Anti-VISTA antibodies and uses thereof

Abstract

The present disclosure provides antibodies against V-domain Ig-containing suppressor of T cell activation (VISTA), and compositions comprising such antibodies. Also provided are methods of using antibodies that specifically bind to VISTA to treat disease, e.g., to treat cancer. In one aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that competes with any one of the antibodies, or antigen-binding fragments thereof, described herein for binding to human VISTA (SEQ ID NO: 377).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 150,995, filed February 18, 2021, the entire contents of which are expressly incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on February 1, 2022, is named 136589-00120_SL.txt and is 784,935 bytes in size.

[0003] The present disclosure provides antibodies against V-domain Ig-containing suppressor of T-cell activation (VISTA), and compositions comprising such antibodies. Also provided are methods of using antibodies that specifically bind to VISTA to treat diseases, such as cancer, autoimmune diseases, and infectious diseases, such as bacterial and fungal infections. [Background technology]

[0004] VISTA is an immunosuppressive cell surface protein of the B7 family that shows similarity to programmed death-ligand 1 (PD-L1). VISTA functions as both a ligand for antigen-presenting cells and a receptor for T cells (Boger et al., Oncoimmunology. 2017;6(4):e1293215(2017)). VISTA is primarily expressed in hematopoietic tissues (e.g., spleen, thymus, and bone marrow) as well as myeloid cells, neutrophils, natural killer (NK) cells, and regulatory T cells (Treg) (Wang et al., J. Exp. Med. Vol. 208 No. 3 577-592).

[0005] In particular, VISTA is highly expressed on myeloid-derived suppressor cells (MDSCs) and Tregs in the tumor microenvironment, where it mediates immunosuppression. Specifically, VISTA inhibits T cell responses by suppressing T cell receptor activation, leading to cell cycle arrest but not apoptosis (Wang et al., J. Exp. Med. Vol. 208 No. 3 577-592). VISTA is highly expressed in "cold tumors" (tumors not infiltrated by T cells), and high VISTA expression is associated with poor survival in cancer patients, e.g., pancreatic cancer (Blando et al., 2019, Proc. Nat. Acad. Sci. 116(5):1692-97). VISTA expression has been found to be increased in prostate cancer and melanoma after treatment with checkpoint inhibitor therapy, suggesting a potential resistance mechanism (Kakavand et al., 2017, Modern Pathology 30:1666-1676; Gao et al., 2017, Nat Med. May;23(5):551-555).

[0006] In antibody engineering, various isotypes and subclasses are important for antibody optimization and function because sequence variations occur at sites that determine affinity and specificity for the neonatal Fc receptor (FcRn), Fc alpha receptor, Fc gamma receptor, and complement protein C1q (Woof, JM, and Burton, D.R. Nat. Rev. Immunol. 4 (2004) 89-99). There are five Fc gamma receptors (FcγRs) that activate effector cells after binding to IgG. Among the activating receptors are FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb (Nimmerjahn, F. and Revetch, J.V. Nat. Rev. Immunol. 8 (2008) 34-47). There is one inhibitory Fc gamma receptor, namely, FcγRIIb. FcγRs are polymorphic, with certain alleles exhibiting higher affinity for Fc than others. Antibodies that bind to these receptors can promote the recruitment of effector cells to opsonized target cells or opsonized pathogens for clearance (Nimmerjahn, F. and Revetch, J.V. Nat. Rev. Immunol. 8 (2008) 34-47). Thus, sequence alterations and post-translational modifications of the Fc and hinge regions of antibodies can manipulate the effector function and circulation of a given antibody or antibody-like protein (Presta, L.G. Curr. Opin. Immunol. 20 (2008) 460-470). In addition to sequence variation, the Fc region also contains an N-linked glycosylation site at residue 297, which is important for Fc structure and function (Dwek, R.R. et al. J. Anat. 187 (1995) 279-292). Mutant Fc regions have been described that have reduced binding to Fc gamma receptors and reduced Fc gamma receptor-mediated activities (US Pat. No. 10,053,513 B2).

[0007] Antibody clearance occurs primarily through intracellular catabolism by uptake via pinocytosis or receptor-mediated endocytosis processes followed by degradation to amino acids by lysosomes (Waldmann, TA and Strober, W. Prog. Allergy 13 (1969) 1-110).

[0008] Receptor-mediated endocytosis of antibodies is mediated by the interaction of cell surface receptors with either the Fc domain or one of the Fab-binding domains of the antibody. This binding event leads to the endocytic internalization of the antibody into vesicles and subsequent lysosomal degradation. When there is binding between an antibody Fab and its antigen, the resulting endocytosis and elimination is called target-mediated drug elimination (TMDD) (Mager, DE and Jusko, WJJ Pharmacokinet. Pharmacodyn. 28 (2001) 507-5320).

[0009] The rate of antibody elimination due to TMDD depends on the expression of the antigen receptor target, the affinity of the mAb for the antigen, the dose of the mAb, the rate of internalization and recycling of the receptor-antibody complex, and the rate of catabolism within the target cell. Antibodies that are primarily eliminated by TMDD are eliminated in a dose-dependent, nonlinear manner. For antibodies with plasma membrane-expressed targets such as VISTA, TMDD is the primary elimination pathway, especially for therapeutic antibodies at low doses and concentrations (Ryman, JT and Meibohm, B. CPT Pharmacometrics Syst. Pharmacol. 6 (2017) 576-588).

[0010] Antibody-receptor complexes on the plasma membrane are internalized into vesicles that fuse with endosomal compartments. The complexes may be sent for degradation in lysosomes or recycled intact to the cell surface and extracellular medium. Evidence suggests that the outcome of this sorting decision is related to antibody-receptor binding affinity, with complexes that remain bound generally being degraded and dissociated ones being recycled (Chimalakonda, AP, et. Al. AAPS J. 15 (2013) 717-727). In general, dissociation of the complex within the endosome promotes antibody recycling. Dissociation of antibody-receptor complexes in endosomes and lysosomes may be regulated by pH-sensitive interactions.

[0011] In most pH-sensitive interactions studied, pH-dependent binding relies on the presence of an ionizable histidine (a "histidine switch") that mediates a structural transition during binding or folding of the interacting proteins (Kulkarni, M. V. At. Al. J. Biol. Chem. 285 (2010) 38524-38533; Maeda, K. et. Al. J. Control. Release 82 (2002) 71-82; Yamamoto, T. et. Al. Biochemistry 47 (2008) 11647-11652). Because changes in electrostatic interactions induced by histidine protonation at lower pH values ​​can reduce binding affinity, protein engineering approaches to incorporate pH sensitivity into proteins typically employ histidine substitution strategies.

[0012] Below is an example of how a histidine switch in a therapeutic protein was successfully engineered to reduce metabolism and improve half-life.

[0013] Igawa T., et al. Nat. Biotechnol. 28 (2010) 1203-1207 describe an engineered antibody against IL-6R that rapidly dissociates from the IL-6 receptor (IL-6R) in the acidic environment of the endosome (pH 6.0) while maintaining binding affinity to the IL-6R in plasma (pH 7.4).

[0014] WO2011 / 111007 discloses antibodies with pH-dependent antigen binding that dissociate from the antigen preferably within endosomes.

[0015] Rother et al. Nat. Biotechnol. 25 (2007) 1256-1264 describe the discovery and development of eculizumab, a complement inhibitor with a histidine switch that mediates endosomal escape, for the treatment of paroxysmal nocturnal hemoglobinuria.

[0016] WO2015 / 134894A1 describes antibodies that bind to C5 that are engineered to rapidly dissociate from their targets at endosomal pH, thereby increasing their half-life.

[0017] US 9,540,449 B2 describes antibodies against PCSK9 with improved serum half-lives that bind to cell surface receptors at neutral pH but readily dissociate from the target at acidic pH.

[0018] Fallon et al., J. Biol. Chem. 275 (1999) 6790-6797, describe mutants of interleukin-2 that exhibit reduced endocytic degradation due to enhanced ligand recycling. The binding affinity of mutant IL-2 to its receptor is higher at neutral than at acidic pH, allowing endosomal sorting and recycling of the ligand-receptor complex.

[0019] Sarkar, CS, et. Al. Nat. Biotech. 20 (2002) 908-913 describe the use of a "histidine switch" to improve cellular transport of granulocyte colony-stimulating factor. Anti-VISTA antibodies have been described (see, e.g., U.S. Pat. Nos. 8,231,872, 8,236,304, 8,501,915, and 10,766,959, as well as U.S. Patent Application Publication No. US2020 / 0407449A1, and International Patent Application Publication Nos. WO2016 / 207717A8, WO2014 / 197849A9, WO2018 / 169993A1, WO2018 / 237287A1, WO2019 / 152810A1, WO2019 / 185879A1, and WO2020 / 016459A1). However, despite all of the above, there remains a need in the art for effective therapeutic antibodies. The citation of any reference in this disclosure should not be construed as an admission that such reference is prior art. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] U.S. Patent No. 10,053,513 [Patent Document 2] International Publication No. 2011 / 111007 [Patent Document 3] International Publication No. 2015 / 134894 [Patent Document 4] U.S. Patent No. 9,540,449 [Patent Document 5] U.S. Patent No. 8,231,872 [Patent Document 6] U.S. Patent No. 8,236,304 [Patent Document 7] U.S. Patent No. 8,501,915 [Patent Document 8] U.S. Patent No. 10,766,959 [Patent Document 9] US Patent Application Publication No. 2020 / 0407449 [Patent Document 10] International Publication No. 2016 / 207717

Non-Patent Document

[0021]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

[0022] Anti-VISTA antibodies known prior to the present disclosure exhibit numerous drawbacks, including an inability to address the fact that multiple ligands bind to the VISTA receptor, the potential for antibody-dependent cellular cytotoxicity (ADCC) of VISTA-expressing immune cells and toxic cytokine release, anti-drug antibodies or immunogenicity, the multiple binding epitopes on VISTA with varying efficacy and toxicity, and suboptimal Fc binding. The inventors of the present application have utilized knowledge of these drawbacks to design novel anti-VISTA antibodies and antigen-binding fragments thereof that overcome these concerns. Each of these aspects is described in detail below.

[0023] First, most known anti-VISTA antibodies bind to one of two major epitopes on the extracellular domain of VISTA. The first epitope, R54 / F62 / Q63, is associated with anti-tumor effects but is also associated with significant cytokine release and inflammation, raising significant concerns regarding therapeutic safety. The second epitope, H121 / H122 / H123, results in pH-sensitive binding and increased PK but abolishes most anti-tumor activity. The inventors of the present application have identified anti-VISTA antibodies and antigen-binding fragments thereof disclosed herein that bind to a third unique epitope and exhibit improved safety and superior single-agent efficacy in tumor models (see Examples herein).

[0024] Second, known anti-VISTA antibodies have been shown to block only one, two, or (at most) three of the known VISTA ligands at either pH 6.0 or pH 7.4. In contrast, the anti-VISTA antibodies and antigen-binding fragments described herein are capable of blocking binding of all five known ligands to VISTA over the pH range of 6.0 to 7.4, providing increased efficacy.

[0025] Furthermore, the anti-VISTA antibodies and antigen-binding fragments thereof described herein are Fc-optimized to reduce Fcγ receptor binding, thereby reducing ADCC, CDC (complement-dependent cytotoxicity), and proinflammatory cytokine release, improving the safety of therapeutic administration. Some anti-VISTA antibodies described herein are further Fc-optimized to increase FcRn binding, thereby increasing antibody recycling and prolonged exposure. Finally, the anti-VISTA antibodies and antigen-binding fragments described herein were developed using a genetically engineered humanized mouse model, providing additional benefits related to reduced immunogenicity in humans. Thus, the anti-VISTA antibodies and antigen-binding fragments thereof described herein exhibit surprising and improved properties over anti-VISTA antibodies and uses thereof known prior to this disclosure.

[0026] Thus, in one aspect, disclosed herein is a human anti-VISTA antibody, or antigen-binding fragment thereof, which (i) blocks binding of all five known ligands for VISTA (VSIG-3 (V-set and immunoglobulin domain containing 3), VSIG-8 (V-set and immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), LRIG1 (leucine-rich repeat and immunoglobulin-like domain 1), and VISTA) at pH 6.0 and / or pH 7.4, and / or (ii) binds to a unique epitope including amino acids tyrosine 37, arginine 54, valine 117, and arginine 127 of the VISTA-ECD (extracellular domain).

[0027] In one aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that binds to V-domain Ig-containing suppressor of T-cell activation (VISTA), wherein the antibody, or antigen-binding fragment thereof, blocks binding of all five known VISTA ligands at pH 6.0 and pH 7.4, the five known VISTA ligands being VSIG-3 (V-set and immunoglobulin domain containing 3), VSIG-8 (V-set and immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), LRIG1 (leucine-rich repeat and immunoglobulin-like domain 1), and VISTA, and the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0028] In another aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that binds to V domain Ig-containing suppressor of T cell activation (VISTA), wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has a binding affinity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% to any one of SEQ ID NOs: 584-597, 227-246, and 383-386. , 98%, or 99% identity to any one of SEQ ID NOs: 84 to 110, and the VL comprises a sequence comprising or consisting of any one of SEQ ID NOs: 84 to 110, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 84 to 110. In one embodiment, the antibody, or antigen-binding fragment thereof, blocks binding of ligands to all five VISTAs at pH 6.0 and pH 7.4, the five known ligands to VISTA being VSIG-3 (V-set and immunoglobulin domain containing 3), VSIG-8 (V-set and immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), LRIG1 (leucine-rich repeat and immunoglobulin-like domain 1), and VISTA.In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH CDR1 of any one of SEQ ID NOs: 247-253 as defined by the Kabat numbering system, any one of SEQ ID NOs: 284-291 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 318-325 as defined by the Paratome numbering system; a VH CDR2 of any one of SEQ ID NOs: 254-265 as defined by the Kabat numbering system, any one of SEQ ID NOs: 292-298 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 326-339 as defined by the Paratome numbering system; a VH CDR3 of any one of SEQ ID NOs: 266-283 as defined by the Kabat numbering system, any one of SEQ ID NOs: 299-317 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 340-359 as defined by the Paratome numbering system. and a VL CDR3, a VL CDR1 of any one of SEQ ID NOS: 111-122 as defined by the Kabat numbering system, any one of SEQ ID NOS: 152-162 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 188-196 or 576-578 as defined by the Paratome numbering system; a VL CDR2 of any one of SEQ ID NOS: 123-131 and 575 as defined by the Kabat numbering system, any one of SEQ ID NOS: 163-167 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 197-206 as defined by the Paratome numbering system; and a VL CDR3 of any one of SEQ ID NOS: 132-151 as defined by the Kabat numbering system, any one of SEQ ID NOS: 168-186 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 207-226 as defined by the Paratome numbering system.

[0029] In one aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that binds to VISTA, the antibody or antigen-binding fragment thereof comprising a VH and a VL, wherein the VH has a VH CDR1 of any one of SEQ ID NOS: 247-253 as defined by the Kabat numbering system, any one of SEQ ID NOS: 284-291 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 318-325 as defined by the Paratome numbering system; a VH CDR2 of any one of SEQ ID NOS: 254-265 as defined by the Kabat numbering system, any one of SEQ ID NOS: 292-298 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 326-339 as defined by the Paratome numbering system; and a VH CDR3 of any one of SEQ ID NOS: 266-283 as defined by the Kabat numbering system, any one of SEQ ID NOS: 299-317 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 340-359 as defined by the Paratome numbering system. and the VL comprises a VL CDR1 of any one of SEQ ID NOS: 111 to 122 as defined by the Kabat numbering system, any one of SEQ ID NOS: 152 to 162 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 188 to 196 or 576 to 578 as defined by the Paratome numbering system; a VL CDR2 of any one of SEQ ID NOS: 123 to 131 and 575 as defined by the Kabat numbering system, any one of SEQ ID NOS: 163 to 167 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 197 to 206 as defined by the Paratome numbering system; and a VL CDR3 of any one of SEQ ID NOS: 132 to 151 as defined by the Kabat numbering system, any one of SEQ ID NOS: 168 to 186 as defined by the IMGT numbering system, or any one of SEQ ID NOS: 207 to 226 as defined by the Paratome numbering system.In one embodiment, the antibody, or antigen-binding fragment thereof, blocks binding of ligands to all five VISTAs at pH 6.0 and pH 7.4, the five known ligands to VISTA being VSIG-3 (V-set and immunoglobulin domain containing 3), VSIG-8 (V-set and immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), LRIG1 (leucine-rich repeat and immunoglobulin-like domain 1), and VISTA.

[0030] In one embodiment, the VH comprises a sequence comprising or consisting of any one of SEQ ID NOs: 584 to 597, 227 to 246, and 383 to 386, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 584 to 597, 227 to 246, and 383 to 386, and the VL comprises a sequence comprising or consisting of any one of SEQ ID NOs: 84 to 110, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 84 to 110.

[0031] In another embodiment, the VH CDR1 comprises SEQ ID NO: 247, the VH CDR2 comprises SEQ ID NO: 254, and the VH CDR3 comprises SEQ ID NO: 266, the VL CDR1 comprises SEQ ID NO: 111, the VL CDR2 comprises SEQ ID NO: 123, and the VL CDR3 comprises SEQ ID NO: 132; or the VH CDR1 comprises SEQ ID NO: 284, the VH CDR2 comprises SEQ ID NO: 292, and the VH CDR3 comprises SEQ ID NO: 299, the VL CDR1 comprises SEQ ID NO: 152, the VL CDR2 comprises SEQ ID NO: 163, and the VL CDR3 comprises SEQ ID NO: 168; or the VH CDR1 comprises SEQ ID NO: 318, the VH CDR2 comprises SEQ ID NO: 326, and the VH CDR3 comprises SEQ ID NO: 340, the VL CDR1 comprises SEQ ID NO: 188, the VL CDR2 comprises SEQ ID NO: 197, and the VL or the VH CDR1 comprises SEQ ID NO: 248, the VH CDR2 comprises SEQ ID NO: 255, the VH CDR3 comprises SEQ ID NO: 267, the VL CDR1 comprises SEQ ID NO: 112, the VL CDR2 comprises SEQ ID NO: 124, and the VL CDR3 comprises SEQ ID NO: 133, or the VH CDR1 comprises SEQ ID NO: 285, the VH CDR2 comprises SEQ ID NO: 293, and the VH CDR3 comprises SEQ ID NO: 300, the VL CDR1 comprises SEQ ID NO: 153, the VL CDR2 comprises SEQ ID NO: 164, and the VL CDR3 comprises SEQ ID NO: 169, or the VH CDR1 comprises SEQ ID NO: 319, the VH CDR2 comprises SEQ ID NO: 327, the VH CDR3 comprises SEQ ID NO: 341, the VL CDR1 comprises SEQ ID NO: 189, and the VL or the VH CDR1 comprises SEQ ID NO: 249, the VH CDR2 comprises SEQ ID NO: 256, the VH CDR3 comprises SEQ ID NO: 268, the VL CDR1 comprises SEQ ID NO: 113, the VL CDR2 comprises SEQ ID NO: 125, and the VL CDR3 comprises SEQ ID NO: 134; or the VH CDR1 comprises SEQ ID NO: 286, the VH CDR2 comprises SEQ ID NO: 294, the VH CDR3 comprises SEQ ID NO: 301, the VL CDR1 comprises SEQ ID NO: 154, and the VLor the VH CDR1 comprises SEQ ID NO: 320, the VH CDR2 comprises SEQ ID NO: 328, the VH CDR3 comprises SEQ ID NO: 342, the VL CDR1 comprises SEQ ID NO: 190, the VL CDR2 comprises SEQ ID NO: 199, and the VL CDR3 comprises SEQ ID NO: 209, or the VH CDR1 comprises SEQ ID NO: 250, the VH CDR2 comprises SEQ ID NO: 257, and the VH CDR3 comprises SEQ ID NO: 269, the VL CDR1 comprises SEQ ID NO: 114, the VL CDR2 comprises SEQ ID NO: 126, and the VL CDR3 comprises SEQ ID NO: 135, or the VH CDR1 comprises SEQ ID NO: 287, the VH CDR2 comprises SEQ ID NO: 295, and the VH CDR3 comprises SEQ ID NO: 302, and the VL wherein CDR1 comprises SEQ ID NO: 155, the VL CDR2 comprises SEQ ID NO: 165, and the VL CDR3 comprises SEQ ID NO: 171; or wherein the VH CDR1 comprises SEQ ID NO: 321, the VH CDR2 comprises SEQ ID NO: 329, and the VH CDR3 comprises SEQ ID NO: 343; wherein the VL CDR1 comprises SEQ ID NO: 191, the VL CDR2 comprises SEQ ID NO: 200, and the VL CDR3 comprises SEQ ID NO: 210; or wherein the VH CDR1 comprises SEQ ID NO: 251, the VH CDR2 comprises SEQ ID NO: 258, and the VH CDR3 comprises SEQ ID NO: 270; wherein the VL CDR1 comprises SEQ ID NO: 115, the VL CDR2 comprises SEQ ID NO: 127, and the VL CDR3 comprises SEQ ID NO: 136; or wherein the VH CDR1 comprises SEQ ID NO: 288, the VH CDR2 comprises SEQ ID NO: 295, and the VH and the VL CDR1 comprises SEQ ID NO: 156, the VL CDR2 comprises SEQ ID NO: 166, and the VL CDR3 comprises SEQ ID NO: 172; or the VH CDR1 comprises SEQ ID NO: 322, the VH CDR2 comprises SEQ ID NO: 330, and the VH CDR3 comprises SEQ ID NO: 344, the VL CDR1 comprises SEQ ID NO: 192, the VL CDR2 comprises SEQ ID NO: 201, and the VL CDR3 comprises SEQ ID NO: 211; or the VH CDR1 comprises SEQ ID NO: 248, the VH CDR2 comprises SEQ ID NO: 259, and the VH CDR3 comprises SEQ ID NO: 271, and the VLCDR1 comprises SEQ ID NO: 116, the VL CDR2 comprises SEQ ID NO: 126, and the VL CDR3 comprises SEQ ID NO: 137; or the VH CDR1 comprises SEQ ID NO: 285, the VH CDR2 comprises SEQ ID NO: 296, and the VH CDR3 comprises SEQ ID NO: 304, the VL CDR1 comprises SEQ ID NO: 157, the VL CDR2 comprises SEQ ID NO: 165, and the VL CDR3 comprises SEQ ID NO: 173; or the VH CDR1 comprises SEQ ID NO: 319, the VH CDR2 comprises SEQ ID NO: 331, and the VH CDR3 comprises SEQ ID NO: 345, the VL CDR1 comprises SEQ ID NO: 193, the VL CDR2 comprises SEQ ID NO: 200, and the VL CDR3 comprises SEQ ID NO: 212.

[0032] In another embodiment, the VH CDR1 comprises SEQ ID NO:247, SEQ ID NO:284, or SEQ ID NO:318, the VH CDR2 comprises SEQ ID NO:254, SEQ ID NO:292, or SEQ ID NO:326, and the VH CDR3 comprises SEQ ID NO:266, SEQ ID NO:299, or SEQ ID NO:340, the VL CDR1 comprises SEQ ID NO:111, SEQ ID NO:152, or SEQ ID NO:188, the VL CDR2 comprises SEQ ID NO:123, SEQ ID NO:163, or SEQ ID NO:197, and the VL CDR3 comprises SEQ ID NO:132, SEQ ID NO:168, or SEQ ID NO:207, or the VH CDR1 comprises SEQ ID NO:248, SEQ ID NO:285, or SEQ ID NO:319, the VH CDR2 comprises SEQ ID NO:255, SEQ ID NO:293, or SEQ ID NO:327, and the VH CDR3 comprises SEQ ID NO:267, SEQ ID NO:300, or SEQ ID NO:341, and the VL wherein CDR1 comprises SEQ ID NO: 112, SEQ ID NO: 153, or SEQ ID NO: 189, the VL CDR2 comprises SEQ ID NO: 124, SEQ ID NO: 164, or SEQ ID NO: 198, and the VL CDR3 comprises SEQ ID NO: 133, SEQ ID NO: 169, or SEQ ID NO: 208; or wherein the VH CDR1 comprises SEQ ID NO: 249, SEQ ID NO: 286, or SEQ ID NO: 320, the VH CDR2 comprises SEQ ID NO: 256, SEQ ID NO: 294, or SEQ ID NO: 328, and the VH CDR3 comprises SEQ ID NO: 268, SEQ ID NO: 301, or SEQ ID NO: 342, and the VL CDR1 comprises SEQ ID NO: 113, SEQ ID NO: 154, or SEQ ID NO: 190, the VL CDR2 comprises SEQ ID NO: 125, SEQ ID NO: 165, or SEQ ID NO: 199, and the VL CDR3 comprises SEQ ID NO: 134, SEQ ID NO: 170, or SEQ ID NO: 209; or wherein the VH1 CDR1 comprises SEQ ID NO: 250, SEQ ID NO: 287, or SEQ ID NO: 321, and the VH wherein the VL CDR1 comprises SEQ ID NO: 114, SEQ ID NO: 155, or SEQ ID NO: 191, the VL CDR2 comprises SEQ ID NO: 126, SEQ ID NO: 165, or SEQ ID NO: 200, and the VL CDR3 comprises SEQ ID NO: 135, SEQ ID NO: 171, or SEQ ID NO: 210;wherein CDR1 comprises SEQ ID NO:251, SEQ ID NO:288, or SEQ ID NO:322, the VH CDR2 comprises SEQ ID NO:258, SEQ ID NO:295, or SEQ ID NO:333, the VH CDR3 comprises SEQ ID NO:270, SEQ ID NO:303, or SEQ ID NO:344, the VL CDR1 comprises SEQ ID NO:115, SEQ ID NO:156, or SEQ ID NO:192, the VL CDR2 comprises SEQ ID NO:127, SEQ ID NO:166, or SEQ ID NO:201, and the VL CDR3 comprises SEQ ID NO:136, SEQ ID NO:172, or SEQ ID NO:211, or wherein VH CDR1 comprises SEQ ID NO:248, SEQ ID NO:185, or SEQ ID NO:319, the VH CDR2 comprises SEQ ID NO:259, SEQ ID NO:296, or SEQ ID NO:331, and the VH CDR3 comprises SEQ ID NO:271, SEQ ID NO:304, or SEQ ID NO:345, the VL CDR1 comprises SEQ ID NO:116, SEQ ID NO:157, or SEQ ID NO:193, and the VL CDR2 comprises SEQ ID NO:126, SEQ ID NO:165 or SEQ ID NO:200, and the VL CDR3 comprises SEQ ID NO:137, SEQ ID NO:173 or SEQ ID NO:212.

[0033] In another embodiment, (i) the VH comprises a sequence comprising or consisting of SEQ ID NO: 592 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 592, and the VL comprises a sequence comprising or consisting of SEQ ID NO: 86 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86; or (ii) the VH comprises a sequence comprising or consisting of SEQ ID NO: 584 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 584. (iii) the VL comprises a sequence comprising or consisting of SEQ ID NO: 84 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 84; or (iv) the VH comprises a sequence comprising or consisting of SEQ ID NO: 585 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 585. 85, wherein the VL comprises a sequence comprising or consisting of SEQ ID NO: 85 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 85; or (iv) the VH comprises a sequence comprising or consisting of SEQ ID NO: 586 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86. or (v) the VH comprises a sequence comprising or consisting of SEQ ID NO: 587 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 587, and the VL has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 87.(vi) the VH comprises a sequence comprising or consisting of SEQ ID NO: 588 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 588 and the VL comprises a sequence comprising or consisting of SEQ ID NO: 88 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88; or (vii) the VH comprises a sequence comprising or consisting of SEQ ID NO: 589 (viii) the VL comprises a sequence comprising or consisting of SEQ ID NO: 589 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 89; or (viii) the VH comprises a sequence comprising or consisting of SEQ ID NO: 238 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 238. or (ix) the VH comprises a sequence comprising or consisting of SEQ ID NO: 590 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 590, and the VL comprises a sequence comprising or consisting of SEQ ID NO: 238 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 84; or or (x) the VH comprises a sequence comprising or consisting of SEQ ID NO: 84 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 84; or (x) the VH comprises a sequence comprising or consisting of SEQ ID NO: 591238 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 85.or (xi) the VH comprises a sequence comprising or consisting of SEQ ID NO: 593 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 593, and the VL comprises a sequence comprising or consisting of SEQ ID NO: 86 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86. (xii) the VH comprises a sequence comprising or consisting of SEQ ID NO: 593238 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 593238 and the VL comprises a sequence comprising or consisting of SEQ ID NO: 87 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 87. (xiii) the VH comprises a sequence comprising or consisting of SEQ ID NO: 594 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 594, and the VL comprises SEQ ID NO: 884 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 884; or or (xiv) the VH comprises a sequence comprising or consisting of SEQ ID NO: 595 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 595, and the VL comprises a sequence comprising or consisting of SEQ ID NO: 89 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 89.

[0034] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain (HC) sequence comprising or consisting of any one of SEQ ID NOs: 407-477, 572-574, and 604-609, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 407-477, 572-574, and 604-609, and a light chain (LC) sequence comprising or consisting of any one of SEQ ID NOs: 387-406, 569-571, and 598-603, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 387-406, 569-571, and 598-603.

[0035] In one embodiment, the antibody, or antigen-binding fragment thereof, binds to human VISTA (amino acids 33-311 of SEQ ID NO: 377). In one embodiment, the antibody, or antigen-binding fragment thereof, specifically binds to human VISTA (amino acids 33-311 of SEQ ID NO: 377). In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope of human VISTA comprising the amino acid sequence HLHHG (amino acids 98-102 of SEQ ID NO: 377) or VVEIRHHHSEHR (amino acids 148-159 of SEQ ID NO: 377). In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope of human VISTA comprising tyrosine 37, arginine 54, valine 117, and arginine 127 of SEQ ID NO: 377.

[0036] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises an Fc region. In one embodiment, the Fc region is a human Fc region or a variant of the human Fc region, which has 1 to 7 amino acid mutations in the Fc region compared to a native human Fc region. In one embodiment, the human Fc region is that of human IgG1, human IgG2, or human IgG4. In another embodiment, the antibody comprises a human IgG1 or human IgG4 constant region, or a variant of the constant region, which has 1 to 7 amino acid mutations in the constant region compared to a native constant region.

[0037] In one embodiment, (a) the Fc region is that of a human IgG1 and the mutations are C220D, D221C, E233P, L234A, L234E, L234Y, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, E318A, K322A, S (b) the Fc region is that of a human IgG2 and the mutations are selected from the group consisting of C220D, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S266A, S267A, S268A, S269A, S270A, S271B, S272B, S273A, S274A, S275B, S276B, S277B, S278B, S279B, S280B, S281B, S282B, S283B, S284B, S285B, S286B, S287B, S288B, S289B, S290B, S291B, S292B, S293B, S294B, S295B, S296B, S297B, S298B, S299B, S300B, S301B, S302B, S303B, S304B, S305B, S310B, S311B, S312B, S313B, S314B, S315B, S316B, S317B, S318B, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU or (c) the Fc region is selected from the group consisting of: 267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system; or gG4, and the mutations are S228P, E233P, F234A, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G,and selected from the group consisting of I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system.

[0038] In one embodiment, the antibody, or antigen-binding fragment thereof, is a bispecific or multispecific antibody. In one embodiment, the antigen-binding fragment is an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a single-chain Fv (scFv).

[0039] In one aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that competes with any one of the antibodies, or antigen-binding fragments thereof, described herein for binding to human VISTA (SEQ ID NO: 377). In one aspect, disclosed herein is an antibody, or antigen-binding fragment thereof, that prevents binding of any one of the antibodies, or antigen-binding fragments thereof, described herein to human VISTA (SEQ ID NO: 377). In one embodiment, the binding is measured by an ELISA assay, surface plasmon resonance, or BLI, for example, BLI with Octet Red 96 (ForteBio System).

[0040] In another aspect, disclosed herein is an antibody-drug conjugate comprising an antibody, or antigen-binding fragment thereof, disclosed herein and a therapeutic agent. In another aspect, disclosed herein is a chimeric antigen receptor (CAR) comprising an scFv disclosed herein.

[0041] In another aspect, disclosed herein is a polynucleotide comprising a nucleotide sequence encoding the VH of an antibody or antigen-binding fragment disclosed herein. In another aspect, disclosed herein is a polynucleotide comprising a nucleotide sequence encoding the VL of an antibody or antigen-binding fragment disclosed herein. In another aspect, disclosed herein is a polynucleotide comprising nucleotide sequences encoding the VH and VL of an antibody or antigen-binding fragment disclosed herein.

[0042] In one aspect, disclosed herein is a cell comprising one or more polynucleotides encoding an antibody, or antigen-binding fragment thereof, disclosed herein.

[0043] In one aspect, disclosed herein is a pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, antibody-drug conjugate, or CAR disclosed herein, and a pharmaceutically acceptable carrier.

[0044] In one aspect, disclosed herein is a method of producing an antibody, or antigen-binding fragment thereof, disclosed herein, the method comprising culturing a cell under conditions such that the one or more polynucleotides are expressed by the cell to produce the antibody, or antigen-binding fragment thereof, encoded by the polynucleotide.

[0045] In one aspect, disclosed herein is a method for treating cancer, autoimmune disease, and / or infectious disease in a subject in need thereof, comprising administering a pharmaceutical composition disclosed herein to the subject. In one embodiment, the method is for treating cancer. In one embodiment, the method is for treating autoimmune disease. In one embodiment, the method is for treating infectious disease. In one embodiment, the cancer is non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, ovarian cancer, neuroblastoma, oral cancer, thyroid cancer, breast cancer, sarcoma, pancreatic cancer, colon cancer, gastric cancer, choriocarcinoma, testicular cancer, mesothelioma, skin cancer, renal cell carcinoma, bladder cancer, blood cancer, or cervical cancer. In one embodiment, the cancer is metastatic cancer. In one embodiment, the cancer is blood cancer, acute myeloid leukemia, or myelodysplastic syndrome. In one embodiment, the method further comprises administering to the subject an additional therapy, optionally wherein the additional therapy is radiation therapy, a chemotherapeutic agent, a targeted therapy, a tyrosine kinase inhibitor, a hormone therapy, and / or an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor is an inhibitor of programmed death-1 (PD-1), or programmed death-ligand 1 (PDL1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0046] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds, e.g., specifically binds, to VISTA, wherein the antibody comprises a variable heavy region (VH) and a variable light region (VL). In one embodiment, the VH comprises (i) a VH complementarity determining region (CDR)1 of SEQ ID NO: 247, a VH CDR2 of SEQ ID NO: 254, and a VH CDR3 of SEQ ID NO: 266, (ii) a VH CDR1 of SEQ ID NO: 284, a VH CDR2 of SEQ ID NO: 292, and a VH CDR3 of SEQ ID NO: 299, or (iii) a VH CDR1 of SEQ ID NO: 318, a VH CDR2 of SEQ ID NO: 326, and a VH CDR3 of SEQ ID NO: 340.

[0047] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 248, a VH CDR2 of SEQ ID NO: 255, and a VH CDR3 of SEQ ID NO: 267, (ii) a VH CDR1 of SEQ ID NO: 285, a VH CDR2 of SEQ ID NO: 293, and a VH CDR3 of SEQ ID NO: 300, or (iii) a VH CDR1 of SEQ ID NO: 319, a VH CDR2 of SEQ ID NO: 327, and a VH CDR3 of SEQ ID NO: 341.

[0048] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 249, a VH CDR2 of SEQ ID NO: 256, and a VH CDR3 of SEQ ID NO: 268, (ii) a VH CDR1 of SEQ ID NO: 286, a VH CDR2 of SEQ ID NO: 294, and a VH CDR3 of SEQ ID NO: 301, or (iii) a VH CDR1 of SEQ ID NO: 320, a VH CDR2 of SEQ ID NO: 328, and a VH CDR3 of SEQ ID NO: 342.

[0049] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 250, a VH CDR2 of SEQ ID NO: 257, and a VH CDR3 of SEQ ID NO: 269, (ii) a VH CDR1 of SEQ ID NO: 287, a VH CDR2 of SEQ ID NO: 295, and a VH CDR3 of SEQ ID NO: 302, or (iii) a VH CDR1 of SEQ ID NO: 321, a VH CDR2 of SEQ ID NO: 329, and a VH CDR3 of SEQ ID NO: 343.

[0050] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 251, a VH CDR2 of SEQ ID NO: 258, and a VH CDR3 of SEQ ID NO: 270, (ii) a VH CDR1 of SEQ ID NO: 288, a VH CDR2 of SEQ ID NO: 295, and a VH CDR3 of SEQ ID NO: 303, or (iii) a VH CDR1 of SEQ ID NO: 322, a VH CDR2 of SEQ ID NO: 330, and a VH CDR3 of SEQ ID NO: 344.

[0051] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 248, a VH CDR2 of SEQ ID NO: 259, and a VH CDR3 of SEQ ID NO: 271, (ii) a VH CDR1 of SEQ ID NO: 285, a VH CDR2 of SEQ ID NO: 296, and a VH CDR3 of SEQ ID NO: 304, or (iii) a VH CDR1 of SEQ ID NO: 319, a VH CDR2 of SEQ ID NO: 331, and a VH CDR3 of SEQ ID NO: 345.

[0052] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein the VH comprises (i) a VH CDR1 of SEQ ID NO: 247, a VH CDR2 of SEQ ID NO: 254, and a VH CDR3 of SEQ ID NO: 277, (ii) a VH CDR1 of SEQ ID NO: 284, a VH CDR2 of SEQ ID NO: 292, and a VH CDR3 of SEQ ID NO: 310, or (iii) a CDR1 of SEQ ID NO: 318, a CDR2 of SEQ ID NO: 326, and a CDR3 of SEQ ID NO: 352.

[0053] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1 of SEQ ID NO: 247, a VH CDR2 of SEQ ID NO: 254, and a VH CDR3 of SEQ ID NO: 266, and the VL comprises a VL CDR1 of SEQ ID NO: 111, a VL CDR2 of SEQ ID NO: 123, and a VL CDR3 of SEQ ID NO: 132; (ii) the VH comprises a VH CDR1 of SEQ ID NO: 284, a VH CDR2 of SEQ ID NO: 292, and a VH CDR3 of SEQ ID NO: 299, and the VL comprises a VL CDR1 of SEQ ID NO: 152, a VL CDR2 of SEQ ID NO: 163, and a VL CDR3 of SEQ ID NO: 168; or (iii) the VH comprises a VH CDR1 of SEQ ID NO: 318, a VH CDR2 of SEQ ID NO: 326, and a VH CDR3 of SEQ ID NO: 340. and the VL comprises a VL CDR1 of SEQ ID NO: 188, a VL CDR2 of SEQ ID NO: 197, and a VL CDR3 of SEQ ID NO: 207.

[0054] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1 of SEQ ID NO: 248, a VH CDR2 of SEQ ID NO: 255, and a VH CDR3 of SEQ ID NO: 267, and the VL comprises a VL CDR1 of SEQ ID NO: 112, a VL CDR2 of SEQ ID NO: 124, and a VL CDR3 of SEQ ID NO: 133; (ii) the VH comprises a VH CDR1 of SEQ ID NO: 285, a VH CDR2 of SEQ ID NO: 293, and a VH CDR3 of SEQ ID NO: 300, and the VL comprises a VL CDR1 of SEQ ID NO: 153, a VL CDR2 of SEQ ID NO: 164, and a VL CDR3 of SEQ ID NO: 169; or (iii) the VH comprises a VH CDR1 of SEQ ID NO: 319, a VH CDR2 of SEQ ID NO: 327, and a VH CDR3 of SEQ ID NO: 341. and the VL comprises a VL CDR1 of SEQ ID NO: 189, a VL CDR2 of SEQ ID NO: 198, and a VL CDR3 of SEQ ID NO: 208.

[0055] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDRl of SEQ ID NO: 249, a VH CDR2 of SEQ ID NO: 256, and a VH CDR3 of SEQ ID NO: 268, and the VL comprises a VL CDRl of SEQ ID NO: 113, a VL CDR2 of SEQ ID NO: 125, and a VL CDR3 of SEQ ID NO: 134; (ii) the VH comprises a VH CDRl of SEQ ID NO: 286, a VH CDR2 of SEQ ID NO: 294, and a VH CDR3 of SEQ ID NO: 301, and the VL comprises a VL CDRl of SEQ ID NO: 154, a VL CDR2 of SEQ ID NO: 165, and a VL CDR3 of SEQ ID NO: 170; or (iii) the VH comprises a VH CDRl of SEQ ID NO: 320, a VH CDR2 of SEQ ID NO: 328, and a VH CDR3 of SEQ ID NO: 342. and the VL comprises a VL CDR1 of SEQ ID NO: 190, a VL CDR2 of SEQ ID NO: 199, and a VL CDR3 of SEQ ID NO: 209.

[0056] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDRl of SEQ ID NO: 250, a VH CDR2 of SEQ ID NO: 257, and a VH CDR3 of SEQ ID NO: 269, and the VL comprises a VL CDRl of SEQ ID NO: 114, a VL CDR2 of SEQ ID NO: 126, and a VL CDR3 of SEQ ID NO: 135; (ii) the VH comprises a VH CDRl of SEQ ID NO: 287, a VH CDR2 of SEQ ID NO: 295, and a VH CDR3 of SEQ ID NO: 302, and the VL comprises a VL CDRl of SEQ ID NO: 155, a VL CDR2 of SEQ ID NO: 165, and a VL CDR3 of SEQ ID NO: 171; or (iii) the VH comprises a VH CDRl of SEQ ID NO: 321, a VH CDR2 of SEQ ID NO: 329, and a VH CDR3 of SEQ ID NO: 343. and the VL comprises a VL CDR1 of SEQ ID NO: 191, a VL CDR2 of SEQ ID NO: 200, and a VL CDR3 of SEQ ID NO: 210.

[0057] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDRl of SEQ ID NO: 251, a VH CDR2 of SEQ ID NO: 258, and a VH CDR3 of SEQ ID NO: 270, and the VL comprises a VL CDRl of SEQ ID NO: 115, a VL CDR2 of SEQ ID NO: 127, and a VL CDR3 of SEQ ID NO: 136; (ii) the VH comprises a VH CDRl of SEQ ID NO: 288, a VH CDR2 of SEQ ID NO: 295, and a VH CDR3 of SEQ ID NO: 303, and the VL comprises a VL CDRl of SEQ ID NO: 156, a VL CDR2 of SEQ ID NO: 166, and a VL CDR3 of SEQ ID NO: 172; or (iii) the VH comprises a VH CDRl of SEQ ID NO: 322, a VH CDR2 of SEQ ID NO: 330, and a VH CDR3 of SEQ ID NO: 344. and the VL comprises a VL CDR1 of SEQ ID NO: 192, a VL CDR2 of SEQ ID NO: 201, and a VL CDR3 of SEQ ID NO: 211.

[0058] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1 of SEQ ID NO: 248, a VH CDR2 of SEQ ID NO: 259, and a VH CDR3 of SEQ ID NO: 271, and the VL comprises a VL CDR1 of SEQ ID NO: 116, a VL CDR2 of SEQ ID NO: 126, and a VL CDR3 of SEQ ID NO: 137; (ii) the VH comprises a VH CDR1 of SEQ ID NO: 285, a VH CDR2 of SEQ ID NO: 296, and a VH CDR3 of SEQ ID NO: 304, and the VL comprises a VL CDR1 of SEQ ID NO: 157, a VL CDR2 of SEQ ID NO: 165, and a VL CDR3 of SEQ ID NO: 173; or (iii) the VH comprises a VH CDR1 of SEQ ID NO: 319, a VH CDR2 of SEQ ID NO: 331, and a VH CDR3 of SEQ ID NO: 345. and the VL comprises a VL CDR1 of SEQ ID NO: 193, a VL CDR2 of SEQ ID NO: 200, and a VL CDR3 of SEQ ID NO: 212.

[0059] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1 of SEQ ID NO: 247, a VH CDR2 of SEQ ID NO: 254, and a VH CDR3 of SEQ ID NO: 277, and the VL comprises a VL CDR1 of SEQ ID NO: 111, a VL CDR2 of SEQ ID NO: 123, and a VL CDR3 of SEQ ID NO: 132; (ii) the VH comprises a VH CDR1 of SEQ ID NO: 284, a VH CDR2 of SEQ ID NO: 292, and a VH CDR3 of SEQ ID NO: 310, and the VL comprises a VL CDR1 of SEQ ID NO: 152, a VL CDR2 of SEQ ID NO: 163, and a VL CDR3 of SEQ ID NO: 168; or (iii) the VH comprises a VH CDR1 of SEQ ID NO: 318, a VH CDR2 of SEQ ID NO: 326, and a VH CDR3 of SEQ ID NO: 352. and the VL comprises a VL CDR1 of SEQ ID NO: 168, a VL CDR2 of SEQ ID NO: 197, and a VL CDR3 of SEQ ID NO: 207.

[0060] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 584. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 84. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 584 and the VL comprises the sequence of SEQ ID NO: 84.

[0061] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 585. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 85. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 585 and the VL comprises the sequence of SEQ ID NO: 85.

[0062] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 586. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 86. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 586 and the VL comprises the sequence of SEQ ID NO: 86.

[0063] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 587. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 87. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 587 and the VL comprises the sequence of SEQ ID NO: 87.

[0064] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 588. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 588 and the VL comprises the sequence of SEQ ID NO: 88.

[0065] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 589. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 89. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 589 and the VL comprises the sequence of SEQ ID NO: 89.

[0066] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 592. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 86. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 592 and the VL comprises the sequence of SEQ ID NO: 86.

[0067] In certain embodiments, the VH comprises the sequence of SEQ ID NO: 238. In certain embodiments, the VL comprises the sequence of SEQ ID NO: 84. In certain embodiments, the VH comprises the sequence of SEQ ID NO: 238 and the VL comprises the sequence of SEQ ID NO: 84.

[0068] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 584. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 84. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 584, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 84.

[0069] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 585. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 85. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 585, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 85.

[0070] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 586. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 86. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 586, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 86.

[0071] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 587. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 87. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 587, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 87.

[0072] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 588. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 88. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 588, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 88.

[0073] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 589. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 89. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 589, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 89.

[0074] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 592. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 86. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 592, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 86.

[0075] In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 238. In certain embodiments, the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 84. In certain embodiments, the VH comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 238, and the VL comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 84.

[0076] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, the VH comprising a VH CDR1, a VH CDR2 and a VH CDR3, the VH CDR1 having the sequence number set forth in Table 1 as the VH CDR1 for the antibody number listed in Table 1, the VH CDR2 having the sequence number set forth in Table 1 as the VH CDR2 of the antibody, and the VH CDR3 having the sequence number set forth in Table 1 as the VH CDR3 of the antibody.

[0077] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, the VH comprising a VH CDR1, a VH CDR2 and a VH CDR3, the VH CDR1 having the sequence number set forth in Table 2 as the VH CDR1 for the antibody number listed in Table 2, the VH CDR2 having the sequence number set forth in Table 2 as the VH CDR2 of the antibody, and the VH CDR3 having the sequence number set forth in Table 2 as the VH CDR3 of the antibody.

[0078] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, the VH comprising a VH CDR1, a VH CDR2 and a VH CDR3, the VH CDR1 having the sequence number set forth in Table 3 as the VH CDR1 for the antibody number listed in Table 3, the VH CDR2 having the sequence number set forth in Table 3 as the VH CDR2 of the antibody, and the VH CDR3 having the sequence number set forth in Table 3 as the VH CDR3 of the antibody.

[0079] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2 and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 1 as the VH CDR1 for the antibody number listed in Table 1, the VH CDR2 has the sequence number set forth in Table 1 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 1 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2 and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 4 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 4 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 4 as the VL CDR3 of the antibody.

[0080] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2 and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 2 as the VH CDR1 for the antibody number listed in Table 2, the VH CDR2 has the sequence number set forth in Table 2 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 2 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2 and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 5 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 5 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 5 as the VL CDR3 of the antibody.

[0081] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2 and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 3 as the VH CDR1 for the antibody number listed in Table 3, the VH CDR2 has the sequence number set forth in Table 3 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 3 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2 and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 6 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 6 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 6 as the VL CDR3 of the antibody.

[0082] In certain embodiments, the VH comprises a SEQ ID NO: listed as the VH of the antibody in Table 7. In certain embodiments, the VH comprises a SEQ ID NO: listed as the VH of the antibody in Table 7, and the VL comprises a SEQ ID NO: listed as the VL of the antibody in Table 8.

[0083] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, the antibody comprising a heavy chain and a light chain, wherein the light chain has the sequence number set forth in Table 9 as the light chain for the antibody number listed in Table 9, and the heavy chain has the sequence number set forth in Table 9 as the heavy chain of the antibody.

[0084] In certain embodiments, the antibody or antigen-binding fragment specifically binds to human VISTA (amino acids 33-311 of SEQ ID NO: 377).

[0085] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to VISTA, wherein the antibody recognizes an epitope of human VISTA comprising the amino acid sequence HLHHG (amino acids 98-102 of SEQ ID NO: 377; numbering starts with the first amino acid after the signal sequence (the signal sequence is underlined and bold; Table 11)) or VVEIRHHHSEHR (amino acids 148-159 of SEQ ID NO: 377; numbering starts with the first amino acid after the signal sequence (underlined and bold; Table 11)). In another embodiment, the antibody recognizes an epitope of human VISTA comprising tyrosine 37, arginine 54, valine 117, and arginine 127 of SEQ ID NO: 377 (numbering starts with the first amino acid after the signal sequence (underlined and bold in Table 11)).

[0086] In another aspect, provided herein is an antibody or antigen-binding fragment thereof that competes with a reference antibody for binding to VISTA, selected from the group consisting of: (a) a first immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 584 and (ii) a VL comprising the sequence of SEQ ID NO: 84; (b) a second immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 585 and (ii) a VL comprising the sequence of SEQ ID NO: 85; (c) a third immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 586 and (ii) a VL comprising the sequence of SEQ ID NO: 86; (d) a VH comprising the sequence of SEQ ID NO: 587; (e) a fourth immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 588 and (ii) a VL comprising the sequence of SEQ ID NO: 88; (f) a sixth immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 589 and (ii) a VL comprising the sequence of SEQ ID NO: 89; (g) a seventh immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 238 and (ii) a VL comprising the sequence of SEQ ID NO: 84; and (h) an eighth immunoglobulin comprising (i) a VH comprising the sequence of SEQ ID NO: 592 and (ii) a VL comprising the sequence of SEQ ID NO: 86.

[0087] In one embodiment, the antibody is a monoclonal antibody. In a specific embodiment, the antibody is a human antibody. In a specific embodiment, the antibody is an immunoglobulin.

[0088] In certain embodiments, the antibody comprises an Fc region or a variant of the Fc region, optionally wherein the Fc region is a human Fc region or a variant of the human Fc region having from 1 to 7 amino acid mutations in the Fc region compared to a native human Fc region, and / or optionally wherein the human Fc region is that of human IgG1, human IgG2, or human IgG4, further optionally wherein the antibody comprises a human IgG1 or human IgG4 constant region or a variant of the constant region having from 1 to 7 amino acid mutations in the constant region compared to a native constant region.

[0089] In certain embodiments, the antibody mediates antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the antibody mediates complement-dependent cell-mediated cytotoxicity (CDC). In certain embodiments, the antibody mediates antibody-dependent cellular phagocytosis (ADCP).

[0090] In certain embodiments, the antibody comprises the variant of the human Fc region. In certain embodiments, (a) the Fc region is that of human IgG1 and the mutations are C220D, D221C, E233P, L234A, L234E, L234Y, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, E318 A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system; or (b) the Fc region is that of a human IgG2 and the mutations are selected from the group consisting of C220D, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T 256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, S383N, M428L, N434S, and N434Y, wherein the residues are selected from the group consisting of: or (c) the Fc region is that of human IgG4 and the mutations are S228P, E233P, F234A, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309L, E318A, K322A,and selected from the group consisting of S324T, K326A, K326M, L328R, P329A, P329G, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system.

[0091] In certain embodiments, the Fc region is (a) that of human IgG1, and the mutations include L234A and L235A, and optionally P329G, where the residues are numbered using the EU numbering system, or (b) that of human IgG4, and the mutations include F234A and L235A, and optionally P329G, where the residues are numbered using the EU numbering system.

[0092] In certain embodiments, the Fc region is of human IgG1, IgG2, or IgG4, and the mutations comprise M252Y, S254T, and T256E, where the residues are numbered using the EU numbering system.

[0093] In a specific embodiment, the Fc region is of human IgG1, IgG2, or IgG4, and the mutations comprise M428L and N434S, wherein the residues are numbered using the EU numbering system.

[0094] In a specific embodiment, the Fc region is that of human IgG4 and the mutations include S228P and L235E, where the residues are numbered using the EU numbering system.

[0095] In one embodiment, the Fc region is human IgG4 and the mutations comprise S228P, M252Y, S254T, and T256E, where the residues are numbered using the EU numbering system.

[0096] In one embodiment, the Fc region is that of human IgG4 and the mutations include S228P, M428L and N434S, where the residues are numbered using the EU numbering system.

[0097] In certain embodiments, the antibody or antigen-binding fragment is a bispecific or trispecific antibody. In other embodiments, the antibody or antigen-binding fragment is BiTE or TriKE.

[0098] In certain embodiments, the antigen-binding fragment is an Fv fragment, a Fab fragment, or an F(ab')2 fragment.

[0099] In certain embodiments, the antibody or antigen-binding fragment thereof is purified. In other embodiments, the antibody or antigen-binding fragment thereof is isolated.

[0100] In another aspect, provided herein is a single-chain Fv (ScFv) comprising a VH and a VL separated by a linker sequence, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has a sequence number set forth in the table selected from the group consisting of Tables 1 to 3 as the VH CDR1 for the antibody number listed in the table, the VH CDR2 has a sequence number set forth in the table as the VH CDR2 of the antibody, and the VH CDR3 has a sequence number set forth in the table as the VH CDR3 of the antibody. In certain embodiments, the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has a SEQ ID NO: set forth in a table selected from the group consisting of Tables 4-6 as the VL CDR1 of the antibody, the VL CDR2 has a SEQ ID NO: set forth in the table as the VL CDR2 of the antibody, and the VL CDR3 has a SEQ ID NO: set forth in the table as the VL CDR3 of the antibody, and the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 are all defined by the same CDR numbering system. In certain embodiments, the VH comprises a SEQ ID NO: set forth in Table 7 as the VH of the antibody. In certain embodiments, the VL comprises a SEQ ID NO: set forth in Table 8 as the VL of the antibody.

[0101] In another aspect, provided herein is a fusion protein comprising an scFv provided herein.

[0102] In another aspect, provided herein is an antibody-drug conjugate comprising the antibody or antigen-binding fragment, scFv, or fusion protein of any of the preceding embodiments linked (optionally covalently linked) to a therapeutic agent.

[0103] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising the scFv of any of the foregoing embodiments. Also provided herein is a cell expressing the CAR.

[0104] In another aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment, scFv, fusion protein, or CAR of any of the preceding embodiments.

[0105] In another aspect, provided herein is an ex vivo cell comprising one or more polynucleotides each comprising a nucleotide sequence encoding the antibody or antigen-binding fragment, scFv, fusion protein, or CAR of any of the preceding embodiments. Also provided herein is a method of producing an antibody or antigen-binding fragment, scFv, or fusion protein, or CAR, comprising culturing the cell under conditions such that the one or more polynucleotides are expressed by the cell to produce the antibody or antigen-binding fragment, scFv, or fusion protein, or CAR, encoded by the polynucleotide.

[0106] In another aspect, provided herein is a pharmaceutical composition comprising (a) a therapeutically effective amount of the antibody or antigen-binding fragment, scFv, fusion protein, CAR, antibody-drug conjugate, or cell of any of the preceding embodiments, and (b) a pharmaceutically acceptable carrier. Also provided herein is a method for treating cancer in a subject in need thereof, comprising administering the pharmaceutical composition to the subject. In certain embodiments, the cancer is non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, blood cancer, hepatocellular carcinoma, ovarian cancer, mesothelioma, neuroblastoma, oral cancer, thyroid cancer, breast cancer, sarcoma, pancreatic cancer, colon cancer, gastric cancer, choriocarcinoma, testicular cancer, skin cancer, renal cell carcinoma, bladder cancer, hematological cancer (e.g., acute myeloid leukemia), or cervical cancer. In one embodiment, the cancer is myelodysplastic syndrome. In certain embodiments, the cancer is metastatic cancer. In certain embodiments, the cancer is acute myeloid leukemia, and the pharmaceutical composition comprises a therapeutically effective amount of the antibody-drug conjugate, wherein the therapeutic agent is optionally a cytotoxic agent.

[0107] In certain embodiments, the method of treating cancer further comprises administering an additional therapy to the subject. In certain embodiments, the additional therapy is for treating the cancer. In certain embodiments, the method of treating cancer further comprises administering to the subject a chemotherapeutic agent, a tyrosine kinase inhibitor, and / or an immune checkpoint inhibitor. In certain embodiments, the method of treating cancer further comprises administering to the subject an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of programmed death-1 (PD-1), or programmed death-ligand 1 (PDL1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, the subject is human. In one embodiment, the additional therapy is radiation therapy. [Brief explanation of the drawings]

[0108] [Figure 1A]Octet sensorgram measuring the affinity of anti-VISTA antibody No. 269.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1B] Octet sensorgram measuring the affinity of anti-VISTA antibody number 321.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1C] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 245.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1D] Octet sensorgram measuring the affinity of anti-VISTA antibody number 465.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1E]Octet sensorgram measuring the affinity of anti-VISTA antibody number 457.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1F] Octet sensorgram measuring the affinity of anti-VISTA antibody number 173.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1G] Octet sensorgram measuring the affinity of anti-VISTA antibody number 833.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1H] Octet sensorgram measuring the affinity of anti-VISTA antibody number 150.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1I]Octet sensorgram measuring the affinity of anti-VISTA antibody number 474.1 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1J] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 80 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1K] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 85 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1L] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 87 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1M]Octet sensorgram measuring the affinity of anti-VISTA antibody No. 91 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 1N] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 92 for human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2A] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 269.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2B] Octet sensorgram measuring the affinity of anti-VISTA antibody number 321.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2C]Octet sensorgram measuring the affinity of anti-VISTA antibody number 245.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2D] Octet sensorgram measuring the affinity of anti-VISTA antibody number 465.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2E] Octet sensorgram measuring the affinity of anti-VISTA antibody number 457.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2F] Octet sensorgram measuring the affinity of anti-VISTA antibody number 173.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 2G]Octet sensorgram measuring the affinity of anti-VISTA antibody number 833.1 for monkey VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged cynomolgus monkey VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3A] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 269.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on the anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3B] Octet sensorgram measuring the affinity of anti-VISTA antibody number 321.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3C] Octet sensorgram measuring the affinity of anti-VISTA antibody No. 245.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3D]Octet sensorgram measuring the affinity of anti-VISTA antibody No. 465.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3E] Octet sensorgram measuring the affinity of anti-VISTA antibody number 457.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3F] Octet sensorgram measuring the affinity of anti-VISTA antibody number 173.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 3G] Octet sensorgram measuring the affinity of anti-VISTA antibody number 833.1 for mouse VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system. Sensorgram showing binding and dissociation of antigen (his-tagged mouse VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). [Figure 4A]Full kinetic characterization of the binding of anti-VISTA antibody number 321.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4B] Full kinetic characterization of the binding of anti-VISTA antibody No. 245.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4C] Full kinetic characterization of the binding of anti-VISTA antibody No. 465.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4D]Full kinetic characterization of the binding of anti-VISTA antibody No. 457.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) to anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4E] Full kinetic characterization of the binding of anti-VISTA antibody number 173.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) to anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4F] Full kinetic characterization of the binding of anti-VISTA antibody No. 150.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 4G]Full kinetic characterization of the binding of anti-VISTA antibody No. 474.1 to human VISTA. Antibody affinity was measured using biolayer interferometry with the Octet Red 96 (ForteBio) system over a range of antigen concentrations. Sensorgrams showing the binding and dissociation of antigen (his-tagged human VISTA Met1-Ala194) on anti-VISTA antibody immobilized on a biosensor (anti-human IgG Fc capture dip and read sensor). Antigen concentrations that gave a response between 0.1 and 0.5 nM were used for the analysis. [Figure 5A] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5B] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5C]Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5D] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5E] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5F]Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA. Using an ELISA assay, binding between immobilized antigen (His-tagged VISTA extracellular domain) on a microtiter plate and anti-VISTA antibody was measured over a concentration range of 0.003 to 10 μg / mL. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents dose-response data for one to three antibodies (antibodies examined are indicated on the x-axis). [Figure 5G] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA at multiple pH levels. Using an ELISA assay, binding between a microtiter plate-immobilized antigen (His-tagged VISTA extracellular domain) and anti-VISTA antibodies was measured at concentrations ranging from 0.16 to 1000 ng / mL at pH 6.0, 6.5, 7.0, and 7.4. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents the dose-response data for a single antibody at each pH tested. Maxisorb ELISA plates were used for this assay. [Figure 5H] Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA at multiple pH levels. Using an ELISA assay, binding between a microtiter plate-immobilized antigen (His-tagged VISTA extracellular domain) and anti-VISTA antibodies was measured at concentrations ranging from 0.16 to 1000 ng / mL at pH 6.0, 6.5, 7.0, and 7.4. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents the dose-response data for a single antibody at each pH tested. Maxisorb ELISA plates were used for this assay. [Figure 5I]Dose-response ELISA analysis of anti-VISTA antibody binding to human VISTA at multiple pH levels. Using an ELISA assay, binding between a microtiter plate-immobilized antigen (His-tagged VISTA extracellular domain) and anti-VISTA antibodies was measured at concentrations ranging from 0.16 to 1000 ng / mL at pH 6.0, 6.5, 7.0, and 7.4. Antigen-antibody complexes were measured using a chromogenic assay, and data are plotted as absorbance at 450 nm versus log-transformed antibody concentration. Curves were fitted by nonlinear regression, and EC50 values ​​were calculated. Each graph represents the dose-response data for a single antibody at each pH tested. Maxisorb ELISA plates were used for this assay. [Figure 6A] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6B] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6C] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6D] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6E]Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6F] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6G] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 6H] Anti-VISTA antibodies are specific for VISTA. Anti-VISTA antibodies were screened for binding to other B7 family members. All of these antibodies were specific for VISTA and did not bind to other related human proteins. The antibodies tested are indicated on the x-axis. [Figure 7A] Anti-VISTA antibodies binding to VISTA stably expressed in CHO K1 cells. Antibodies were evaluated for their ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Cells were incubated with 0.05, 1, or 150 nM of each anti-VISTA antibody. Cell-bound antibodies were detected with a PE-conjugated secondary antibody, and the mean fluorescence intensity (MFI) of binding was measured by FACS analysis. Data are plotted as log-transformed MFI. Each antibody was evaluated against CHO cells expressing human ("Hs"), cynomolgus monkey ("Mf"), and mouse ("Mm") VISTA. Circles represent MFI at an antibody concentration of 150 nM, squares represent MFI at an antibody concentration of 1 nM, and triangles represent MFI at an antibody concentration of 0.05 nM. The antibodies tested are indicated on the x-axis. [Figure 7B]Anti-VISTA antibodies binding to VISTA stably expressed in CHO K1 cells. Antibodies were evaluated for their ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Cells were incubated with 0.05, 1, or 150 nM of each anti-VISTA antibody. Cell-bound antibodies were detected with a PE-conjugated secondary antibody, and the mean fluorescence intensity (MFI) of binding was measured by FACS analysis. Data are plotted as log-transformed MFI. Each antibody was evaluated against CHO cells expressing human ("Hs"), cynomolgus monkey ("Mf"), and mouse ("Mm") VISTA. Circles represent MFI at an antibody concentration of 150 nM, squares represent MFI at an antibody concentration of 1 nM, and triangles represent MFI at an antibody concentration of 0.05 nM. The antibodies tested are indicated on the x-axis. [Figure 7C] Anti-VISTA antibodies binding to VISTA stably expressed in CHO K1 cells. Antibodies were evaluated for their ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Cells were incubated with 0.05, 1, or 150 nM of each anti-VISTA antibody. Cell-bound antibodies were detected with a PE-conjugated secondary antibody, and the mean fluorescence intensity (MFI) of binding was measured by FACS analysis. Data are plotted as log-transformed MFI. Each antibody was evaluated against CHO cells expressing human ("Hs"), cynomolgus monkey ("Mf"), and mouse ("Mm") VISTA. Circles represent MFI at an antibody concentration of 150 nM, squares represent MFI at an antibody concentration of 1 nM, and triangles represent MFI at an antibody concentration of 0.05 nM. The antibodies tested are indicated on the x-axis. [Figure 8A] Multipoint dose-response binding data for cell surface-expressed VISTA. Human IgG1 controls were evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for their ability to bind to human, mouse, and cynomolgus VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8B] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #269.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8C] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #321.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8D] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #245.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8E]Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #465.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8F] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #457.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8G] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #173.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8H]Multipoint dose-response binding data for cell surface-expressed VISTA. The anti-VISTA antibody VSTB174 was evaluated for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or at 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8I] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #474.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8J] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #150.1 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8K]Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #80 was evaluated for its ability to bind to human, mouse, and cynomolgus VISTA stably expressed in CHO K1 cells at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8L] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #92 was evaluated for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or at 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8M] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #87 was evaluated for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or at 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8N]Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #91 was evaluated for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8O] Multipoint dose-response binding data for cell surface-expressed VISTA. Human IgG4 controls were evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for their ability to bind to human, mouse, and cynomolgus VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8P] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #245.4 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8Q]Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #465.4 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8R] Multipoint dose-response binding data for cell surface-expressed VISTA. Anti-VISTA antibody #173.4 was evaluated at 0.006, 0.024, 0.098, 0.391, 1.563, 6.25, 25, and 100 nM, or 0.003, 0.012, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 nM for its ability to bind to human, mouse, and cynomolgus monkey VISTA stably expressed in CHO K1 cells. Data were plotted as log-transformed MFI versus log-transformed antibody concentration. Curves were fitted by nonlinear regression with variable slope, and EC50 values ​​were calculated. [Figure 8S] Assessment of binding of mutant VISTA-ECD-Fc to antibody no. 474.1. Binding of antibody no. 474.1 was investigated using human VISTA-ECD mutants. Antibody no. 474.1 titrated from 100 μg / mL to 0.003 μg / mL was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody no. 474.1 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8T]Assessment of binding of mutant VISTA-ECD-Fc to antibody no. 150.1. Binding of antibody no. 150.1 was investigated using human VISTA-ECD mutants. Antibody no. 150.1 titrated from 100 μg / mL to 0.003 μg / mL was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody no. 150.1 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8U] Assessment of binding of mutant VISTA-ECD-Fc to antibody #85. Binding of antibody #85 was investigated using human VISTA-ECD mutants. Antibody #85, titrated from 100 μg / mL to 0.003 μg / mL, was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody #85 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8V] Assessment of binding of mutant VISTA-ECD-Fc to antibody #87. Binding of antibody #87 was investigated using human VISTA-ECD mutants. Antibody #87, titrated from 100 μg / mL to 0.003 μg / mL, was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody #87 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8W]Assessment of binding of mutant VISTA-ECD-Fc to antibody no. 91. Binding of antibody no. 91 was investigated using human VISTA-ECD mutants. Antibody no. 91, titrated from 100 μg / mL to 0.003 μg / mL, was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody no. 91 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8X] Assessment of binding of mutant VISTA-ECD-Fc to antibody no. 92. Binding of antibody no. 92 was investigated using human VISTA-ECD mutants. Antibody no. 92, titrated from 100 μg / mL to 0.003 μg / mL, was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. Antibody no. 92 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 8Y] Evaluation of binding of mutant VISTA-ECD-Fc to antibody VSTB174. VSTB174 binding was investigated using human VISTA-ECD mutants. VSTB174 titrated from 100 μg / mL to 0.003 μg / mL was captured onto 96-well plates coated with mutant VISTA-ECD at 3 μg / mL. VSTB174 was detected using biotinylated anti-human Ig light chain kappa followed by streptavidin-HRP and TMB substrate. Optical density at 450 nm was measured using a CLARIOstar plate reader. [Figure 9A]Effect of antibody #269.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of 3, 0.3, or 0.03 μg / ml of anti-VISTA or control antibody and 5 ng / ml of Staphylococcal enterotoxin B (SEB) for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 9B] Effect of antibody #833.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of 3, 0.3, or 0.03 μg / ml anti-VISTA or control antibody and 5 ng / ml Staphylococcal enterotoxin B (SEB) for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 9C] Effect of antibodies 474.1 (WT), 150.1 (YTE), and 246.4 compared to VSTB174 on SEB-induced stimulation of NK cell-depleted human PBMCs on day 4. NK cell-depleted human PBMCs were plated at 2x10 cells / well in the presence of tested anti-VISTA or control antibodies at 30, 3, 0.3, and 0.03 μg / ml and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog number 88-7316-88) according to the manufacturer's instructions. [Figure 10] Effect of antibody #321.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of tested anti-VISTA or control antibodies at 3, 0.3, or 0.03 μg / ml and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 11]Effect of antibody #245.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of 3, 0.3, or 0.03 μg / ml of anti-VISTA or control antibody and 5 ng / ml of SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 12] Effect of antibody #465.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of 3, 0.3, or 0.03 μg / ml of anti-VISTA or control antibody and 5 ng / ml of SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 13] Effect of antibody #457.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of tested anti-VISTA or control antibodies at 3, 0.3, or 0.03 μg / ml and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 14] Effect of antibody #173.1 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of 3, 0.3, or 0.03 μg / ml of anti-VISTA or control antibody and 5 ng / ml of SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. [Figure 15]Effect of antibody #245.1 and antibody #245.4 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of the listed anti-VISTA or control antibodies (3 μg / ml) and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. The antibodies tested are indicated on the x-axis. [Figure 16] Effect of antibody #465.1 and antibody #465.4 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of the listed anti-VISTA or control antibodies (3 μg / ml) and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog #88-7316-88) according to the manufacturer's instructions. The antibodies tested are indicated on the x-axis. [Figure 17] Effect of antibody no. 173.1 and antibody no. 173.4 on SEB-induced stimulation of NK-depleted human PBMCs on day 4. Human NK cell-depleted PBMCs were plated at 2 x 10 cells / well in the presence of the listed anti-VISTA or control antibodies (3 μg / ml) and 5 ng / ml SEB for 4 days at 37°C. IFN-γ production on day 4 was quantified by ELISA (Invitrogen catalog no. 88-7316-88) according to the manufacturer's instructions. The antibody numbers examined are indicated on the x-axis. [Figure 18]A and B. Effect of anti-VISTA antibody #269.1 on reversal of VISTA-mediated suppression of T cell activation. 2 x 10 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on plates coated with an antibody to CD3 ("Ab") in the presence of VISTA-coated beads (bead-to-cell ratio 2:1) and 100 μg / ml of antibody #269.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 19] A and B. Effect of anti-VISTA antibody #321.1 on reversal of VISTA-mediated suppression of T cell activation. 2 x 10 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on anti-CD3 Ab-coated plates in the presence of VISTA-coated beads (bead-to-cell ratio 2:1) and 100 μg / ml of antibody #321.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 20A] Effect of anti-VISTA antibody #245.1 on reversal of VISTA-mediated suppression of T cell activation. 2x105 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on anti-CD3 Ab-coated plates in the presence of VISTA-coated beads (bead-to-cell ratio 2:1) and 100µg / ml of antibody #245.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 20B]Effect of anti-VISTA antibody #245.1 on reversal of VISTA-mediated suppression of T cell activation. 2x105 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on anti-CD3 Ab-coated plates in the presence of VISTA-coated beads (bead-to-cell ratio 2:1) and 100µg / ml of antibody #245.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 21] A and B. Effect of anti-VISTA antibody #457.1 on reversal of VISTA-mediated suppression of T cell activation. 2 x 10 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on anti-CD3 Ab-coated plates in the presence of VISTA-coated beads (bead-to-cell ratio 2:1) and 100 μg / ml of antibody #457.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 22] A and B. Effect of anti-VISTA antibody #173.1 on reversal of VISTA-mediated suppression of T cell activation. 2 x 10 Cell Trace Violet-labeled human pan-T cells were cultured for 24 hours on anti-CD3 Ab-coated plates in the presence of VISTA-coated beads and 100 μg / ml of antibody #173.1 or control Ab. T cell proliferation was quantified by flow cytometry, and IFNγ was quantified by ELISA (R&D Systems, catalog #DY285B-05). [Figure 23A]Effect of anti-VISTA antibody #269.1 on upregulation of activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #269.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 23B] Effect of anti-VISTA antibody #269.1 on upregulation of activation marker CD86 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #269.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD86 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 23C] Effect of anti-VISTA antibody #269.1 on upregulation of activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #269.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 23D]Effect of anti-VISTA antibody #269.1 on CXCL10 secretion during 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #269.1 or control Ab. CXCL10 secretion during 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 24A] Effect of anti-VISTA antibody #833.1 on upregulation of activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #833.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 24B] Effect of anti-VISTA antibody #833.1 on upregulation of activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #833.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 24C] Effect of anti-VISTA antibody #833.1 on upregulation of CXCL10 secretion in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #833.1 or control Ab. CXCL10 secretion in 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 25A] Effect of anti-VISTA antibody #321.1 on upregulation of activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #321.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 25B] Effect of anti-VISTA antibody #321.1 on upregulation of activation marker CD86 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #321.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD86 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 25C] Effect of anti-VISTA antibody #321.1 on upregulation of activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #321.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 25D]Effect of anti-VISTA antibody #321.1 on CXCL10 secretion during 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #321.1 or control Ab. CXCL10 secretion during the 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 26A] Effect of anti-VISTA antibody #245.1 on upregulation of activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 26B] Effect of anti-VISTA antibody #245.1 on upregulation of activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 26C] Effect of anti-VISTA antibody #245.1 on upregulation of CXCL10 secretion in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 or control Ab. CXCL10 secretion in the 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 27] A-C. Effect of anti-VISTA antibody #465.1 on upregulation of activation markers CD80 and HLA-DR on CD14+ monocytes and CXCL10 secretion during 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of 10 μg / ml of antibody #465.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. Expression of CD80 and HLA-DR on CD14+ cells was quantified by mean fluorescence intensity (MFI). CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 28] A-D. Effect of anti-VISTA antibody #457.1 on upregulation of activation markers CD80, CD86, and HLA-DR on CD14+ monocytes and CXCL10 secretion during 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of 10 μg / ml of antibody #457.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. Expression of CD80, CD86, and HLA-DR on CD14+ cells was quantified by mean fluorescence intensity (MFI). CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 29]A-D. Effect of anti-VISTA antibody #173.1 on upregulation of activation markers CD80, CD86, and HLA-DR on CD14+ monocytes and CXCL10 secretion during 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of 10 μg / ml of antibody #173.1 or control Ab. Upregulation of activation markers on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. Expression of CD80, CD86, and HLA-DR on CD14+ cells was quantified by mean fluorescence intensity (MFI). CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-05). [Figure 30] A-C. Dose-response effects of anti-VISTA antibody #269.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour coculture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #269.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 31]A-C. Dose-response effects of anti-VISTA antibody #833.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour coculture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #833.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 32] A-C. Dose-response effects of anti-VISTA antibody #321.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour coculture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #321.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 33]A-C. Dose-response effects of anti-VISTA antibody #245.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour coculture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #245.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 34] A-C. Dose-response effects of anti-VISTA antibody #465.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour culture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were co-cultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #465.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of co-culture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 35]A-C. Dose-response effects of anti-VISTA antibody #457.1 on upregulation of activation markers HLA-DR and CD80 on CD14+ monocytes during 24-hour coculture, and on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of antibody #457.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 36A] Dose-response effect of anti-VISTA antibody #173.1 on upregulation of the activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of antibody #173.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. [Figure 36B] Dose-response effect of anti-VISTA antibody #173.1 on upregulation of the activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of antibody #173.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. [Figure 36C]Dose-response effect of anti-VISTA antibody #173.1 on CXCL10 secretion in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of antibody #173.1 or control Ab 30, 3, 0.3, 0.03, or 0.003 μg / ml. CXCL10 secretion in 24-hour coculture was quantified by ELISA (R&D Systems, catalog no. DY266-15). [Figure 36D] Dose-response effect of antibody #150.1 on the upregulation of the activation marker CD80 on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of the tested anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 36E] Dose-response effect of antibody #150.1 on CXCL10 secretion at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03µg / ml of the tested anti-VISTA Ab or isotype control Ab. CXCL10 chemokine secretion was quantified using the R&D Systems DuoSet ELISA Human CXCL10 Kit (R&D Systems, Product #DY266). [Figure 36F]Dose-response effect of antibody #150.1 on the upregulation of the activation marker HLA-DR on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of the tested anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 36G] Dose-response effect of antibody 474.1 (control VSTB174) on the upregulation of the activation marker CD80 on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of the tested anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 36H] Dose-response effect of antibody 474.1 (control VSTB174) on the upregulation of the activation marker HLA-DR on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of the tested anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 36I]Dose-response effect of antibody 474.1 (VSTB174 as control) on CXCL10 secretion at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3, 0.3, or 0.03µg / ml of the tested anti-VISTA Ab or isotype control Ab. CXCL10 chemokine secretion was quantified using the R&D Systems DuoSet ELISA Human CXCL10 Kit (R&D Systems, Product No. DY266). [Figure 37A] Effect of anti-VISTA antibodies on upregulation of the activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 and #245.4 or control Ab. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. Antibodies tested are indicated on the x-axis. [Figure 37B] Effect of anti-VISTA antibodies on upregulation of the activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 and #245.4 or control Ab. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. The antibodies tested are indicated on the x-axis. [Figure 37C]Effect of anti-VISTA antibodies on CXCL10 secretion during 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human PBMCs from the same donor for 24 hours in the presence of 10µg / ml of antibody #245.1 and antibody #245.4 or control Ab. CXCL10 secretion during 24-hour coculture was quantified by ELISA (R&D Systems, Cat. #DY266-05). Antibodies tested are indicated on the x-axis. [Figure 37D] Effect of antibody #474.1 and antibody #246.4 and their IgG4 counterparts on the upregulation of the induced activation marker CD80 on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3µg / ml of anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 37E] Effect of antibody #474.1 and antibody #246.4 and their IgG4 counterparts on CXCL10 secretion at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3µg / ml of anti-VISTA Ab or isotype control Ab. CXCL10 chemokine secretion was quantified using the R&D Systems DuoSet ELISA Human CXCL10 Kit (R&D Systems, Product #DY266). [Figure 37F]Effect of antibody No. 474.1 and antibody No. 246.4 and their IgG4 counterparts on the upregulation of the induced activation marker HLA-DR on CD14+ monocytes at 24 hours. 0.5x105 CD14+ enriched human PBMCs were co-cultured with 2x105 human PBMCs for 24 hours in the presence of 3µg / ml of anti-VISTA Ab or isotype control Ab. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. HLA-DR expression on CD14+ cells was quantified by mean fluorescence intensity (MFI). [Figure 38] A-C. Effect of anti-VISTA antibody on upregulation of activation markers CD80 and HLA-DR on CD14+ monocytes during 24-hour coculture, and effect of anti-VISTA antibody on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of 10 μg / ml of antibody #465.1 and #465.4 or control Ab. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). The antibodies tested are indicated on the x-axis. [Figure 39]A-C. Effect of anti-VISTA antibody on upregulation of activation markers CD80 and HLA-DR on CD14+ monocytes during 24-hour coculture, and effect of anti-VISTA antibody on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human PBMCs from the same donor for 24 hours in the presence of 10 μg / ml of antibody #173.1 and #173.4 or control Ab. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CXCL10 secretion during 24 hours of coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). The antibodies tested are indicated on the x-axis. [Figure 40] A–F. Dose-adjusted effects of anti-VISTA antibody No. 269.1 on upregulation of activation markers CD80 and HLA-DR on CD14+ monocytes during 24-hour coculture, and the role of NK cells and anti-VISTA antibody effects on CXCL10 secretion. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human total PBMCs (A–C) or NK-depleted human PBMCs (D–F) from the same donor for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of antibody No. 269.1, an IgG1 control, or negative control antibody No. 18.1. Upregulation of activation markers CD80 and HLA-DR on CD14+ cells at 24 hours was quantified by MFI measured by flow cytometry on gated live CD14+ cells. CXCL10 chemokine secretion during 24 h of coculture was quantified by ELISA (R&D Systems, catalog no. DY266-05). [Figure 41A]Dose-adjusted effect of anti-VISTA antibody No. 173.1 on upregulation of the activation marker CD80 on CD14+ monocytes in 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human whole PBMCs from the same donor for 24 hours in the presence of tested antibody No. 173.1 Ab at 3, 0.3, or 0.03 μg / ml, IGG1 control, or negative control antibody No. 18.1 Ab. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by MFI measured by flow cytometry on gated live CD14+ cells. [Figure 41B] Dose-adjusted effect of anti-VISTA antibody No. 173.1 on upregulation of the activation marker HLA-DR on CD14+ monocytes in 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 human whole PBMCs from the same donor for 24 hours in the presence of antibody No. 173.1 Ab, IGG1 control, or negative control antibody No. 18.1 Ab, tested at 3, 0.3, or 0.03 μg / ml. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by MFI measured by flow cytometry on gated live CD14+ cells. [Figure 41C] Effect of anti-VISTA antibodies on CXCL10 secretion during 24-hour coculture. 0.5x105 CD14+ enriched human PBMCs were cocultured with 2x105 human whole PBMCs from the same donor for 24 hours in the presence of antibody #173.1 Ab, IGG1 control, or negative control antibody #18.1 Ab, tested at 3, 0.3, or 0.03 μg / ml. CXCL10 chemokine secretion during 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 41D]Dose-adjusted effect of anti-VISTA antibody #173.1 and the role of NK cells on upregulation of the activation marker CD80 on CD14+ monocytes during 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 NK-depleted human PBMCs from the same donor for 24 hours in the presence of antibody #173.1 Ab, IGG1 control, or negative control antibody #18.1 Ab, tested at 3, 0.3, or 0.03 μg / ml. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by MFI measured by flow cytometry on gated live CD14+ cells. [Figure 41E] Dose-adjusted effect of anti-VISTA antibody No. 173.1 on upregulation of the activation marker HLA-DR on CD14+ monocytes during 24-hour coculture and the role of NK cells. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 NK-depleted human PBMCs from the same donor for 24 hours in the presence of antibody No. 173.1 Ab, IGG1 control, or negative control antibody No. 18.1 Ab, tested at 3, 0.3, or 0.03 μg / ml. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by MFI measured by flow cytometry on gated live CD14+ cells. [Figure 41F] Effect of anti-VISTA antibodies and the role of NK cells on CXCL10 secretion during 24-hour coculture. 0.5 x 105 CD14+ enriched human PBMCs were cocultured with 2 x 105 NK-depleted human PBMCs from the same donor for 24 hours in the presence of antibody #173.1 Ab, IGG1 control, or negative control antibody #18.1 Ab, tested at 3, 0.3, or 0.03 μg / ml. CXCL10 chemokine secretion during the 24-hour coculture was quantified by ELISA (R&D Systems, catalog #DY266-05). [Figure 41G]Dose-response effects of antibody #474.1 and antibody #150.1 on the upregulation of the activation marker CD80 on CD14+ monocytes at 24 hours and the contribution of NK cells. 0.5x105 CD14+ enriched human PBMCs were co-cultured with either 2x105 human PBMCs from the same donor or 2x105 NK-depleted human PBMCs for 24 hours in the presence of the tested anti-VISTA Ab or isotype control Ab at 3, 0.3, or 0.03 μg / ml. Upregulation of the activation marker CD80 on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. CD80 expression on CD14+ cells in the presence of total human PBMCs (left) or NK-depleted human PBMCs (right) was quantified by mean fluorescence intensity (MFI). [Figure 41H] Dose-response effects of antibody #474.1 and antibody #150.1 on CXCL10 secretion and NK cell contribution. 0.5x105 CD14+ enriched human PBMCs were co-cultured with either 2x105 human PBMCs or 2x105 NK-depleted human PBMCs from the same donor for 24 hours in the presence of 3, 0.3, or 0.03 μg / ml of the tested anti-VISTA Ab or isotype control Ab. CXCL10 chemokine secretion was quantified using the R&D Systems DuoSet ELISA Human CXCL10 Kit (R&D Systems, Product #DY266). [Figure 41I]Dose-response effects of antibody #474.1 and antibody #150.1 on the upregulation of the activation marker HLA-DR on CD14+ monocytes at 24 hours and the contribution of NK cells. 0.5x105 CD14+ enriched human PBMCs were co-cultured with either 2x105 human PBMCs from the same donor or 2x105 NK-depleted human PBMCs for 24 hours in the presence of the tested anti-VISTA Ab or isotype control Ab at 3, 0.3, or 0.03 μg / ml. Upregulation of the activation marker HLA-DR on CD14+ cells at 24 hours was quantified by flow cytometry on gated live CD14+ cells. Expression of HLA-DR on CD14+ cells in the presence of total human PBMCs (left) or NK-depleted human PBMCs (right) was quantified by mean fluorescence intensity (MFI). [Figure 42] A and B. Effect of anti-VISTA antibody #321.1 on the suppressive activity of cytokine-induced MDSCs. Human PBMCs from healthy donors were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng / ml) and IL-6 (10 ng / ml). After 7 days, the ability of CD11b+ cells (MDSCs) to suppress anti-CD3 Ab-induced PBMC proliferation was measured in the presence of antibody #321.1 or an isotype control Ab. PBMCs (2 x 105 cells / well) were labeled with 5 mM CellTrace Violet and then added to MDSCs (2 x 105 cells / well) in the presence of anti-CD3 Ab (2 μg / ml). After 4 days of incubation, T cell proliferation was measured by flow cytometry analysis (Attune Nxt), and IFNγ production was measured by ELISA (ProQuantum). Bars represent single specimens. [Figure 43]A and B. Effect of anti-VISTA antibodies 245.1 and 245.4 on the suppressive activity of cytokine-induced MDSCs. Human PBMCs from healthy donors were cultured for 7 days in the presence of GM-CSF (10 ng / ml) and IL-6 (10 ng / ml). After 7 days, the ability of CD11b+ cells (MDSCs) to suppress anti-CD3 Ab-induced PBMC proliferation was measured in the presence of antibodies 245.1 and 245.4 or an isotype control Ab. PBMCs (2 x 105 cells / well) were labeled with 5 mM CellTrace Violet and then added to MDSCs (2 x 105 cells / well) in the presence of anti-CD3 Ab (2 μg / ml). After 4 days of incubation, T cell proliferation was measured by flow cytometry (Attune Nxt) and IFNγ production was measured by ELISA (ProQuantum). Bars represent single specimens. The antibodies tested are indicated on the x-axis. [Figure 44] A and B. Effect of anti-VISTA antibody #465.1 on the suppressive activity of cytokine-induced MDSCs. Human PBMCs or CD11b+ cells from healthy donors were cultured for 7 days in the presence of GM-CSF (10 ng / ml) and IL-6 (10 ng / ml). After 7 days, the ability of CD11b+ cells (MDSCs) to suppress anti-CD3 Ab-induced proliferation of PBMCs was measured in the presence of antibody #465.1 or an isotype control Ab. PBMCs (2 x 105 cells / well) were labeled with 5 mM CellTrace Violet and then added to MDSCs (2 x 105 cells / well) in the presence of anti-CD3 Ab (2 μg / ml). After 4 days of incubation, T cell proliferation was measured by flow cytometry (Attune Nxt) and IFNγ production was measured by ELISA (ProQuantum). Bars represent single specimens. [Figure 45]A and B. Effect of anti-VISTA antibody #457.1 on the suppressive activity of cytokine-induced MDSCs. Human PBMCs or CD11b+ cells from healthy donors were cultured for 7 days in the presence of GM-CSF (10 ng / ml) and IL-6 (10 ng / ml). After 7 days, the ability of CD11b+ cells (MDSCs) to suppress anti-CD3 Ab-induced proliferation of PBMCs was measured in the presence of antibody #457.1 or an isotype control Ab. PBMCs (2 x 105 cells / well) were labeled with 5 mM CellTrace Violet and then added to MDSCs (2 x 105 cells / well) in the presence of anti-CD3 Ab (2 μg / ml). After 4 days of incubation, T cell proliferation was measured by flow cytometry (Attune Nxt) and IFNγ production was measured by ELISA (ProQuantum). Bars represent single specimens. [Figure 46] A and B. Effect of anti-VISTA antibody #173.1 on the suppressive activity of cytokine-induced MDSCs. Human PBMCs or CD11b+ cells from healthy donors were cultured for 7 days in the presence of GM-CSF (10 ng / ml) and IL-6 (10 ng / ml). After 7 days, the ability of CD11b+ cells (MDSCs) to suppress anti-CD3 Ab-induced proliferation of PBMCs was measured in the presence of antibody #173.1 or an isotype control Ab. PBMCs (2 x 105 cells / well) were labeled with 5 mM CellTrace Violet and then added to MDSCs (2 x 105 cells / well) in the presence of anti-CD3 Ab (2 μg / ml). After 4 days of incubation, T cell proliferation was measured by flow cytometry (Attune Nxt) and IFNγ production was measured by ELISA (ProQuantum). Bars represent single specimens. [Figure 47A]Evaluation of Fc variants in FcRn binding assays. Antibody No. 173 wild-type (WT) in IgG1 or IgG4 backbones, as well as antibody No. 289 (with M428L and N434S substitutions according to EU numbering) and antibody No. 420 (with M252Y, S254T, and T256E substitutions according to EU numbering), were tested for their ability to bind to the FcRn receptor using the AlphaLisa binding kit (Perkin Elmer #AL3095C). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcRn was prepared at 800 ng / ml (4x the final concentration), and streptavidin donor and huIgG binding acceptor beads were prepared at 20 mg / ml (2x the final concentration) followed by 90 minutes of incubation in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. Antibodies tested are indicated in the legend. [Figure 47B] Evaluation of Fc variants in the FcRn binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (antibody #246.4), and the YTE variant (SEQ ID NO:150.1) in the IgG1 backbone were tested for their ability to bind to the FcRn receptor using the AlphaLisa binding kit (Perkin Elmer #AL3095C). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcRn was prepared at 800 ng / ml (4x), and streptavidin donor and huIgG binding acceptor beads were prepared at 40 mg / ml (2x) followed by 90 minutes of incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 48A]Evaluation of Fc variants in an immunoglobulin gamma Fc receptor 1 (FcγR1) binding assay. Antibody #173WT and the LS (antibody #289) and YTE (antibody #420) variants, respectively, in IgG1 or IgG4 backbones, were tested for their ability to bind to the FcγR1 receptor using the AlphaLisa binding kit (Perkin Elmer #AL3081C). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcγR1 was prepared at 200 ng / ml (4x the final concentration), and streptavidin donor and huIgG binding acceptor beads were prepared at 40 mg / ml (2x the final concentration) and incubated for 90 minutes in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. The antibodies tested are indicated in the legend. [Figure 48B] Evaluation of Fc variants in an FcγR1 binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (#246.4), and the YTE variant (#150.1) in an IgG1-based framework were tested for their ability to bind to the FcγR1 receptor using the AlphaLisa binding kit (Perkin Elmer #AL3081C). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcγR1 was prepared at 200 ng / ml (4x), and streptavidin donor and huIgG binding acceptor beads were prepared at 40 μg / ml (2x) followed by 90 min incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 49A]Evaluation of Fc variants in an immunoglobulin gamma Fc receptor IIa (FcγR2a) (allele 167R) binding assay. Antibody #173WT, LS (antibody #289), and YTE (antibody #420) variants, respectively, in IgG1 or IgG4 backbones, were tested for their ability to bind to the FcγR2a receptor (allele 167R) using AlphaLisa binding kits (Perkin Elmer #AL3086C and #AL3087C). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcγR2a (167R) was prepared at 200 ng / ml (4x the final concentration), and streptavidin donor and huIgG binding acceptor beads were prepared at 20 mg / ml (2x the final concentration) and incubated for 90 minutes in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. Antibodies tested are indicated in the legend. [Figure 49B] Evaluation of Fc variants in an immunoglobulin gamma Fc receptor IIa (FcγR2a) (allele 167H) binding assay. Antibody #173WT, in either an IgG1 or IgG4 backbone, and the LS (antibody #289) and YTE (antibody #420) variants, respectively, were tested for their ability to bind to the FcγR2a receptor (allele 167H) using AlphaLisa binding kits (Perkin Elmer #AL3086C and #AL3087C). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcγR2a (167H) was prepared at 120 ng / ml (4x the final concentration) and streptavidin donor and huIgG binding acceptor beads were prepared at 20 mg / ml (2x the final concentration) followed by 90 minutes of incubation in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. Antibodies tested are indicated in the legend. [Figure 49C]Evaluation of Fc variants in the FcγR2a(167R) binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (#246.4), and the YTE variant (#150.1) in the IgG1 backbone were tested for their ability to bind to the FcγR2a receptor (allele 167R) using AlphaLisa binding kits (Perkin Elmer #AL3086C and #AL3087C). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcγR2a(167R) was prepared at 200 ng / ml (4x) and streptavidin donor and huIgG binding acceptor beads were prepared at 20 μg / ml (2x) followed by 90 min incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 49D] Evaluation of Fc variants in the FcγR2a (167H) binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (#246.4), and the YTE variant (#150.1) in the IgG1 backbone were tested for their ability to bind to the FcγR2a receptor (allele 167H) using AlphaLisa binding kits (Perkin Elmer #AL3086C and #AL3087C). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcγR2a (167H) was prepared at 120 ng / ml (4x) and streptavidin donor and huIgG binding acceptor beads were prepared at 20 μg / ml (2x) followed by 90 min incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 50A]Evaluation of Fc variants in an immunoglobulin gamma Fc receptor IIIa (FcγR3a) (allele 176F) binding assay. Antibody #173WT, in either an IgG1 or IgG4 backbone, and the LS (antibody #289) and YTE (antibody #420) variants, respectively, were tested for their ability to bind to the FcγR3a receptor (allele 176F) using AlphaLisa binding kits (Perkin Elmer #AL347HV and #AL348HV). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcγR3a (176F) was prepared at 8 nM (4x the final concentration) and streptavidin donor and huIgG binding acceptor beads were prepared at 40 mg / ml (2x the final concentration) and incubated for 90 minutes in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. Antibodies tested are indicated in the legend. [Figure 50B] Evaluation of Fc variants in an immunoglobulin gamma Fc receptor IIIa (FcγR3a) (allele 176V) binding assay. Antibody #173WT and the LS (antibody #289) and YTE (antibody #420) variants, respectively, in IgG1 or IgG4 backbones, were tested for their ability to bind to the FcγR3a receptor (allele 176V) using AlphaLisa binding kits (Perkin Elmer #AL347HV and #AL348HV). Anti-VISTA antibodies were prepared at 1.2 mg / ml (4x the final concentration) followed by half-log serial dilutions. FcγR3a (176V) was prepared at 1.2 nM (4x the final concentration) and streptavidin donor and huIgG binding acceptor beads were prepared at 40 mg / ml (2x the final concentration) and incubated for 90 minutes in the dark. Emission was read at 615 nm on a CLARIOstar spectrometer. Antibodies tested are indicated in the legend. [Figure 50C]Evaluation of Fc variants in the FcγR3a (176F) binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (#246.4), and the YTE variant (#150.1) in the IgG1 backbone were tested for their ability to bind to the FcγR3a receptor (allele 176F) using AlphaLisa binding kits (Perkin Elmer #AL347HV and #AL348HV). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcγR3a (176F) was prepared at 8 nM (4x) and streptavidin donor and huIgG binding acceptor beads were prepared at 40 μg / ml (2x) followed by 90 min incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 50D] Evaluation of Fc variants in the FcγR3a(176V) binding assay. The IgG1-based antibody #474.1 (WT) or IgG4 S228P-based antibody (antibody #246.4), and the YTE variant (antibody #150.1) in the IgG1 backbone were tested for their ability to bind to the FcγR3a receptor (allele 176V) using AlphaLisa binding kits (Perkin Elmer #AL347HV and #AL348HV). Anti-VISTA antibodies were prepared at 1 mg / ml (4x) followed by half-log serial dilutions. FcγR3a(176V) was prepared at 1.2 nM (4x) and streptavidin donor and huIgG binding acceptor beads prepared at 40 μg / ml (2x) followed by 90 min incubation in the dark. Emission was read at 680 nm / 615 nm (excitation / emission) on a CLARIOstar spectrometer. [Figure 50E]Kinetics of FcRn binding to VISTA mAb bound to a FAB2G biosensor on an Octet K2 system. Antibody number 150.1 was loaded onto an anti-human Fab-CH1 (FAB2G) dip and read biosensor (ForteBio) at a concentration of 1 μg / ml for 120 seconds (loading step). The loaded biosensor was immersed in 0, 50, 200, and 800 nanomolar concentrations of FcRn (R&D Systems) for 240 seconds (binding step). The bound biosensor was immersed in phosphate assay buffer (PAB) solution for 360 seconds (dissociation step). All steps, including baseline, association, and dissociation, were performed in PAB (pH 6.0) to mimic the intracellular biology of FcRn-Fc activity. A 1:1 global curve-fitting analysis was performed to determine the k, k, and K across all concentrations of FcRn analyte. Only the first 10 seconds of the biphasic dissociation curve was analyzed. Kinetic experiments were performed on a ForteBio Octet K2 system and analyzed using ForteBio Data Analysis HT software version 12.0.2.59. [Figure 50F]Kinetics of FcRn binding to VISTA mAb bound to a FAB2G biosensor on an Octet K2 system. Antibody number 474.1 was loaded onto an anti-human Fab-CH1 (FAB2G) dip and read biosensor (ForteBio) at a concentration of 1 μg / ml for 120 seconds (loading step). The loaded biosensor was immersed in 0, 50, 200, and 800 nanomolar concentrations of FcRn (R&D Systems) for 240 seconds (binding step). The bound biosensor was immersed in phosphate assay buffer (PAB) solution for 360 seconds (dissociation step). All steps, including baseline, association, and dissociation, were performed in PAB (pH 6.0) to mimic the intracellular biology of FcRn-Fc activity. A 1:1 global curve-fitting analysis was performed to determine the k, k, and K across all concentrations of FcRn analyte. Only the first 10 seconds of the biphasic dissociation curve was analyzed. Kinetic experiments were performed on a ForteBio Octet K2 system and analyzed using ForteBio Data Analysis HT software version 12.0.2.59. [Figure 50G]Kinetics of FcRn binding to VISTA mAb bound to a FAB2G biosensor on an Octet K2 system. VSTB174 was loaded onto an anti-human Fab-CH1 (FAB2G) dip and read biosensor (ForteBio) at a concentration of 1 μg / ml for 120 seconds (loading step). The loaded biosensor was immersed in 0, 50, 200, and 800 nanomolar concentrations of FcRn (R&D Systems) for 240 seconds (binding step). The bound biosensor was immersed in phosphate assay buffer (PAB) solution for 360 seconds (dissociation step). All steps, including baseline, association, and dissociation, were performed in PAB (pH 6.0) to mimic the intracellular biology of FcRn-Fc activity. A 1:1 global curve-fitting analysis was performed to determine the k, k, and K across all concentrations of FcRn analyte. Only the first 10 seconds of the biphasic dissociation curve was analyzed. Kinetic experiments were performed on a ForteBio Octet K2 system and analyzed using ForteBio Data Analysis HT software version 12.0.2.59. [Figure 51] Dose-response effect of anti-VISTA antibody #421.1 and effector PBMCs on ADCC activity against target Raji cells expressing human VISTA (Raji-hVISTA cells) at 3 hours. Effector cells (5 x 10 human PBMCs) were cocultured with target cells (1 x 10 Raji-hVISTA cells labeled with BATDA) for 3 hours in the presence of antibody #421.1 or control Ab at 100, 30, 10, 3.0, 1.0, or 0.3 ng / ml. Induced ADCC activity was quantified by short-term time-resolved fluorescence detection of the DELFIA® EuTDA cytotoxicity reagent on a CLARIOstar Plus. [Figure 52]Dose-response effects of anti-VISTA antibodies Nos. 245.1 and 475.1 and effector PBMCs on ADCC activity against target Raji cells expressing human VISTA (Raji-hVISTA cells) at 3 hours. Effector cells (5 x 10 human PBMCs) were cocultured with target cells (1 x 10 Raji-hVISTA cells labeled with BATDA) for 3 hours in the presence of antibody No. 245.1 (WT), antibody No. 475.1 (LS), or control Ab at 100, 30, 10, 3.0, 1.0, or 0.3 ng / ml. Induced ADCC activity was quantified by short-term time-resolved fluorescence detection of the DELFIA® EuTDA cytotoxicity reagent on a CLARIOstar Plus. The antibodies tested are indicated in the legend. [Figure 53] Dose-response effect of anti-VISTA antibody #465.1 and effector PBMCs on ADCC activity against target Raji cells expressing human VISTA (Raji-hVISTA cells) at 3 hours. Effector cells (5 x 10 human PBMCs) were cocultured with target cells (1 x 10 Raji-hVISTA cells labeled with BATDA) for 3 hours in the presence of antibody #465.1 or control Ab at 100, 30, 10, 3.0, 1.0, or 0.3 ng / ml. Induced ADCC activity was quantified by short-term time-resolved fluorescence detection of the DELFIA® EuTDA cytotoxicity reagent on a CLARIOstar Plus. [Figure 54] Dose-response effect of anti-VISTA antibody #457.1 and effector PBMCs on ADCC activity against target Raji cells expressing human VISTA (Raji-hVISTA cells) at 3 hours. Effector cells (5 x 10 human PBMCs) were cocultured with target cells (1 x 10 Raji-hVISTA cells labeled with BATDA) for 3 hours in the presence of antibody #457.1 or control Ab at 100, 30, 10, 3.0, 1.0, or 0.3 ng / ml. Induced ADCC activity was quantified by short-term time-resolved fluorescence detection of the DELFIA® EuTDA cytotoxicity reagent on a CLARIOstar Plus. [Figure 55A]Dose-response effects of anti-VISTA antibodies Nos. 173.1, 173.4, and 420.1 and effector PBMCs on ADCC activity against target Raji cells expressing human VISTA (Raji-hVISTA cells) at 3 hours. Effector cells (5 x 10 human PBMCs) were cocultured with target cells (1 x 10 Raji-hVISTA cells labeled with BATDA) for 3 hours in the presence of 173.1 (WT, IgG1), 420.1 (YTE), 173.4 (WT, IgG4), or control Ab at 100, 30, 10, 3.0, 1.0, or 0.3 ng / ml. Induced ADCC activity was quantified by short-term time-resolved fluorescence detection of the DELFIA® EuTDA cytotoxicity reagent on a CLARIOstar Plus. The antibodies tested are indicated in the legend. [Figure 55B] Cell death was measured by luminescence in the presence of CytoTox-Glo reagent. 5x105 / well PBMCs were incubated overnight in X-VIVO-15 medium containing 200u / mL IL-2 in a 96-well plate. Antibodies and 4x104 / well hVISTA-Raji cells were added to the 96-well plate, mixed, and incubated for 4 hours using an effector:target (E:T) ratio of 12:1 (n=2). Data from one representative donor of six healthy donors. [Figure 55C] Cell death was measured by luminescence in the presence of CytoTox-Glo reagent. 5x105 / well PBMCs were incubated overnight in X-VIVO-15 medium containing 200u / mL IL-2 in a 96-well plate. Antibodies and 4x104 / well hVISTA-Raji cells were added to the 96-well plate, mixed, and incubated for 4 hours using an effector:target (E:T) ratio of 12:1 (n=2). Data from one representative donor of six healthy donors. [Figure 55D]CDC assay. Cell death was measured by luminescence in the presence of CytoTox-Glo reagent. 1x105 Raji+ hVISTA cells / well were plated in X-VIVO-15 medium in a 96-well plate. Antibodies and 25% human serum were added to the 96-well plate, mixed, and incubated for 6 hours. [Figure 56A] In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in the MC38 tumor model in human VISTA knock-in (KI) mice. VISTA KI mice were subcutaneously injected with 1 x 10 MC38 cells and randomized after tumor volumes reached approximately 70-100 mm. Mice were then treated intraperitoneally (IP) with vehicle control, 10 mg / kg antibody No. 245.2 (three times weekly), 5 mg / kg anti-PD1 antibody (twice weekly), or a combination of antibody No. 245.2 and anti-PD1 antibody at the respective monotherapy dose frequencies and doses. Tumor volume was measured in two dimensions twice weekly using calipers and expressed in mm using the formula: V = (L x W x W) / 2. Here, V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). [Figure 56B] In vivo antitumor efficacy of antibody No. 474.1 and antibody No. 150.1 in an MB49 tumor model in VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70-100 mm. Mice were treated with 20 mg / kg of antibody No. 474.1, antibody No. 150.1, or human IgG1 by intraperitoneal (IP) injection twice weekly for 3 weeks, beginning on day 5. Tumor volume was measured in two dimensions using calipers three times weekly, and the volume is expressed in mm using the formula V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Error bars represent SEM. [Figure 56C]In vivo antitumor efficacy of antibody No. 150.1 in the MB49 tumor model in VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70-100 mm. Mice were treated with 20 mg / kg antibody No. 150.1, 5 mg / kg anti-mPD1, or combination therapy by intraperitoneal (IP) injection twice weekly for 3 weeks, beginning on day 5. Tumor volume was measured in two dimensions using calipers three times weekly, and the volume is expressed in mm using the formula V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Error bars represent SEM. [Figure 56D] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). [Figure 56E] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). [Figure 56F]Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). [Figure 56G] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). [Figure 56H] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). [Figure 56I]Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). gMDSC, granulocyte-derived suppressor cells. [Figure 56J] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). M1 TAM, M1 tumor-associated macrophage. [Figure 56K] Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). M2 TAM, M2-type tumor-associated macrophages. [Figure 56L]Tumor samples were collected 24 hours after the third dose on day 12 from hVISTA KI C57B / 6 mice (n=3) treated with hIgG (20 mg / kg), antibody #150.1 (20 mg / kg), mPD1 (5 mg / kg), and the combination of antibody #150.1 and mPD1. Tumors were dissociated into single-cell suspensions and stained with a lymphoid panel, followed by RBC lysis with Biolegend RBC lysis buffer. Cells were fixed with BD cytofix and analyzed by flow cytometry (Attune Nxt). M1 TAM, M1 tumor-associated macrophages; M2 TAM, M2 tumor-associated macrophages. [Figure 57A] Individual anti-VISTA antibody concentrations in serum over time after a single intraperitoneal (IP) dose in human VISTA KI mice (samples in duplicate). Antibodies tested are indicated in the legend. [Figure 57B] Individual anti-VISTA antibody concentrations in serum over time after a single intraperitoneal (IP) dose in human VISTA KI mice (samples in duplicate). Antibodies tested are indicated in the legend. [Figure 57C] Individual concentrations of anti-VISTA antibodies over time in serum after a single IP dose in human VISTA KI female mice (samples in duplicate). [Figure 57D] Individual concentrations of anti-VISTA antibodies over time in serum after single or repeated IP administration in human VISTA KI female mice (samples in duplicate). [Figure 57E] Individual concentrations of anti-VISTA antibodies over time in serum after single or repeated IP administration in human VISTA KI female mice (samples in duplicate). [Figure 57F] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 10 mg / kg in cynomolgus monkeys. Shown are group average animal data. 10 mg / kg: Legend A. Group average. Dotted line = 100% receptor occupancy. [Figure 57G]Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 30 mg / kg in cynomolgus monkeys. Shown are group average animal data. 30 mg / kg: Legend B. Group average. Dotted line = 100% receptor occupancy. [Figure 57H] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 100 mg / kg in cynomolgus monkeys. Shown are group average animal data. 100 mg / kg: legend C. Group average. Dotted line = 100% receptor occupancy. [Figure 57I] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 10 mg / kg in cynomolgus monkeys. Shown are data for male (M) animals assigned to each group. 10 mg / kg: Legend: D. Animal 2001 (M). Dotted line = 100% receptor occupancy. [Figure 57J] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 30 mg / kg in cynomolgus monkeys. Shown are data for male (M) animals assigned to each group. 30 mg / kg: Legend: E. Animal 3001 (M). Dotted line = 100% receptor occupancy. [Figure 57K] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 100 mg / kg in cynomolgus monkeys. Shown are data for male (M) animals assigned to each group. 100 mg / kg: Legend: F. Animal 4001 (M). Dotted line = 100% receptor occupancy. [Figure 57L]Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum over time following a single (filled symbols) or four weekly (open symbols) IV dose of 10 mg / kg in cynomolgus monkeys. Shown are data for a female (F) animal assigned to each group. 10 mg / kg: Legend: G. Animal 2601 (F). Dotted line = 100% receptor occupancy. [Figure 57M] Antibody concentration (ug / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 versus time in serum after a single (filled symbols) or four weekly (open symbols) IV dose of 30 mg / kg in cynomolgus monkeys. Shown are data for a female (F) animal assigned to each group. 30 mg / kg: Legend: H. Animal 3501 (G). Dotted line = 100% receptor occupancy. [Figure 57N] Antibody concentration (µg / mL, gray) and receptor occupancy (%, black) of antibody number 150.1 in serum versus time after a single (filled symbols) or four weekly (open symbols) IV dose of 100 mg / kg in cynomolgus monkeys. Shown are data for female (F) animals assigned to each group. 100 mg / kg: Legend: I. Animal 4501 (F). Dotted line = 100% receptor occupancy. [Figure 58A]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs was obtained in technical duplicate, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58B]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs was obtained in technical duplicate, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58C]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58D]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs was obtained in technical duplicate, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58E]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs was obtained in technical duplicate, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58F]Changes in myeloid dendritic cell (mDC) activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58G]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58H]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58I]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58J]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58K]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58L]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58M]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58N]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58O]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.1 (WT). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58P]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58Q]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58R]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 173.4 (WT). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58S]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58T]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58U]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 289.1 (LS). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 58V]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD80 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker CD80. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58W]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated against CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). CD86 MFI of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFI from each Fluorescence Minus One (FMO) sample was subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker CD86. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies tested are indicated on the x-axis. [Figure 58X]Changes in CD14+ monocyte activation markers during in vivo exposure of non-human primates (NHPs) to anti-VISTA antibodies. Graph of the % change in mean fluorescence intensity (MFI) of activation markers compared to pre-dose, as determined by ex vivo flow cytometry analysis. To measure myeloid population size and activation markers, samples from all time points (0 h (pre-dose), 72 h, 168 h (pre-second dose), 240 h, and 336 h) were thawed, stained, and analyzed on the same day. CD45+CD66-CD3-CD8-CD20-, side-scatter intermediate myeloid populations were gated on CD14+ monocytes or CD14-HLA-DR+CD1c+ or CD11c+CD123- myeloid dendritic cells (mDCs). HLA-DR MFIs of CD14+ monocytes and mDCs were obtained in technical duplicates, and the MFIs from each Fluorescence Minus One (FMO) sample were subtracted. (Fluorescence Minus One (FMO) controls are samples in which cells are stained with all but one fluorochrome used in the experiment; one FMO control is used for each fluorochrome to identify the cutoff point between background fluorescence and the positive population.) The percentage change in MFI relative to pre-dose levels was calculated as % change = 100 * (MFI x day - MFI day 0) / MFI day 0. Results from samples from in vivo treated animals are shown for antibody number 420.1 (YTE). The graph shows the change in expression levels of the activation marker HLA-DR. Arrows indicate iv administration of 10 mg / kg at 0 and 168 hours. The antibodies examined are indicated on the x-axis. [Figure 59A]In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in an MB49 tumor model in human VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70 mm. Mice were treated twice weekly with six intraperitoneal (IP) injections of 30 mg / kg of mouse IgG2a control antibody, 30 mg / kg of antibody No. 245.2, or 30 mg / kg of antibody No. 245.2 with L234A and L235A substitutions ("LALA") according to EU numbering. Tumor volume was measured in two dimensions twice weekly using calipers, and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Mean tumor volumes ± SEM are shown for IgG2a control (squares), 245.2 (open circles) and 245.2LALA (filled circles). [Figure 59B] In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in the MB49 tumor model in human VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70 mm. Mice were treated with 30 mg / kg of mouse IgG2a control antibody by intraperitoneal (IP) injection twice weekly for a total of six injections. Tumor volume was measured twice weekly in two dimensions using calipers, and the volume was expressed in mm using the formula: V = (LxWxW) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Individual tumor measurements over time by animal and group are shown. [Figure 59C]In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in the MB49 tumor model in human VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70 mm. Mice were treated with 30 mg / kg of antibody No. 245.2 by intraperitoneal (IP) injection twice weekly for a total of six doses. Tumor volume was measured twice weekly in two dimensions using calipers, and the volume was expressed in mm using the formula: V = (LxWxW) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Individual tumor measurements over time by animal and group are shown. [Figure 59D] In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in the MB49 tumor model in human VISTA KI mice. VISTA KI mice were subcutaneously injected with 5x10 MB49 cells and randomized after tumor volumes reached approximately 70 mm. Mice were treated with 30 mg / kg of antibody No. 245.2 with L234A and L235A substitutions ("LALA") according to EU numbering via intraperitoneal (IP) injection twice weekly for a total of six doses. Tumor volume was measured twice weekly in two dimensions using calipers, and the volume was expressed in mm using the formula: "V = (L x W x W) / 2," where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Individual tumor measurements over time by animal and group are shown. [Figure 59E] Tumor-infiltrating lymphocytes—lymphoid panel. Tumors were harvested from hVISTA KI C57B / 6 mice (n=3) treated with mIgG 2a (30 mg / kg), antibody #245.2-LALA (30 mg / kg), and antibody #245.2 (30 mg / kg) three days after the last dose on day 24 (dose #6). Tumors were isolated using a Miltenyi tumor isolation kit (catalog #130-096-730) and an octoMACS. Cells were then stained with a lymphoid panel, followed by RBC lysis with Biolegend RBS lysis buffer (catalog #420301) and analysis by flow cytometry on an Attune Nxt instrument. [Figure 59F] Tumor-infiltrating lymphocytes—lymphoid panel. Tumors were harvested from hVISTA KI C57B / 6 mice (n=3) treated with mIgG 2a (30 mg / kg), antibody #245.2-LALA (30 mg / kg), and antibody #245.2 (30 mg / kg) three days after the last dose on day 24 (dose #6). Tumors were isolated using a Miltenyi tumor isolation kit (catalog #130-096-730) and an octoMACS. Cells were then stained with a lymphoid panel, followed by RBC lysis with Biolegend RBS lysis buffer (catalog #420301) and analysis by flow cytometry on an Attune Nxt instrument. [Figure 59G] Tumor-infiltrating lymphocytes—lymphoid panel. Tumors were harvested from hVISTA KI C57B / 6 mice (n=3) treated with mIgG 2a (30 mg / kg), antibody #245.2-LALA (30 mg / kg), and antibody #245.2 (30 mg / kg) three days after the last dose on day 24 (dose #6). Tumors were isolated using a Miltenyi tumor isolation kit (catalog #130-096-730) and an octoMACS. Cells were then stained with a lymphoid panel, followed by RBC lysis with Biolegend RBS lysis buffer (catalog #420301) and analysis by flow cytometry on an Attune Nxt instrument. [Figure 59H] Tumor-infiltrating lymphocytes—lymphoid panel. Tumors were harvested from hVISTA KI C57B / 6 mice (n=3) treated with mIgG 2a (30 mg / kg), antibody #245.2-LALA (30 mg / kg), and antibody #245.2 (30 mg / kg) three days after the last dose on day 24 (dose #6). Tumors were isolated using a Miltenyi tumor isolation kit (catalog #130-096-730) and an octoMACS. Cells were then stained with a lymphoid panel, followed by RBC lysis with Biolegend RBS lysis buffer (catalog #420301) and analysis by flow cytometry on an Attune Nxt instrument. [Figure 60]A-D. In vivo antitumor efficacy of anti-VISTA antibody No. 245.2 in the E.G7-OVA tumor model in human VISTA KI mice. VISTA KI mice were subcutaneously injected with 1 x 106 E.G7-OVA cells and randomized after tumor volume reached approximately 70 mm3. Mice were treated with 30 mg / kg control antibody (mouse IgG2a), 30 mg / kg antibody No. 245.2, or 30 mg / kg antibody No. 245.2LALA by intraperitoneal (IP) injection twice weekly for a total of six doses. Tumor volume was measured in two dimensions twice weekly using calipers, and the volume was expressed in mm3 using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). A shows the mean tumor volume ± SEM for IgG2a control (squares), 245.2 (open circles), and 245.2LALA (filled circles). B-D show individual tumor measurements over time by animal and group. DETAILED DESCRIPTION OF THE INVENTION

[0109] Provided herein are antibodies and antigen-binding fragments thereof that specifically bind to VISTA; these antibodies and fragments are also referred to herein as "anti-VISTA antibodies" and "anti-VISTA antigen-binding fragments," respectively. As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are used interchangeably in the context of antibodies and antigen-binding fragments thereof, and, as will be understood by those skilled in the art, refer to binding by antibodies and antigen-binding fragments to an antigen via the antigen-binding site of the antibody, and do not exclude cross-reactivity of the antibody or antigen-binding fragment with other antigens. For example, an antibody or antigen-binding fragment provided herein may immunospecifically bind to both human VISTA and cynomolgus monkey VISTA, but may not bind to mouse VISTA. Any method known in the art can be used to confirm whether immunospecific binding to VISTA occurs.

[0110] The antibodies described herein may be monoclonal or polyclonal, preferably monoclonal. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA is an immunoglobulin, a tetrameric antibody comprising two heavy chains and two light chains, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, a single domain antibody, a monovalent antibody, a single chain antibody, a single chain Fv (scFv), or a disulfide-linked Fv. For the purposes of this disclosure, an scFv shall be considered an antigen-binding fragment because it comprises a VH domain and a VL domain (connected by a linker).

[0111] In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are bivalent. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are multispecific or bispecific. In certain embodiments, the antibodies that specifically bind to VISTA described herein are bispecific monoclonal antibodies. In certain embodiments, the antibodies described herein are monovalent. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are bivalent. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are monospecific. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are recombinantly produced. In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are purified. In certain embodiments, the antibodies that specifically bind to VISTA described herein are synthetic antibodies. In certain embodiments, the antibodies that specifically bind to VISTA described herein are human antibodies. In certain embodiments, the antibodies that specifically bind to VISTA described herein are murine antibodies.

[0112] In certain embodiments of the antibodies of the invention, the antibodies are immunoglobulins. The antibodies described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgW, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ). In certain embodiments, the antibody that specifically binds to VISTA described herein is an IgG antibody, or a class or subclass thereof. In certain embodiments, the antibody that specifically binds to VISTA described herein is a monoclonal antibody. In certain embodiments, the antibody that specifically binds to VISTA described herein is an IgG antibody. In certain embodiments, the antibody that specifically binds to VISTA described herein is an IgG1 antibody. In another specific embodiment, the antibody that specifically binds to VISTA described herein is an IgG2 antibody. In another specific embodiment, the antibody that specifically binds to VISTA described herein is an IgG3 antibody. In another specific embodiment, the antibody that specifically binds to VISTA described herein is an IgG4 antibody.

[0113] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA as described herein is formed by the association of a heavy chain and a light chain, or by the association of a heavy chain variable region and a light chain variable region.

[0114] Antigen-binding fragments include portions of antibody molecules that contain amino acid residues surrounded by framework regions that bind to an antigen and confer specificity to the antibody molecule for that antigen (e.g., complementarity-determining regions (CDRs)). The CDRs can be derived from any animal species, including rodents (e.g., mice, rats, or hamsters), chickens, cows, camels, and humans. Exemplary antigen-binding fragments include Fab fragments, F(ab')2 fragments, and other antigen-binding fragments of any of the antibodies described herein.

[0115] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. Variable region typically refers to a portion of an antibody, typically a light or heavy chain, which differs significantly in sequence between antibodies and is used in the binding and specificity of a particular antibody for a particular antigen. Sequence variability is concentrated in the CDRs, while the more highly conserved regions within variable domains are called framework regions. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen.

[0116] CDRs are defined in various ways in the art, including the Kabat, Chothia, AbM, contact, IMGT, and Paratome numbering systems. Any of the CDR numbering systems known in the art can be used to define the CDRs of the anti-VISTA antibodies disclosed herein. The Kabat numbering system is based on sequence variability and is the most commonly used definition for predicting CDR regions (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). The Chothia numbering system is based on the location of structural loop regions (Chothia et al., (1987) J Mol Biol 196:901-917). The AbM numbering system is a compromise between the Kabat and Chothia numbering systems and is a set of integrated programs for antibody structure modeling created by the Oxford Molecular Group (bioinf.org.uk / abs) (Martin ACR et al., (1989) PNAS 86:9268-9272). The contact numbering system is based on the analysis of available complex crystal structures (bioinf.org.uk / abs) (see MacCallum RM et al., (1996) J Mol Biol 5:732-745). The IMGT numbering system is from IMGT ("IMGT®, the international ImMunoGeneTics information system® website imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France). The Paratome numbering system predicts the antigen-binding region of an antibody based on a set of consensus regions obtained from the structural alignment of a non-redundant set of all known antibody-antigen complexes.This algorithm is based on the premise that the majority of antigen-binding residues are located in structural consensus regions between antibodies, forming six sequence stretches within the antibody sequence that roughly correspond to the six CDRs (see Kunit et al., Nucleic Acids Research, 2012, Vol. 40, Web Server issue W521-W524).

[0117] The present disclosure provides antibodies and antigen-binding fragments and other subjects that comprise the sequences disclosed herein (e.g., scFvs, CDRs, variable regions, etc.), as well as antibodies and antigen-binding fragments and other subjects that consist of or consist essentially of the sequences disclosed herein.

[0118] In another aspect, provided herein are multispecific antibodies and heteroconjugate antibodies. In certain embodiments, provided herein are bispecific antibodies comprising two different antigen-binding regions, one of which binds to VISTA and comprises the variable heavy chain region, variable light chain region, or both, of an antibody described herein, and the other binding region binds to another antigen of interest. In certain aspects, provided herein are bispecific antibodies comprising two different antigen-binding regions, one of which specifically binds to VISTA and the other binding region binds to another antigen of interest, wherein the binding region specifically binds to VISTA is an antibody or antigen-binding fragment thereof described herein that specifically binds to VISTA. In certain aspects, provided herein are bispecific antibodies comprising a first variable region (i.e., bivalent) comprising two antigen-binding sites that specifically bind to VISTA, and a second variable region (i.e., bivalent) comprising two binding sites that specifically bind to different antigens, wherein the first and second variable regions are linked by an Fc fragment, and in certain embodiments of this aspect, the variable regions are each a Fab region.

[0119] In certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein are bispecific or trispecific antibodies. In certain embodiments, provided herein are bispecific antibodies comprising two different antigen-binding regions, one of which specifically binds to VISTA and the other of which binds to another antigen of interest, wherein the binding region that specifically binds to VISTA comprises the variable heavy chain region of an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein. In certain embodiments, provided herein are bispecific antibodies comprising two different antigen-binding regions, one of which specifically binds to VISTA and the other of which binds to another antigen of interest, wherein the binding region that specifically binds to VISTA comprises the VH, and optionally the VL, of an antibody listed in Table 7. In another specific embodiment, provided herein are bispecific antibodies comprising two different antigen-binding regions, one of which specifically binds to VISTA and the other of which binds to another antigen of interest, wherein the binding region that specifically binds to VISTA comprises the VH of an antibody listed in Table 7 and the VL of the same antibody listed in Table 8. In one embodiment, the binding region that specifically binds to VISTA is an scFv. In certain embodiments, the target antigen that the other binding region of the bispecific antibody described herein binds to is an antigen present on immune cells (e.g., T cells, NK cells, or dendritic cells). In certain embodiments, the antibody provided herein is a bispecific T cell engager (BiTE). Several different formats of multispecific antibodies have been described. See, for example, Brinkman and Kontermann, MABS 2017,9(2):182-212.

[0120] Also provided herein is a fusion protein comprising an antibody or antigen-binding fragment that specifically binds to VISTA as described herein. In certain embodiments, the fusion protein comprises the VH and VL of an antibody or antigen-binding fragment that specifically binds to VISTA as described herein. Also provided herein is a fusion protein comprising an scFv comprising an antibody or antigen-binding fragment that specifically binds to VISTA as described herein. In certain embodiments, the scFv comprises a VH and VL separated by a linker sequence, the VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in the table as the VH CDR1 for the antibody number listed in the table selected from the group consisting of Tables 1 to 3, the VH CDR2 has the sequence number set forth in the table as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in the table as the VH CDR3 of the antibody. In certain embodiments, the scFv comprises a VH and a VL separated by a linker sequence, wherein (i) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has a sequence number set forth in a table selected from the group consisting of Tables 1 to 3 as the VH CDR1 for the antibody number listed in the table, the VH CDR2 has a sequence number set forth in the table as the VH CDR2 of the antibody, and the VH CDR3 has a sequence number set forth in the table as the VH CDR3 of the antibody; and (ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has a sequence number set forth in a table selected from the group consisting of Tables 4 to 6 as the VL CDR1 of the antibody, the VL CDR2 has a sequence number set forth in the table as the VL CDR2 of the antibody, and the VL CDR3 has a sequence number set forth in the table as the VL CDR3 of the antibody. and the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 are all defined by the same CDR numbering system. In a specific embodiment, the scFv comprises a VH and a VL, and the VH comprises the SEQ ID NO listed in Table 7 as the VH of the antibody.In certain embodiments, the scFv comprises a VH and a VL, wherein (i) the VH comprises a sequence number set forth in Table 7 for the VH of the antibody, and (ii) the VL comprises a sequence number set forth in Table 8 for the VL of the antibody.

[0121] Further provided herein are chimeric antigen receptors (CARs) comprising scFvs comprising antibodies or antigen-binding fragments that specifically bind to VISTA as described herein, and cells comprising nucleic acids encoding the CARs, including cells that express the CARs. In certain embodiments, the cells comprise recombinant nucleic acids encoding the CARs such that the cells express the CARs. In certain embodiments, the cells are ex vivo.

[0122] The structures of "first generation," "second generation," and "third generation" CARs have been described in the art (e.g., Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al. al., Curr. Opin. Immunol. 21(2):215-223 (2009), see Hollyman et al., J. Immunother. 32: 169-180 (2009)).

[0123] In certain embodiments, the CAR is a "first generation" CAR comprising an extracellular antigen-binding fragment that specifically binds to VISTA described herein (e.g., an scFv that specifically binds to VISTA described herein) fused to a transmembrane domain fused to the cytoplasmic / intracellular domain of a T cell receptor complex chain. In certain embodiments, the cytoplasmic domain is the intracellular domain of the CD3ζ chain.

[0124] In certain embodiments, the CAR is a "second-generation" CAR that includes an antigen-binding fragment that specifically binds to VISTA described herein (e.g., an scFv that specifically binds to VISTA described herein) fused to an intracellular signaling domain (e.g., the intracellular domain of the CD3ζ chain) that can activate an immune cell, fused to a costimulatory domain to increase the potency and persistence of an immune cell (e.g., a T cell), and fused to a transmembrane domain (see Sadelain et al., Cancer Discov. 3:388-398 (2013)). The costimulatory domain of the "second-generation" CAR can be an intracellular domain derived from any of a variety of costimulatory molecules, e.g., the costimulatory domain of CD28, 4-IBB, ICOS, or OX40. Thus, a "second-generation" CAR provides both costimulation (e.g., by the CD28 or 4-IBB intracellular domain) and activation (e.g., by the CD3ζ signaling domain).

[0125] A "third generation" CAR includes the structure of a second generation CAR, except that it has multiple (e.g., two) costimulatory domains. Thus, a third generation CAR provides multiple costimulation, e.g., by including both the CD28 and 4-1BB intracellular domains, and activation, e.g., by including a CD3ζ activation domain.

[0126] In certain embodiments, the CAR of the present invention comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, as described above, wherein the extracellular antigen-binding domain is an scFv comprising an antibody or antigen-binding fragment that specifically binds to VISTA as described herein. In certain embodiments, the intracellular domain is a CD3ζ signaling domain.

[0127] In certain embodiments, the cell comprising the CAR is a T cell. In other specific embodiments, the cell comprising the CAR is a natural killer (NK) cell. In other specific embodiments, the cell comprising the CAR is a macrophage. Examples of cells that can express CAR are described. For example, Basar et al., Hematology 2020 ASH Education Program pp.570-578, Villanueva 2020, Nat.Rev.Drug Discov.Vol.20:300, Mukhopadhyay 2020, Nat.Methods Vol.17:561, Anderson et al.,2017, Cancer Res(published online November 17,2020), Cortez-Selva et al.,2021,Trends in Pharmacological Sciences 42(1):45-59, Klichinsky et al.,2020,Nat.Biotech.38:947-959,Vacca et al.,2020,Front.Immunol.10:3013 doi:10.3389 / fimmu.2019.03013, and Xie et al. See al., 2020, EBioMedicine 59:102975.

[0128] Also provided herein is an antibody-drug conjugate comprising an antibody or antigen-binding fragment or scFv that specifically binds to VISTA as described herein linked (e.g., covalently linked) to a therapeutic agent. In certain embodiments, the therapeutic agent is a cytotoxic agent. In certain embodiments, provided herein is an antibody-drug conjugate comprising a fusion protein comprising an antibody or antigen-binding fragment that specifically binds to VISTA as described herein.

[0129] In another specific embodiment, provided is an antibody-drug conjugate comprising an antibody or antigen-binding fragment or scFv that specifically binds to VISTA as described herein, conjugated to a label or imaging agent, for use in detecting and / or measuring levels and / or localizing VISTA in vivo or ex vivo (e.g., in a patient biopsy tumor sample) by contacting an ex vivo cell sample (e.g., a biopsy tumor sample) or administering the antibody to the patient, thereby detecting binding via the label or imaging agent. In a specific embodiment, such a method is used to determine whether a patient is a candidate for cancer treatment with the anti-VISTA antibodies and antigen-binding fragments and antibody-drug conjugates (conjugated to a therapeutic agent) of the present invention by identifying the patient's cancer as VISTA-positive or expressing VISTA at a desired level.

[0130] 1.1 Antibodies For each of the antibody numbers used to identify a particular antibody in this disclosure (each "antibody [or Ab] number"), the number after the period in the antibody number indicates the IgG class of that antibody. Thus, for example, antibody number 173.1 is an IgG1 antibody, while antibody number 173.4 is an IgG4 antibody.

[0131] Sequences and Variants Tables 1-6 below provide the VH and VL CDRs of antibodies or antigen-binding fragments thereof that specifically bind to VISTA, as defined using different numbering systems (Kabat, IMGT, and Paratome). Tables 1-3 below provide VH CDRs defined using different systems, which may be combined with VL CDRs in Tables 4-6. In certain embodiments, the VH CDR of an antibody from Table 1 (e.g., antibody No. 269.1) is combined with the VL CDR of the same antibody from Table 4 (e.g., 269.1). In certain embodiments, the VH CDR of an antibody from Table 2 (e.g., antibody No. 269.1) is combined with the VL CDR of the same antibody from Table 5 (e.g., antibody No. 269.1). In certain embodiments, the VH CDR of an antibody from Table 3 (e.g., antibody No. 269.1) is combined with the VL CDR of the same antibody from Table 6 (e.g., antibody No. 269.1).

[0132] In a particular embodiment, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 1 as the VH CDR1 for the antibody number listed in Table 1, the VH CDR2 has the sequence number set forth in Table 1 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 1 as the VH CDR3 of the antibody. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 1 as the VH CDR1 for the antibody number listed in Table 1, the VH CDR2 has the sequence number set forth in Table 1 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 1 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 4 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 4 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 4 as the VH CDR3 of the antibody.

[0133] In a particular embodiment, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 2 as the VH CDR1 for the antibody number listed in Table 2, the VH CDR2 has the sequence number set forth in Table 2 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 2 as the VH CDR3 of the antibody. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 2 as the VH CDR1 for the antibody number listed in Table 2, the VH CDR2 has the sequence number set forth in Table 2 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 2 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 5 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 5 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 5 as the VL CDR3 of the antibody.

[0134] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein (i) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the sequence number set forth in Table 3 as the VH CDR1 for the antibody number listed in Table 3, the VH CDR2 has the sequence number set forth in Table 3 as the VH CDR2 of the antibody, and the VH CDR3 has the sequence number set forth in Table 3 as the VH CDR3 of the antibody, and (ii) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has the sequence number set forth in Table 6 as the VL CDR1 of the antibody, the VL CDR2 has the sequence number set forth in Table 6 as the VL CDR2 of the antibody, and the VL CDR3 has the sequence number set forth in Table 6 as the VL CDR3 of the antibody. [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2]

[0135] Also provided herein is an antibody or antigen-binding fragment that specifically binds to VISTA, comprising a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of the SEQ ID NOs: listed in Table 7 for the VH of an antibody. In certain embodiments, the VH CDRs are defined according to the Kabat numbering system. In certain embodiments, the VH CDRs are defined according to the Chothia numbering system. In certain embodiments, the VH CDRs are defined according to the AbM numbering system. In certain embodiments, the VH CDRs are defined according to the IMGT numbering system. In certain embodiments, the VH CDRs are defined according to the Paratome numbering system. In certain embodiments, the VH CDRs are defined according to the Contact numbering system.

[0136] In certain embodiments, the antibody or antigen-binding fragment specifically binds to VISTA, comprising a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of the SEQ ID NOs: listed in Table 7 as the VH of an antibody, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 of a SEQ ID NO: listed in Table 8 as the VL of the antibody. In certain embodiments, the VH CDRs are defined by the Kabat numbering system. In certain embodiments, the VH CDRs are defined by the Chothia numbering system. In certain embodiments, the VH CDRs are defined by the AbM numbering system. In certain embodiments, the VH CDRs are defined by the IMGT numbering system. In certain embodiments, the VH CDRs are defined by the Paratome numbering system. In certain embodiments, the VH CDRs are defined by the Contact numbering system.

[0137] The CDRs of the antibodies or antigen-binding fragments provided herein that specifically bind to VISTA can be modified, for example, by introducing one or more mutations into one or more of the CDRs. Such mutations can, for example, change the binding affinity of the antibody or its antigen-binding fragment at a certain pH value, thereby affecting the biological half-life of the antibody. Thus, by redesigning an antibody that has reduced binding affinity to VISTA in endosomes after internalization, antibody depletion can be reduced and half-life can be extended.

[0138] In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein bind to VISTA with higher affinity at neutral pH than at acidic pH (i.e., binding affinity is reduced at acidic pH). Anti-VISTA antibodies that have reduced binding affinity at acidic pH may have various improved / enhanced biological properties compared to antibodies that do not exhibit reduced binding affinity at acidic pH. For example, in certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein that have reduced binding affinity at acidic pH may have a longer half-life in circulation compared to anti-VISTA antibodies that do not exhibit reduced binding affinity at acidic pH. In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein that have reduced binding affinity at acidic pH may be cleared from the circulation more slowly than anti-VISTA antibodies that lack pH-dependent binding. Slower antibody clearance (i.e., longer circulating half-life) should correlate with extended biological activity. Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein can be administered to a subject less frequently and / or in lower doses than antibodies that do not have a reduced binding affinity at acidic pH, while still exhibiting comparable (or better) efficacy.

[0139] Without wishing to be bound by theory or mechanism, it is believed that anti-VISTA antibodies, which have lower binding affinity at acidic pH compared to neutral pH, dissociate from antigen in the acidic environment of the endosome and are recycled to plasma where they undergo further therapeutic antigen binding. Neonatal Fc receptor (FcRn) binding modifications in the Fc region of the antibody (e.g., modifications such as those described below), coupled with the introduction of a histidine switch in the antigen-binding region, result in antibodies that bind VISTA with high affinity at the cell surface, dissociate from VISTA after transport into the acidic endosomal environment, and are captured and recycled into the extracellular space by the FcRn receptor.

[0140] In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein comprise one or more histidine substitutions at any Y, D, E, N, or Q amino acid occurring in the light chain CDRs or heavy chain CDRs. In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to VISTA provided herein comprise one histidine substitution at a Y, D, E, N, or Q amino acid occurring in the light chain CDRs or heavy chain CDRs. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA provided herein comprises the VH CDRs of antibody No. 474.1 described in Table 1, Table 2, or Table 3, and the VL CDRs of antibody No. 474.1 described in Table 4, Table 5, or Table 6 except for one or more histidine substitutions in the VL CDRs, which substitutions may be selected from Y31H (e.g., present in antibody No. 373.1), Y32H (e.g., present in antibody No. 467.1), D50H (e.g., present in antibody No. 908.1), N53H (e.g., present in antibody No. 386.1), Q89H (e.g., present in antibody No. 268.1), Q90H (e.g., present in antibody No. 342.1), and N93H (e.g., present in antibody No. 259.1), each numbered according to the Kabat numbering system.

[0141] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA provided herein comprises the VH CDRs of antibody No. 474.1 described in Table 1, Table 2, or Table 3, and the VL CDRs of antibody No. 474.1 described in Table 4, Table 5, or Table 6, except for one or more histidine substitutions in the VH CDRs. The substitutions, including CDRs, may be selected from Y32H (e.g., present in antibody No. 338.1), Y33H (e.g., present in antibody No. 419.1), Y50H (e.g., present in antibody No. 277.1), Y52H (e.g., present in antibody No. 946.1), Y53H (e.g., present in antibody No. 322.1), N58H (e.g., present in antibody No. 346.1), Y59H (e.g., present in antibody No. 304.1), N60H (e.g., present in antibody No. 814.1), D95H (e.g., present in antibody No. 210.1), and D101H (e.g., present in antibody No. 460.1), each according to the Kabat numbering system.

[0142] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA provided herein comprises the VH CDRs of antibody No. 173.1 described in Table 1, Table 2, or Table 3, and the VL CDRs of antibody No. 173.1 described in Table 4, Table 5, or Table 6, except for one or more histidine substitutions in the VL CDRs, each of which may be selected from D47H (e.g., present in antibody No. 374.1), D69H (e.g., present in antibody No. 394.1), D111H (e.g., present in antibody No. 213.1), and E74H (e.g., present in antibody No. 219.1), according to the Kabat numbering system.

[0143] In certain embodiments, the antibodies described herein can be described by specifying their VH domain alone, their VL domain alone, or a set of three CDRs of a VH or VL. See, for example, Clackson T et al., (1991) Nature 352:624-628, incorporated herein by reference in its entirety, which describes methods for generating antibodies that bind to a specific antigen by using a particular VH domain (or VL domain) and screening a library of complementary variable domains. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, incorporated herein by reference in its entirety, which describes methods for generating antibodies that bind to a specific antigen by using a particular VH domain and screening a library of complementary VL domains (e.g., a human VL library). The selected VL domain can then be used to guide the selection of additional complementary (e.g., human) VH domains.

[0144] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH, wherein the VH comprises a SEQ ID NO: set forth in Table 7 as the VH of the antibody. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VL, wherein the VL comprises a SEQ ID NO: set forth in Table 8 as the VL of the antibody. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH, wherein the VH comprises a VH comprising a SEQ ID NO: set forth in Table 7 as the VH of the antibody, and a VL. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein (i) the VH comprises a SEQ ID NO: set forth in Table 7 as the VH of the antibody, and (ii) the VL comprises a SEQ ID NO: set forth in Table 8 as the VL of the antibody.

[0145] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the VH set forth in Table 7. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VH has at least 95% sequence identity to the amino acid sequence of the VH set forth in Table 7. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the VL set forth in Table 8. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VL that has at least 95% sequence identity to the amino acid sequence of a VL listed in Table 8.

[0146] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein (i) the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of a VH of an antibody listed in Table 7, and (ii) the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of a VL of the antibody listed in Table 8. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a VH and a VL, wherein the VH has at least 95% sequence identity to the amino acid sequence of a VH of an antibody listed in Table 7, and (ii) the VL has at least 95% sequence identity to the VL of the antibody listed in Table 8. [Table 7-1] [Table 7-2] [Table 7-3] [Table 8-1] [Table 8-2]

[0147] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain and a light chain, wherein the light chain has a sequence number set forth in Table 9 as the light chain of the antibody number listed in Table 9, and the heavy chain has a sequence number set forth in Table 9 as the heavy chain of the antibody (e.g., antibody number 269.1, antibody number 321.1, antibody number 245.1, antibody number 465.1, antibody number 457.1, antibody number 173.1, antibody number 833.1, antibody number 245.4, antibody number 465.4, antibody number 173.4, antibody number 245.2, antibody number 289.1 or antibody number 420.1).

[0148] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain and a light chain, wherein the heavy chain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a SEQ ID NO: set forth in Table 9 as the heavy chain of the antibody number listed in Table 9. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain and a light chain, wherein the light chain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a SEQ ID NO: set forth in Table 9 as the light chain of the antibody number listed in Table 9, and the heavy chain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a SEQ ID NO: set forth in Table 9 as the heavy chain of the antibody.

[0149] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain and a light chain, wherein the heavy chain has at least 95% sequence identity to the SEQ ID NO: set forth in Table 9 as the heavy chain of the antibody number listed in Table 9. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain and a light chain, wherein the light chain has at least 95% sequence identity to the SEQ ID NO: set forth in Table 9 as the light chain of the antibody number listed in Table 9, and the heavy chain has at least 95% sequence identity to the SEQ ID NO: set forth in Table 9 as the heavy chain of the antibody.

[0150] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms known in the art. The percent identity between two sequences can be determined with or without allowing gaps. In calculating percent identity, typically, only exact matches are counted. A non-limiting example of a mathematical algorithm utilized to compare two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, as modified by Karlin S & Altschul SF (1993) PNAS 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al. (1990) J Mol Biol 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide program, and protein searches can be performed using the XBLAST program. To obtain gapped alignments for comparison purposes, Gapped BLAST and / or PSI BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389 3402. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) internet at ncbi.nlm.nih.gov). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

[0151] In certain aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to VISTA, comprising an antibody heavy chain and / or light chain, e.g., a heavy chain alone, a light chain alone, or both a heavy and light chain. With respect to the light chain, in certain embodiments, the light chain of an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein is a kappa light chain. In another specific embodiment, the light chain of an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein is a lambda light chain. In yet another specific embodiment, the light chain of an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein is a human kappa light chain or a human lambda light chain.

[0152] As used herein, the terms "constant region" and "constant domain" are synonymous and have the meaning commonly used in the art. The constant region is a portion of an antibody, e.g., the carboxyl-terminal portion of the light chain and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors in the case of the heavy chain. The constant region of an immunoglobulin molecule has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0153] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA as described herein comprises a light chain, wherein the light chain comprises a VL and a human kappa or human lambda light chain constant region, wherein the VL comprises a sequence set forth in Table 8. Non-limiting examples of human constant region sequences are described in the art, see, for example, Kabat EA et al., (1991).

[0154] With respect to heavy chains, in certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein may be a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises a heavy chain, wherein the heavy chain comprises a constant region or portion thereof (e.g., C H 1. C H 2 or C H 3 or a combination thereof), and a variable heavy chain region (VH), wherein the VH comprises a sequence set forth in Table 7. In certain embodiments, the constant region is that of a human gamma heavy chain.

[0155] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising any of the amino acid sequences described herein, and the constant region comprises the amino acid sequence of the constant region of a human IgG, IgE, IgM, IgD, IgA, IgW, or IgY immunoglobulin molecule. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA comprises an IgG constant region, e.g., an IgG1, IgG2, or IgG4 constant region described in Table 10. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein comprises any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG 2a and IgG 2b ) the constant region of a human IgG, IgE, IgM, IgD, IgA IgW, or IgY immunoglobulin molecule. [Table 10-1] [Table 10-2] [Table 10-3]

[0156] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein comprises a heavy chain and / or a light chain, wherein the heavy chain comprises (a) the VH of an antibody described in Table 7 and (b) a constant heavy chain domain comprising the amino acid sequence of the constant domain of human IgG.

[0157] In another specific embodiment, an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a constant heavy chain domain comprising (a) the VH of an antibody described in Table 7 and (b) the amino acid sequence of the constant domain of human IgG, and (ii) the light chain comprises a VL of the same antibody described in Table 8 and (b) a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.

[0158] Fc regions and variant Fc regions In certain embodiments, the antibodies described herein comprise an Fc region. In certain embodiments, the antibodies described herein comprise an Fc region of human IgG1. In certain embodiments, the Fc region is a human Fc region. In further particular embodiments, the human Fc region is that of human IgG1, human IgG2, or human IgG4. In even more particular embodiments, the Fc region is a variant human Fc region comprising one or more mutations (e.g., 1, 2, 3, 4, or 5 amino acid mutations) in the Fc region compared to a native human Fc region. In certain embodiments, the one or more mutations are insertions, substitutions, and / or deletions.

[0159] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA has in its constant region, compared to the wild-type constant region, only one of the specific amino acid mutations or a combination of specific amino acid mutations identified in this disclosure, and does not contain any other amino acid mutations compared to the wild-type constant region. In another specific embodiment, an antibody or antigen-binding fragment thereof that specifically binds to VISTA has in its constant region, compared to the wild-type constant region, a specific amino acid mutation or a combination of specific amino acid mutations identified in this disclosure, and has a total of seven or fewer (i.e., 1, 2, 3, 4, 5, 6, or 7) amino acid mutations compared to the wild-type constant region.

[0160] Thus, in certain embodiments where the Fc region comprises 1, 2, 3, 4, or 5 amino acid mutations in the Fc region compared to a native human Fc region, the mutations are independently selected from the group consisting of a deletion of one amino acid, a substitution of one amino acid, or an insertion of one amino acid, and in certain embodiments may be any of the mutations described herein.

[0161] In certain embodiments, antibodies provided herein that specifically bind to VISTA comprise a heavy chain constant region of an IgG1, IgG2, IgG3, or IgG4 isotype / class. In certain embodiments, antibodies provided herein that specifically bind to VISTA comprise a human Fc region of an IgG1, IgG2, IgG3, or IgG4 isotype / class. In certain embodiments, the constant region of an antibody or antigen-binding fragment provided herein that specifically binds to VISTA comprises 1, 2, 3, 4, or 5 amino acid mutations compared to the native constant region. In certain embodiments of the anti-VISTA antibodies and antigen-binding fragments of the invention, the antibodies comprise an Fc region or a variant of the Fc region, optionally wherein the Fc region is a human Fc region or a variant of the human Fc region having 1, 2, 3, 4 or 5 amino acid mutations in the Fc region compared to a native human Fc region, and / or optionally wherein the human Fc region is that of human IgG1, human IgG2 or human IgG4, and further optionally wherein the antibodies comprise the constant region of human IgG1 or human IgG4 or a variant of the constant region having 1, 2, 3, 4 or 5 amino acid mutations in the constant region compared to a native constant region.

[0162] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody or antigen-binding fragment thereof that specifically binds to VISTA as described herein to alter one or more functional properties of the antibody.

[0163] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region and / or hinge region of an antibody or antigen-binding fragment thereof that specifically binds to VISTA as described herein. Mutations in the Fc region of an antibody or antigen-binding fragment thereof that specifically binds to VISTA may modify the affinity of the antibody for an Fc receptor and / or a complement receptor. Techniques for introducing such mutations into the Fc receptor or fragment thereof are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody or antigen-binding fragment thereof that specifically binds to VISTA that can be made to alter the affinity of the antibody or antigen-binding fragment thereof for an Fc receptor are described, for example, in Smith P et al., (2012) Proc. Natl. Acad. Sci 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO02 / 060919, WO98 / 23289, and WO97 / 34631.

[0164] In certain embodiments, the antibodies provided herein comprise a constant region as set forth in Table 10. Without wishing to be bound by theory, the constant region of the antibody contributes to the sequence variation of the heavy chain. The variable region of the heavy chain recombines with the constant region of the heavy chain to generate a full-length heavy chain (Dreyer, WJ, and JC Bennett Proc. Natl. Acad. Sci. USA 54 (1965) 864-869). The antibody can have different isotypes depending on whether an alpha, mu, gamma, epsilon, or delta constant region gene segment is recombined with the variable region (Kataoka, T., et. Al. Proc. Natl. Acad. Sci. USA 77 (1980) 919-923). Within the human gamma gene segment, there are four distinct subclasses, designated gamma 1, 2, 3, and 4, which are approximately 90% identical to each other. Any of these different isotypes or subclasses may be combined with the variable regions of the antibodies described herein to produce a full-length heavy chain, which can then be paired with a complementary light chain to produce the antibody of the present invention.

[0165] In the case of antibody engineering of the antibodies provided herein, altering the sequence and / or post-translational modification of the Fc and hinge regions of the antibody allows for manipulation of the effector function and circulation of a given antibody or antibody-like protein (Presta, LG Curr. Opin. Immunol. 20 (2008) 460-470). In addition to sequence variation, the Fc region also contains an N-linked glycosylation site at residue 297 (EU numbering system), which is important for Fc structure and function (Dwek, RR et al. J. Anat. 187 (1995) 279-292), which can be mutated, for example, to alanine, glycine, or glutamic acid to disrupt the glycosylation site and affect the properties of the antibody.

[0166] The glycan present at N297 typically consists of two N-acetylglucosamines (GlcNAc), three mannoses, and two more GlcNAc linked to the mannose to form a biantennary complex glycan (Liu, LJ Pharm Sci. 104 (2015) 1866-84). These two GlcNAc are linked to the mannose via either a β1,2 linkage to the α-3 or α-6 of the mannose. Thus, each arm of the glycan can be distinguished as an α1,3 or α1,6 arm, depending on how the mannose is linked to GlcNAc2 (Liu, LJ Pharm Sci. 104 (2015) 1866-84). Additional fucose, galactose, sialic acid, and GlcNAc can be added to the core glycan structure, and the glycan composition directly influences FcγR binding.

[0167] For example, expression of β(1,4)-N-acetylglucosaminyltransferase III during IgG expression results in antibodies glycosylated at N297, which have biantennary glycans and higher ADCC activity (Umana, P. et. Al. Nat. Biotech. 17 (1999) 176-180). Antibodies with reduced fucose content have been reported to have increased affinity for Fc receptors, such as FcγRIIIa. Antibodies lacking fucose have been shown to bind to FcγRIIIa 50-fold more strongly and have enhanced ADCC activity (Shields, R.L. et. Al. J. Biol. Chem. 277 (2002) 26733-26740). Galactosylation of N297 enhances C1q binding and CDC activity (Dekkers, G., et. Al. Front. Immunol. 8 (2017) 877). Any of these N297 glycosylation manipulations can be performed on the antibodies of the present invention.

[0168] In certain embodiments, the antibodies described herein mediate ADCC, ADCP, and / or CDC. Mutations in the Fc region of the antibody can enhance effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) (Saunders, KO Front. Immunol. 10 (2019) 1-20).

[0169] Certain combinations of mutations that increase affinity for FcγRIIIa, FcγRIIa and / or FcγRIa, enhance ADCC and / or ADCP, and may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein. Examples of combinations of mutations that may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein that increase affinity for FcγRIIIa and / or FcγRIIa and enhance ADCC and / or ADCP include any of the following: (1) a combination of S298A, E333A, and K334A; (2) a combination of S239D, A330L, and I332E; (3) a combination of S239D and I332E; (4) a combination of G236A, S239D, A330L, and I332E; (5) a combination of S239D, I332E, and G236A; and (6) a combination of L234Y, G236W, and S298A, where these residues are numbered using the EU numbering system. The "EU numbering system", also known as the "EU index" (i.e., the EU index as reported in Kabat et al., 1991, National Institutes of Health (US) Office of the Director, Sequences of Proteins of Immunological Interest (5th ed., DIANE Publishing: Collingdale, PA 1991)) is commonly used when referring to residues within immunoglobulin heavy chain constant regions.

[0170] Certain mutations or combinations of mutations increase C1q binding and CDC and may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein. Examples of mutations or combinations of mutations that increase C1q binding and CDC that may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein include any of: (1) the combination of K326A and E333A, (2) the combination of K326M and E333S, (3) the combination of C221D and D222C, (4) the combination of S267E, H268F and S324T, (5) the combination of H268F and S324T, and (6) E345R, where these residues are numbered using the EU numbering system.

[0171] In vivo, IgG catabolism is regulated by the interaction of IgG with neonatal Fc receptor (FcRn) (Sockolosky JT, et. Al. Adv. Drug Deliv. Rev. 91 (2015) 109-124). IgG is taken up by cells, where it can be transported to lysosomes or recycled to the cell surface (Roopenian, DC, and Akilesh, S. Nat. Rev. Immunol. 7 (2007) 715-725). When IgG binds to FcRn at low pH (pH < 6.5) in endosomes, the antibody can be transported with FcRn back to the cell surface. At pH < 6.5, insufficient binding to FcRn results in the antibody being transported to lysosomes for degradation. At physiological pH in the extracellular environment, IgG has weak affinity for FcRn, resulting in its release from FcRn and its return to circulation. Thus, mutations that may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein may enhance FcRn binding at pH<6.5, thus increasing antibody recycling and improving PK.

[0172] Examples of mutations and combinations of mutations that may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein that increase FcRn binding and thus extend the half-life of the antibody include any of: (1) R435H; (2) N434A; (3) a combination of M252Y, S254T, and T256E; (4) a combination of M428L and N434S; (5) a combination of E294 deletion, T307P, and N434Y; (6) a combination of T256N, A378V, S383N, and N434Y; and (7) an E294 deletion, where these residues are numbered using the EU numbering system.

[0173] Mutations that reduce Fc receptor binding, complement receptor binding, or antibody effector function may also be desirable and may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein. Such mutations may reduce inflammation and cell death mediated by the antibody or antigen-binding fragment. Examples of mutations and combinations of mutations that may be present in the anti-VISTA antibodies and antigen-binding fragments provided herein that reduce binding to FcγRI, FcγRII, FcγRIII, and / or C1q, thereby reducing ADCC, ADCP, and / or CDC, include: (1) L235E; (2) the combination of L234A and L235A; and (3) the combination of S228P and L235E (this combination is referred to as an SPLE mutation; the S228P mutation prevents class switching to IgG4). Schlothauer et al., Protein Eng Des Sel (2016) 29:457-466), (4) a combination of L234A, L235A, and P329G, (5) a combination of P331S, L234E, and L235F, (6) D265A, (7) G237A, (8) E318A, (9) E233P, (10) a combination of G236R and L328R, (11) a combination of H268Q, V309L, A330S, and P331S, (12) a combination of L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (13 ) any one, two, three, four, five, or six of L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (14) A330L, (15) D270A, (16) K322A, (17) P329A, (18) P331A, (19) V264A, (20) F241A, N297A, N297G, N297E, and (21) any combination of S228P, F234A, and L235A, where these residues are numbered using the EU numbering system.

[0174] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of a human IgG1, and the Fc region has one or more amino acid mutations in the Fc region compared to a native human Fc region, for example, 1, 2, 3, 4, 5, 6, or 7 amino acid mutations, such as C220D, D221C, E233P, L234A, L234E, L234Y, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, and selected from the group consisting of T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein these residues are numbered using the EU numbering system.

[0175] In certain embodiments, an antibody provided herein that specifically binds to VISTA comprises a variant of a human Fc region, wherein the human Fc region is that of a human IgG2, and the Fc region has one or more amino acid mutations in the Fc region compared to a native human Fc region, for example, 1, 2, 3, 4, 5, 6, or 7 amino acid mutations, such as C220D, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H26 8F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, S383N, M428L, N434S, and N434Y, wherein these residues are numbered using the EU numbering system.

[0176] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and the Fc region has one or more amino acid mutations in the Fc region compared to a native human Fc region, for example, 1, 2, 3, 4, 5, 6, or 7 amino acid mutations, such as S228P, E233P, F234A, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T and N434Y, wherein the residues are numbered using the EU numbering system.

[0177] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, said human Fc region being that of a human IgG1, said Fc region having one or more amino acid mutations compared to a native human Fc region, said mutations being L234A and L235A, and optionally P329G, wherein these residues are numbered using the EU numbering system. In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, said human Fc region being that of a human IgG1, said Fc region having one or more amino acid mutations compared to a native human Fc region, said mutations being F234A and L235A, and optionally P329G, wherein these residues are numbered using the EU numbering system.

[0178] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG1, IgG2, or IgG4, and wherein the Fc region has one or more amino acid mutations compared to a native human Fc region, the mutations being M252Y, S254T, and T256E, where these residues are numbered using the EU numbering system.

[0179] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG1, IgG2, or IgG4, and wherein the Fc region has one or more amino acid mutations compared to a native human Fc region, the mutations being M428L and N434S, and wherein these residues are numbered using the EU numbering system.

[0180] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and wherein the Fc region has one or more amino acid mutations compared to a native human Fc region, the mutations being S228P and L235E, and wherein these residues are numbered using the EU numbering system.

[0181] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and wherein the Fc region has one or more amino acid mutations compared to a native human Fc region, the mutations being S228P, M252Y, S254T, and T256E, wherein these residues are numbered using the EU numbering system.

[0182] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and wherein the Fc region has one or more amino acid mutations compared to a native human Fc region, the mutations being S228P, M428L and N434S, and wherein these residues are numbered using the EU numbering system.

[0183] In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, the human Fc region of a human IgG1, wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including L234A and L235A, and optionally P329G, and these residues are numbered using the EU numbering system. In certain embodiments, an antibody that specifically binds to VISTA provided herein comprises a variant of a human Fc region, the human Fc region of a human IgG1, wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including F234A and L235A, and optionally P329G, and these residues are numbered using the EU numbering system.

[0184] In certain embodiments, antibodies that specifically bind to VISTA provided herein comprise a variant of a human Fc region, wherein the human Fc region is that of human IgG1, IgG2, or IgG4, and wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including M252Y, S254T, and T256E, where these residues are numbered using the EU numbering system.

[0185] In certain embodiments, antibodies that specifically bind to VISTA provided herein comprise a variant of a human Fc region, wherein the human Fc region is that of human IgG1, IgG2, or IgG4, and wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including the mutations M428L and N434S, where these residues are numbered using the EU numbering system.

[0186] In certain embodiments, antibodies that specifically bind to VISTA provided herein comprise a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including S228P and L235E, where these residues are numbered using the EU numbering system.

[0187] In certain embodiments, antibodies that specifically bind to VISTA provided herein comprise a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including S228P, M252Y, S254T, and T256E, where these residues are numbered using the EU numbering system.

[0188] In certain embodiments, antibodies that specifically bind to VISTA provided herein comprise a variant of a human Fc region, wherein the human Fc region is that of human IgG4, and wherein the Fc region has between 1 and 10 amino acid mutations compared to a native human Fc region, the mutations including S228P, M428L and N434S, where these residues are numbered using the EU numbering system.

[0189] In certain embodiments, antibodies that specifically bind to VISTA described herein comprise a glycosylated constant region. In certain embodiments, an antibody comprises an aglycosylated constant region. Thus, in certain embodiments, antibodies that specifically bind to VISTA described herein have reduced fucose content or are fucose-free.

[0190] In certain embodiments, antibodies that specifically bind to VISTA provided herein are mixed-isotype antibodies, ie, antibodies that comprise heavy chain constant regions from two or more different isotypes.

[0191] In certain embodiments, antibodies provided herein that specifically bind to VISTA comprise a biantennary glycan (GlcNAc2Man3GlcNAc2) attached to Asn297 (as identified by the EU numbering system) of the IgG-Fc of the antibody. In certain embodiments, antibodies provided herein that specifically bind to VISTA are deglycosylated, defucosylated, or galactosylated antibodies.

[0192] In certain embodiments, antibodies that specifically bind to VISTA provided herein and contain one or more amino acid mutations described herein exhibit reduced target-mediated drug elimination, e.g., receptor-mediated endocytosis and subsequent lysosomal degradation, compared to antibodies that do not contain the one or more amino acid substitutions.

[0193] In certain embodiments, antibodies provided herein that specifically bind to VISTA exhibit pH-dependent binding to VISTA.

[0194] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA provided herein comprises any of the histidine mutations described in the preceding paragraph in combination with any mutation to the constant region described herein, including mutations that increase or decrease binding to an Fc receptor or C1q receptor. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA provided herein comprises a histidine substitution that enhances target dissociation at acidic pH and a mutation that enhances FcRn binding.

[0195] Binding properties In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to human VISTA (e.g., expressed from a nucleic acid encoding SEQ ID NO: 377), particularly mature human VISTA lacking the signal sequence. Mature human VISTA is amino acids 33-311 of SEQ ID NO: 377, which lacks the signal sequence that is amino acids 1-32 of SEQ ID NO: 377. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to the extracellular domain (ECD) of human VISTA (e.g., amino acids 33-194 of SEQ ID NO: 378). In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to both human VISTA and cynomolgus monkey VISTA (e.g., mature monkey VISTA expressed from a nucleic acid encoding SEQ ID NO: 380 or lacking the signal sequence). In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA does not bind to mouse VISTA (e.g., mature mouse VISTA expressed from a nucleic acid encoding SEQ ID NO: 369 or lacking the signal sequence). Exemplary VISTA sequences are shown below in Table 11, with the signal sequence underlined and in bold. [Table 11-1] [Table 11-2]

[0196] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to the ECD of human VISTA at a K D It binds at about 0.5 nM to about 1 nM, about 1 nM to about 1.5 nM, about 1.5 nM to about 2 nM, about 2 nM to about 2.5 nM, about 2.5 nM to about 3 nM, about 3 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 20 nM, or about 20 nM to about 100 nM.

[0197] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to the ECD of cynomolgus monkey VISTA at a K D It binds at about 0.5 nM to about 1 nM, about 1 nM to about 1.5 nM, about 1.5 nM to about 2 nM, about 2 nM to about 2.5 nM, about 2.5 nM to about 3 nM, about 3 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 20 nM, or about 20 nM to about 100 nM.

[0198] The affinity is determined by the equilibrium dissociation constant (K D ), and the equilibrium binding constant (K A ) can be measured and / or expressed in several ways known in the art, including, but not limited to, D can be measured by techniques known to those skilled in the art, such as biolayer interferometry or surface plasmon resonance, for example, as described below.

[0199] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to human VISTA at a median effective concentration (EC 50 ) binds at about 0.5 nM, about 0.9 nM, about 1.6 nM, about 1.7 nM, about 2.1 nM, about 2.7 nM, about 3.2 nM, about 4.2 nM, about 5.5 nM, or about 11.7 nM.

[0200] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VISTA binds to human VISTA on the cell surface, and 50It binds at about 0.05 nM, about 0.06 nM, about 0.09 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.6 nM, about 0.8 nM or about 1.2 nM.

[0201] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA does not bind to B7-1 (also known as CD80), B7-2 (also known as CD86), B7-H2 (also known as ICOS ligand), B7-H1 (also known as PD-L1 or CD274), B7-DC (also known as PD-L2 or CD273), B7-H4 (also known as B7S1), and / or B7-H3 (also known as CD276).

[0202] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA blocks the interaction between V-set and Ig domain-containing protein 3 (VSIG3) and VISTA. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA blocks VISTA dimerization or another homotypic interaction. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA blocks the interaction between P-selectin glycoprotein ligand 1 (PSGL1) and VISTA. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA blocks the interaction between V-set Ig domain-containing protein 8 (VSIG8) and VISTA. In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA blocks the interaction between leucine-rich repeat and immunoglobulin-like domain protein 1 (LRIG1) and VISTA.

[0203] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to VISTA has the sequence 66 HLHHG 70 (amino acids 98-102 of SEQ ID NO: 377) or 116 VVEIRHHHSEHR 127(amino acids 148-159 of SEQ ID NO: 377) (the numbering of the VISTA residues starts with the first amino acid after the signal peptide). The VISTA signal sequence is bolded and underlined in Table 11. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope of human VISTA comprising tyrosine 37, arginine 54, valine 117, and arginine 127 of SEQ ID NO: 377.

[0204] In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that bind to the same or overlapping epitope of VISTA as the antibodies described herein. As used herein, "epitope" is a term used in accordance with its meaning known in the art and refers to a localized region of an antigen to which an antibody can specifically bind via its antigen-binding domain. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, assembled from two or more non-contiguous regions of a polypeptide(s) (a conformational, non-linear, discontinuous, or discontinuous epitope).

[0205] Antibodies or antigen-binding fragments thereof that bind to the same or overlapping epitope of VISTA as the antibodies described herein may be human, humanized, chimeric, or bispecific. Humanized antibodies generally contain variable regions comprising human constant regions and human framework regions, but the CDRs are from a non-human species (e.g., murine CDRs). Chimeric antibodies generally contain constant regions of human origin and variable regions from a non-human species (e.g., murine variable regions).

[0206] In certain embodiments, the epitope of an antibody can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., MALDI mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping), or by the methods described below: For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals may be examined using well-known X-ray diffraction techniques and may be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al., (1995) and Cunningham BC & Wells JA (1989).

[0207] Antibodies that recognize and bind to the same or overlapping epitope of VISTA as the antibodies described herein can also be identified using routine techniques, such as immunoassays, to demonstrate, for example, the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as VISTA. Numerous types of competitive binding assays are available, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253), solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9), solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using I-125 labels (see Morel GA et al., (1988) Mol Immunol 25(1):7-15), solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52), direct label RIA. (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), and biolayer interferometry ("BLI"), such as the Octet Red 96 (ForteBio) system, are known. Typically, such assays involve the use of purified antigen (e.g., VISTA) or cells bearing either of these bound to a solid surface, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess.Typically, when a competing antibody is present in excess, it inhibits specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzymatic labels. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315, Wagener C et al., (1984) J Immunol Methods 68:269-274, Kuroki M et al., (1990) Cancer Res 50:4872-4879, Kuroki M et al., (1992) Immunol Invest 21:523-538, Kuroki M et al., (1992) Hybridoma 11:391-407, and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors, supra, pp. 386-389.

[0208] In certain aspects, competitive binding assays can be used to determine whether an antibody is competitively blocked by another antibody, for example, in a dose-dependent manner. In certain embodiments, the competitive binding assay is a competitive ELISA, which can be configured in several different formats, using either labeled antigen or labeled antibody. In certain embodiments, antibodies can be tested in competitive binding assays with the antibodies described herein.

[0209] In certain embodiments, provided herein are antibodies that compete (e.g., in a dose-dependent manner) with an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein for binding to VISTA, as measured using any of the assays known to those of skill in the art or described herein, for example, an ELISA competition assay, BLI (e.g., BLI with an Octet Red 96 (ForteBio) system, or surface plasmon resonance). In certain embodiments, provided herein are antibodies that competitively inhibit (e.g., in a dose-dependent manner) an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein from binding to VISTA, as measured using any of the assays known to those of skill in the art or described herein, for example, an ELISA competition assay, suspension array, BLI (e.g., BLI with an Octet Red 96 (ForteBio) system, or surface plasmon resonance).

[0210] In certain embodiments, provided herein are antibodies that compete for binding to VISTA with an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein to the same extent as the antibody described herein self-competes for binding to VISTA. In certain embodiments, provided herein are first antibodies that compete for binding to VISTA with an antibody or antigen-binding fragment thereof that specifically binds to VISTA described herein, wherein the competition is indicated by a greater than 80% (e.g., 85%, 90%, 95%, or 98%, or 80%-85%, 80%-90%, 85%-90%, or 85%-95%) reduction in binding of the first antibody to VISTA.

[0211] Antibodies or antigen-binding fragments thereof that compete for binding to VISTA with the antibodies or antigen-binding fragments thereof that specifically bind to VISTA described herein may be human, humanized or chimeric antibodies.

[0212] In certain aspects, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to VISTA with an antibody comprising the VH of an antibody listed in Table 7 and the VL of the same antibody listed in Table 8. In certain embodiments, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to VISTA with an immunoglobulin comprising the light chain of an antibody listed in Table 9 and the heavy chain of the same antibody listed in Table 9.

[0213] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to the same or overlapping epitope of an antibody comprising a VH of an antibody listed in Table 7 and a VL of the same antibody listed in Table 8. Assays known to those of skill in the art or described herein (e.g., X-ray crystallography, ELISA assays, etc.) can be used to determine whether two antibodies bind to the same epitope.

[0214] In certain embodiments, an antibody or antigen-binding fragment thereof that competes for binding to VISTA with an antibody or antigen-binding fragment thereof that specifically binds to VISTA as...

Claims

1. 1. An antibody, or antigen-binding fragment thereof, that binds to V domain Ig-containing suppressor of T cell activation (VISTA), the antibody, or antigen-binding fragment thereof, blocks binding of ligands to all five VISTA at pH 6.0 and pH 7.4, the five known ligands to VISTA being VSIG-3 (V-set and immunoglobulin domain containing 3), VSIG-8 (V-set and immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), LRIG1 (leucine-rich repeat and immunoglobulin-like domain 1) and VISTA; The antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL).

2. An antibody or antigen-binding fragment thereof that binds to VISTA, the antibody or antigen-binding fragment thereof comprising a VH and a VL, The VH is a VH CDR1 of any one of SEQ ID NOs: 249, 247, 248, or 250-253 as defined by the Kabat numbering system, any one of SEQ ID NOs: 286, 284, 285, or 287-291 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 320, 318, 319, or 321-325 as defined by the Paratome numbering system; a VH CDR2 of any one of SEQ ID NOs: 256, 254, 255, or 257-265 as defined by the Kabat numbering system, any one of SEQ ID NOs: 294, 292, 293, or 295-298 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 328, 326, 327, or 329-339 as defined by the Paratome numbering system; comprising a VH CDR3 of any one of SEQ ID NOs: 268, 266, 267, or 269-283 as defined by the Kabat numbering system, any one of SEQ ID NOs: 301, 299, 300, or 302-317 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 342, 340, 341, or 343-359 as defined by the Paratome numbering system; The VL is a VL CDR1 of any one of SEQ ID NOs: 113, 111, 112, or 114-122 as defined by the Kabat numbering system, any one of SEQ ID NOs: 154, 152, 153, or 155-162 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 190, 188, 189, 191-196, or 576-578 as defined by the Paratome numbering system; a VL CDR2 of any one of SEQ ID NOs: 125, 123, 124, or 126-131 and 575 as defined by the Kabat numbering system, any one of SEQ ID NOs: 165, 163, 164, or 166-167 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 199, 197, 198, or 200-206 as defined by the Paratome numbering system; and The antibody, or antigen-binding fragment thereof, comprises a VL CDR3 of any one of SEQ ID NOs: 134, 132, 133, or 135-151 as defined by the Kabat numbering system, any one of SEQ ID NOs: 170, 168, 169, or 171-186 as defined by the IMGT numbering system, or any one of SEQ ID NOs: 209, 207, 208, or 210-226 as defined by the Paratome numbering system.

3. 3. The antibody, or antigen-binding fragment thereof, of claim 1 or claim 2, the VH comprises a sequence comprising, or consisting of, any one of SEQ ID NOs: 592, 584-591, 593-597, 227-246, and 383-386 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 592, 584-591, 593-597, 227-246, and 383-386; The antibody, or antigen-binding fragment thereof, wherein the VL comprises a sequence comprising, or consisting of, any one of SEQ ID NOs: 86, 84, 85, or 87-110 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 86, 84, 85, or 87-110.

4. The antibody or antigen-binding fragment thereof of claim 2, the VH CDR1 comprises SEQ ID NO:249, the VH CDR2 comprises SEQ ID NO:256, the VH CDR3 comprises SEQ ID NO:268, the VL CDR1 comprises SEQ ID NO:113, the VL CDR2 comprises SEQ ID NO:125, and the VL CDR3 comprises SEQ ID NO:134; or the VH CDR1 comprises SEQ ID NO:286, the VH CDR2 comprises SEQ ID NO:294, the VH CDR3 comprises SEQ ID NO:301, the VL CDR1 comprises SEQ ID NO:154, the VL CDR2 comprises SEQ ID NO:165, and the VL CDR3 comprises SEQ ID NO:170; or the VH CDR1 comprises SEQ ID NO: 320, the VH CDR2 comprises SEQ ID NO: 328, the VH CDR3 comprises SEQ ID NO: 342, the VL CDR1 comprises SEQ ID NO: 190, the VL CDR2 comprises SEQ ID NO: 199, and the VL CDR3 comprises SEQ ID NO: 209, or an antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, (i) the VH comprises a sequence comprising, or consisting of, SEQ ID NO:592, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:592; the VL comprises a sequence comprising or consisting of SEQ ID NO: 86, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86; or (ii) the VH comprises a sequence comprising, or consisting of, SEQ ID NO: 586, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 586; The antibody, or antigen-binding fragment thereof, wherein the VL comprises a sequence comprising, or consisting of, SEQ ID NO: 86, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

86.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, a heavy chain (HC) sequence comprising, or consisting of, any one of SEQ ID NOs: 573, 407-477, 572, 574, and 604-609 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 573, 407-477, 572, 574, and 604-609; The antibody, or an antigen-binding fragment thereof, comprising a light chain (LC) sequence comprising, or consisting of, any one of SEQ ID NOs: 391, 387-390, 392-406, 569-571, and 598-603 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 391, 387-390, 392-406, 569-571, and 598-603.

7. binds to human VISTA (amino acids 33-311 of SEQ ID NO:377); Optionally, binds to an epitope of human VISTA comprising the amino acid sequence 66 HLHHG 70 (amino acids 98-102 of SEQ ID NO:377) or 116 VVEIRHHHSEHR 127 (amino acids 148-159 of SEQ ID NO:377); or Optionally, an antibody or antigen-binding fragment thereof according to any one of the preceding claims, which binds to an epitope of human VISTA including tyrosine 37, arginine 54, valine 117, and arginine 127 of SEQ ID NO:377 (amino acids 69, 86, 149, and 159, respectively, of SEQ ID NO:377).

8. 10. The antibody, or antigen-binding fragment thereof, of any one of the preceding claims, comprising an Fc region, Optionally, the Fc region is a human Fc region or a variant of the human Fc region having from 1 to 7 amino acid mutations in the Fc region compared to a native human Fc region; and / or Optionally, the human Fc region is of human IgG1, human IgG2 or human IgG4, and further optionally, the antibody comprises a human IgG1 or human IgG4 constant region, or a variant of said constant region having from 1 to 7 amino acid mutations in said constant region compared to a native constant region, and optionally (a) the Fc region is that of a human IgG1 and the mutations are C220D, D221C, E233P, L234A, L234E, L234Y, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion; N297A, N297G, N297E, S298A, T307P, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system; (b) the Fc region is of human IgG2 and the mutations are C220D, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N297G, N297E, S298A, T307P, V309 L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, A330L, A330S, P331A, P331S, I332E, E333A, E333S, K334A, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system; or (c) the Fc region is that of human IgG4 and the mutations are S228P, E233P, F234A, L235A, L235E, L235F, G236A, G236W, G236R, G237A, P238S, S239D, F241A, M252Y, S254T, T256E, T256N, V264A, D265A, S267E, H268F, H268A, D270A, H268Q, E294 deletion, N297A, N2 97G, N297E, S298A, T307P, V309L, E318A, K322A, S324T, K326A, K326M, L328R, P329A, P329G, I332E, E333A, E333S, K334A, A378V, S383N, M428L, N434S, and N434Y, wherein the residues are numbered using the EU numbering system.

9. a bispecific or multispecific antibody; or the antigen-binding fragment is an Fv fragment, a Fab fragment, an F(ab')2 fragment, or a single chain Fv (scFv); 10. An antibody, or an antigen-binding fragment thereof, according to any one of the preceding claims.

10. An antibody, or antigen-binding fragment thereof, that competes with any one of the antibodies, or antigen-binding fragments thereof, of the preceding claims for binding to human VISTA (sequence number 377).

11. An antibody-drug conjugate comprising the antibody, or antigen-binding fragment thereof, of any one of the preceding claims and a therapeutic agent.

12. A chimeric antigen receptor (CAR) comprising the scFv of claim 9.

13. A polynucleotide comprising a nucleotide sequence encoding the VH, the VL, or the VH and the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 10.

14. A cell comprising one or more polynucleotides encoding the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 10.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the antibody-drug conjugate according to claim 11, or the CAR according to claim 12, and a pharma- ceutical acceptable carrier.

16. A method for producing an antibody, or antigen-binding fragment thereof, comprising culturing a cell described in claim 14 under conditions such that one or more polynucleotides are expressed by the cell to produce the antibody, or antigen-binding fragment thereof, encoded by the polynucleotides.

17. 16. A pharmaceutical composition according to claim 15 for use in the treatment of cancer, an autoimmune disease and / or an infectious disease in a subject in need thereof, comprising: Optionally, the pharmaceutical composition for use, wherein the cancer is non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, ovarian cancer, neuroblastoma, oral cancer, thyroid cancer, breast cancer, sarcoma, pancreatic cancer, colon cancer, gastric cancer, choriocarcinoma, testicular cancer, mesothelioma, skin cancer, renal cell carcinoma, bladder cancer, blood cancer, cervical cancer, blood cancer, acute myeloid leukemia, myelodysplastic syndrome, and / or metastatic cancer.

18. 18. The pharmaceutical composition for use according to claim 17, characterized in that the subject is further administered an additional therapy, optionally wherein the additional therapy is radiation therapy, a chemotherapeutic agent, a targeted therapy, a tyrosine kinase inhibitor, a hormone therapy, and / or an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is an inhibitor of programmed death-1 (PD-1), or programmed death-ligand 1 (PDL1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or lymphocyte-activation gene 3 (LAG-3).