide and V of SEQ ID NO:218 Ｌ comprising a polypeptide, (xiii) V of SEQ ID NO: 226 Ｈ Polypeptide and V of SEQ ID NO:228 Ｌ comprising a polypeptide, (xiv) V of SEQ ID NO: 236 Ｈ Polypeptide and V of SEQ ID NO:238 Ｌ comprising a polypeptide, (xv) V of SEQ ID NO: 246 Ｈ Polypeptide and V of SEQ ID NO:248 Ｌ comprising a polypeptide, (xvi) V of SEQ ID NO: 256 Ｈ Polypeptide and V of SEQ ID NO:258 Ｌ comprising a polypeptide, (xvii) V of SEQ ID NO: 266 Ｈ Polypeptide and V of SEQ ID NO:268 Ｌ comprising a polypeptide, (xviii) V of SEQ ID NO: 276 Ｈ polypeptide and a VL polypeptide of SEQ ID NO: 278, (xix) V of SEQ ID NO: 286 Ｈ Polypeptide and V of SEQ ID NO:288 Ｌ comprising a polypeptide, (xx) V of SEQ ID NO: 296 Ｈ Polypeptide and V of SEQ ID NO:298 Ｌ comprising a polypeptide, (xxi) V of SEQ ID NO: 306 Ｈ Polypeptide and V of SEQ ID NO:308 Ｌ comprising a polypeptide, (xxii) V of SEQ ID NO: 316 Ｈ Polypeptide and V of SEQ ID NO:318 Ｌ comprising a polypeptide, (xxiii) V of SEQ ID NO: 326 Ｈ Polypeptide and V of SEQ ID NO:328 Ｌ comprising a polypeptide, (xxiv) V of SEQ ID NO: 336 Ｈ Polypeptide and V of SEQ ID NO:338 Ｌ comprising a polypeptide, (xxv) V of SEQ ID NO: 346 Ｈ Polypeptide and V of SEQ ID NO:348 Ｌ comprising a polypeptide, (xxvi) V of SEQ ID NO: 356 Ｈ Polypeptide and V of SEQ ID NO:358 Ｌ comprising a polypeptide, (xxvii) V of SEQ ID NO: 366 Ｈ Polypeptide and V of SEQ ID NO:368 Ｌ comprising a polypeptide, (xxviii) V of SEQ ID NO: 376 Ｈ Polypeptides and V of SEQ ID NO:378 Ｌ comprising a polypeptide, (xxix) V of SEQ ID NO: 386 Ｈ Polypeptide and V of SEQ ID NO:388 Ｌ comprising a polypeptide, (xxx) V of SEQ ID NO: 396 Ｈ Polypeptide and V of SEQ ID NO:398 Ｌ comprising a polypeptide, (xxxi) V of SEQ ID NO: 406 Ｈ Polypeptide and V of SEQ ID NO:408 Ｌ comprising a polypeptide, (xxxii) V of SEQ ID NO: 416 Ｈ Polypeptide and V of SEQ ID NO:418 Ｌ comprising a polypeptide, (xxxiii) V of SEQ ID NO: 426 Ｈ Polypeptide and V of SEQ ID NO:428 Ｌ comprising a polypeptide, (xxxiv) V of SEQ ID NO: 436 Ｈ Polypeptide and V of SEQ ID NO:438 Ｌ comprising a polypeptide, (xxxv) V of SEQ ID NO: 446 Ｈ Polypeptide and V of SEQ ID NO:448 Ｌ comprising a polypeptide, (xxxvi) V of SEQ ID NO: 456 Ｈ Polypeptide and V of SEQ ID NO:458 Ｌ comprising a polypeptide, (xxxvii) V of SEQ ID NO: 466 Ｈ Polypeptide and V of SEQ ID NO:468 Ｌ comprising a polypeptide, (xxxviii) V of SEQ ID NO: 476 Ｈ Polypeptide and V of SEQ ID NO:478 Ｌ comprising a polypeptide, (xxxix) V of SEQ ID NO: 486 Ｈ Polypeptide and V of SEQ ID NO:488 Ｌ comprising a polypeptide, (xl) V of SEQ ID NO: 496 Ｈ Polypeptide and V of SEQ ID NO:498 Ｌ comprising a polypeptide, (xli) V of SEQ ID NO:506 Ｈ Polypeptide and V of SEQ ID NO:508 Ｌ comprising a polypeptide, (xlii) V of SEQ ID NO:516 Ｈ Polypeptide and V of SEQ ID NO:518 Ｌ comprising a polypeptide, (xliii) V of SEQ ID NO: 526 Ｈ Polypeptide and V of SEQ ID NO:528 Ｌ comprising a polypeptide, (xliv) V of SEQ ID NO:536 Ｈ Polypeptides and V of SEQ ID NOs: 533, 534, and 535 Ｌ comprising a polypeptide, (xlv) V of SEQ ID NO:546 Ｈ Polypeptide and V of SEQ ID NO:548 Ｌ comprising a polypeptide, (xlvi) V of SEQ ID NO:556 Ｈ Polypeptide and V of SEQ ID NO:558 Ｌ comprising a polypeptide, (xlvii) V of SEQ ID NO: 566 Ｈ Polypeptide and V of SEQ ID NO:568 Ｌ comprising a polypeptide, (xlviii) V of SEQ ID NO: 576 Ｈ Polypeptide and V of SEQ ID NO:578 Ｌ comprising a polypeptide, (xlix) V of SEQ ID NO:586 Ｈ Polypeptide and V of SEQ ID NO:588 Ｌ comprising a polypeptide, (l) V of SEQ ID NO: 596 Ｈ Polypeptide and V of SEQ ID NO:598 Ｌ comprising a polypeptide, (li) V of SEQ ID NO: 606 Ｈ Polypeptide and V of SEQ ID NO:608 Ｌ comprising a polypeptide, (lii) V of SEQ ID NO: 616 Ｈ Polypeptide and V of SEQ ID NO:618 Ｌ comprising a polypeptide, (liii) V of SEQ ID NO: 626 Ｈ Polypeptide and V of SEQ ID NO:628 Ｌ comprising a polypeptide, (liv) V of SEQ ID NO: 636 Ｈ Polypeptide and V of SEQ ID NO:638 Ｌ comprising a polypeptide, (lv) V of SEQ ID NO: 646 Ｈ Polypeptide and V of SEQ ID NO:648 Ｌ comprising a polypeptide, (lvi) V of SEQ ID NO: 656 Ｈ Polypeptide and V of SEQ ID NO:658 Ｌ comprising a polypeptide, (lvii) V of SEQ ID NO: 666 Ｈ Polypeptide and V of SEQ ID NO:668 Ｌ comprising a polypeptide, (lviii) V of SEQ ID NO: 676 Ｈ Polypeptides and V of SEQ ID NO:678 Ｌ comprising a polypeptide, (lix) V of SEQ ID NO: 686 Ｈ Polypeptide and V of SEQ ID NO:688 Ｌ comprising a polypeptide, (lx) V of SEQ ID NO: 696 Ｈ Polypeptides and V of SEQ ID NO:698 Ｌ comprising a polypeptide, (lxi) V of SEQ ID NO: 706 Ｈ Polypeptides and V of SEQ ID NO:708 Ｌ comprising a polypeptide, (lxii) V of SEQ ID NO: 716 Ｈ Polypeptides and V of SEQ ID NO:718 Ｌ comprising a polypeptide, (lxiii) V of SEQ ID NO: 726 Ｈ Polypeptide and V of SEQ ID NO:728 Ｌ comprising a polypeptide, (lxiv) V of SEQ ID NO:736 Ｈ Polypeptides and V of SEQ ID NO:738 Ｌ comprising a polypeptide, (lxv) V of SEQ ID NO:746 Ｈ Polypeptides and V of SEQ ID NO:748 Ｌ comprising a polypeptide, (lxvi) V of SEQ ID NO:756 Ｈ Polypeptide and V of SEQ ID NO:758 Ｌ comprising a polypeptide, (lxvii) V of SEQ ID NO: 766 Ｈ Polypeptides and V of SEQ ID NO:768 Ｌ comprising a polypeptide, (lxviii) V of SEQ ID NO: 776 Ｈ Polypeptides and V of SEQ ID NO:778 Ｌ comprising a polypeptide, (lxix) V of SEQ ID NO:786 Ｈ Polypeptide and V of SEQ ID NO:788 Ｌ comprising a polypeptide, (lxx) V of SEQ ID NO:796 Ｈ Polypeptides and V of SEQ ID NO:798 Ｌ comprising a polypeptide, (lxxi) V of SEQ ID NO: 806 Ｈ Polypeptide and V of SEQ ID NO:808 Ｌ comprising a polypeptide, (lxxii) V of SEQ ID NO: 816 Ｈ Polypeptide and V of SEQ ID NO:818 Ｌ 34. The antibody or antibody fragment of any one of claims 1 to 33, comprising a polypeptide.

34. 34. The antibody of claim 32 or 33, comprising a human IgG2 constant domain, optionally with at least one cysteine ​​residue deleted or changed to another amino acid.

35. The following immunosuppressive effects: (i) reduced immune response, (ii) reduced T cell activation, (iii) reduced cytotoxic T cell activity, (iv) reduced natural killer (NK) cell activity, (v) reduced T cell activity, (vi) reduced proinflammatory cytokine secretion, (vii) reduced IL-2 secretion, (viii) reduced interferon-γ production, (ix) reduced Th1 response, (x) reduced Th2 response, (xi) reduced cell number and / or activity of regulatory T cells, (xii) reduced regulatory cell activity and (xiii) increased regulatory cell activity and / or increased activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes; (xiii) increased M2 macrophages; (xiv) increased M2 macrophage activity; (xv) increased N2 neutrophils; (xvi) increased N2 neutrophil activity; (xvii) increased inhibition of T cell activation; (xviii) increased regulatory cell activity and / or increased activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes; (xviii) increased M2 macrophages; (xviii) increased M2 macrophage activity; (xviii) increased N2 neutrophils; (xviii) increased N2 neutrophil activity; (xviii) increased inhibition of T cell activation; (xx) increased inhibition of CTL activation, (xix) increased inhibition of NK cell activation, (xx) increased T cell depletion, (xxi) decreased T cell response, (xxii) decreased cytotoxic cell activity, (xxiii) decreased antigen-specific memory response, (xxiv) inhibition of cell apoptosis or lysis, (xxv) decreased cytotoxic or cytostatic effects on cells, (xxvi) decreased direct cell killing, (xxvii) decreased Th17 activity, and / or (xxviii) increased complement dependent cytotoxicity and / or increased T cell depletion.

35. The agonistic anti-human VISTA antibody or antibody fragment of any one of claims 1 to 34, wherein the antibody or antibody fragment mediates any one of the following: induction of inflammatory responses and / or reduction of antibody-dependent cell-mediated cytotoxicity, or a combination of at least one of them, with the proviso that the anti-VISTA antibody or antigen-binding fragment may induce the opposite effect of one or more of (i) to (xxviii), optionally for use in treating autoimmunity, allergy, inflammation, transplantation, or sepsis.

36. A pharmaceutical or diagnostic composition comprising at least one antagonistic or agonistic antibody or antibody fragment according to any one of claims 1 to 35.

37. A therapeutic and / or diagnostic method or use of a composition containing at least one antagonistic antibody or antibody fragment according to any one of claims 1 to 36 for use in diagnosis or therapy, said method or use comprising administering to a subject in need of treatment and / or diagnosis at least one dose or a therapeutically or diagnostically effective amount of at least one composition comprising at least one antagonistic antibody or antibody fragment according to any one of claims 1 to 36 or a composition containing according to any one of claims 1 to 36.

38. 38. The method of claim 37 for treating cancer or an infectious disorder.

39. 39. The method of claim 38, wherein the cancer is a hematological cancer or a solid tumor, e.g., surrounded by a tumor stroma comprising myeloid cells, T cells, or a combination of myeloid cells and T cells.

40. 39. The method of claim 38 for treating a cancer selected from leukemia, lymphoma, myelodysplastic syndrome or myeloma, lung cancer, or a combination thereof.

41. The leukemia may be acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myelogenous) leukemia (AML), or chronic myeloid leukemia (AML).

41. The method of claim 40, comprising myelogenous leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, or adult T-cell leukemia.

42. 42. A therapeutic and / or diagnostic method or use of a composition containing at least one agonistic antibody or antibody fragment according to any one of claims 1 to 41 for use in diagnosis or therapy, said method or use comprising administering to a subject in need of treatment and / or diagnosis at least one dose or a therapeutically or diagnostically effective amount of at least one composition comprising at least one agonistic antibody or antibody fragment according to any one of claims 1 to 41 or a composition containing according to any one of claims 1 to 41.

43. The following immunosuppressive effects: (i) reduced immune response; (ii) reduced T cell activation; (iii) reduced cytotoxic T cell activity; (iv) reduced natural killer (NK) cell activity; (v) reduced T cell activity; (vi) reduced proinflammatory cytokine secretion; (vii) reduced IL-2 secretion; (viii) reduced interferon-γ production; (ix) reduced Th1 response; (x) reduced Th2 response; (xi) reduced cell number and / or activity of regulatory T cells; (xii) reduced regulatory cell activity and / or bone marrow (xiii) increased regulatory cell activity and / or increased activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, and TIE2-expressing monocytes; (xiii) increased M2 macrophages; (xiv) increased M2 macrophage activity; (xv) increased N2 neutrophils; (xvi) increased N2 neutrophil activity; (xvii) increased inhibition of T cell activation; (xviii) increased inhibition of CTL activation; (xix) increased inhibition of NK cell activation, (xx) increased T cell depletion, (xxi) decreased T cell response, (xxii) decreased cytotoxic cell activity, (xxiii) decreased antigen-specific memory response, (xxiv) inhibition of cell apoptosis or lysis, (xxv) decreased cytotoxic or cytostatic effects on cells, (xxvi) decreased direct cell killing, (xxvii) decreased Th17 activity, and / or (xxviii) complement dependent cytotoxicity and / or antibody dependent cell-mediated cytotoxicity.

43. The method of any one of claims 1 to 42, or use of any agonistic antibody or antibody fragment thereof, to effect in vitro and / or in vivo any one of: a reduction in the level of inflammatory cytokines, inflammatory bowel disease, or a combination of at least one of them, with the proviso that the anti-VISTA antibody or antigen-binding fragment may induce an adverse effect to one or more of (i) to (xxviii), optionally for use in treating autoimmunity, allergy, inflammation, transplantation, or sepsis.

44. 44. The method or use of any one of claims 37 to 43 for use in the treatment or prevention of allergy, autoimmunity, transplantation, gene therapy, inflammation, cancer, GVHD, or sepsis, or for use in treating or preventing inflammation, autoimmunity, or allergic side effects associated with any of the foregoing in a human subject.

45. 45. The anti-VISTA antibody or antigen-binding fragment or composition, or method or use, of any one of claims 1 to 44, further comprising another immunomodulatory antibody or fusion protein selected from an immune inhibitory antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and / or an agonistic antibody or fusion protein targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS.

46. 46. ​​The method or use of any one of claims 1 to 45, comprising assaying VISTA protein in cells or in a body fluid of the individual before, simultaneously with and / or after treatment.

47. 47. The method or use of any one of claims 1 to 46, comprising assaying VISTA levels on hematopoietic cells.

48. 48. The method or use of any one of claims 1 to 47, comprising assaying VISTA levels on hematopoietic cells selected from myeloid lineage cells and / or any one or more of lymphocytes, monocytes, or neutrophils, T cells, B cells, natural killer (NK) cells, or natural killer T (NKT) cells.

49. 49. The method or use of any one of claims 1 to 48, wherein the agonist anti-human VISTA antibody or fragment comprises the same CDRs as an antibody selected from VSTB49 to VSTB116 and an optionally mutated human IgG2 Fc region.

50. 50. The method or use of claim 49, wherein the IgG2 constant or Fc region retains the ability to bind a native FcR and / or to bind CD32A.

51. The anti-human VISTA antibody or fragment has an affinity or K for human VISTA of 50 M or less as determined by surface plasmon resonance at 37° C. Ｄ The antibody, composition, method or use of any one of claims 1 to 50, comprising:

52. The anti-human VISTA antibody or fragment has an affinity, or K, for human VISTA of 1 nM or less as determined by surface plasmon resonance at 37° C. Ｄ The antibody, composition, method or use of any one of claims 1 to 51, comprising:

Images