Antibodies binding to vista at acidic ph

Abstract

To provide an isolated antibody that specifically binds to human VISTA and its use in cancer treatment.SOLUTION: An isolated antibody is provided that has been engineered by replacing from 1 to 5 glutamic acid, aspartic acid, or histidine residues in VH CDRs of antibody P1-068744 or antibody P1-068748 with the amino acid residue found at the same position in antibody P1-061015, wherein the amino acid sequences of HV CDR1, VH CDR2, and VH CDR3 of the isolated antibody, considered as a whole, differ from those of antibody P1-061015. In some embodiments, the antibodies bind specifically to human VISTA at acidic pH, but do not significantly bind to human VISTA at neutral or physiological pH.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application relates to antibodies that specifically bind to V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA) at acidic pH and their use in the treatment of cancer. [Background technology]

[0002] V-domain Ig-containing suppressor of T-cell activation, or VISTA, is a co-inhibitory member of the B7 family of immune receptors expressed by myelomonocytic cells and other leukocytes. However, the mechanism by which VISTA suppresses immune responses is not well understood. Summary of the Invention [Problem to be solved by the invention]

[0003] The present inventors have found that, unlike other known immune receptors, VISTA binds to its counterreceptor and functions selectively at acidic pH, with only limited activity at physiological pH (e.g., 7.3-7.4). Therefore, VISTA can suppress immune responses in acidic microenvironments, such as tumor tumors or sites of inflammation, without disrupting cell circulation in the blood or in non-inflamed, non-acidic tissues. Furthermore, the present inventors have found that anti-VISTA antibodies can be engineered to selectively bind to VISTA at acidic pH and exhibit little or no binding at physiological pH, mimicking VISTA's own acidic pH selectivity. These acidic pH-selective antibodies may offer desirable properties for treating diseases such as cancer, compared to antibodies that bind to VISTA at physiological pH. [Means for solving the problem]

[0004] The present disclosure relates to antibodies that specifically bind to the extracellular domain (ECD) of VISTA, such as human VISTA ("hVISTA" or "huVISTA"), at acidic pH (e.g., in acidic conditions). The present disclosure also relates to antibodies that specifically bind to the extracellular domain (ECD) of VISTA, such as hVISTA, at acidic pH, but have little or no binding at neutral or physiological pH. The inventors have noted herein that the hVISTA-ECD amino acid sequence contains several conserved and non-conserved histidine residues, and that the frequency of histidine residues in the ECD of VISTA is exceptionally high compared to other B7 family members and other immunoglobulin superfamily members (see Figures 1A and 1B). In solution, the amino acid histidine has a pK of approximately 6.5. a This means that at pH 6.5 and below, histidine residues within proteins are often protonated and therefore positively charged, whereas at pHs above 6.5, they become increasingly unprotonated and neutrally charged. Tumor microenvironments and inflamed tissues are often acidic, and therefore VISTA proteins found in these microenvironments may be partially protonated at least at their histidine residues. As discussed herein, the inventors hypothesized that histidine protonation may affect the conformation, surface structure, and / or charge density of VISTA, which may then create pH-specific or pH-selective epitopes for both receptor-ligand interactions and antibody binding. Targeting VISTA with antibodies that bind at acidic pH but not at neutral or physiological pH may prevent target-mediated pharmacokinetics via myelomonocytic cells resident in circulatory and lymphoid organs, improving antibody PK, receptor occupancy, and activity in the tumor microenvironment. Acidic pH selective antibodies may also improve the specificity of VISTA antibodies for intratumoral target cells rather than circulating target cells for therapeutic modalities such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and delivery of payloads (antibody-drug conjugates).

[0005] This disclosure relates to the following: P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E 102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32 Described are anti-hVISTA Abs including P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, and P1-068748_D57K_D100S. These antibodies may contain the heavy chain CDRs of either the P1-068744 or P1-068748 Abs modified to restore at least one H, D, or E residue to the residue found at the same position in the P1-061015 parent antibody.

[0006] More generally, the disclosure includes anti-hVISTA Abs comprising a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1YAMH (wherein X1 is E or S) (SEQ ID NO: 690), a VH CDR2 comprising X2IWYDGSNKYX3ADSVKG (wherein X2 is H or I and X3 is E or Y) (SEQ ID NO: 691), and / or a VH CDR3 comprising DSGFYX4SYYFDX5 (wherein X4 is E or S and X5 is E or Y) (SEQ ID NO: 692) (see also Figure 29C). In Ab P1-061015, these positions are S, I, Y, S, and Y, respectively, while in P1-068744, these positions are E, H, E, E, and E, respectively. In such cases, the antibody may comprise a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1YAMH (wherein X1 is E or S) (SEQ ID NO: 690), a VH CDR2 comprising X2IWYDGSNKYX3ADSVKG (wherein X2 is H or I and X3 is E or Y) (SEQ ID NO: 691), and / or a VH CDR3 comprising DSGFYX4SYYFDX5 (wherein X4 is E or S and X5 is E or Y) (SEQ ID NO: 692). In Ab P1-061015, these positions are S, I, Y, S, and Y, respectively, while in P1-068744, these positions are E, H, E, E, and E, respectively. In such cases, the antibody may comprise a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1YAMH (wherein X1 is E or S) (SEQ ID NO: 690), a VH CDR2 comprising X2IWYDGSNKYX3ADSVKG (wherein X2 and P1-68744_E31S (i.e., containing the CDRs of P1-068744 except when X1 is S), P1-68744_H50I (i.e., X2 is I), P1-68744_E59Y (i.e., X3 is Y), P1-068744_E100S (i.e. X4 is S), P1-068744_E102Y (i.e. X5 is Y), P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, or P1-068744_E59Y_E102Y. Note that the nomenclature for these antibodies used above is somewhat based on the Kabat numbering scheme, which numbers residues by their structural position rather than by their sequence position. Because antibodies may have CDRs of different lengths, the numbering scheme may not necessarily correspond to the number of residue positions in the antibody sequence.The above substitutions at X1, X2, X3, X4, and X5 are located at positions 31, 50, 60, 104, and 110 of the heavy chain sequences of SEQ ID NOs: 95 and 103 herein, as can be readily seen from reviewing Tables 11 and 27. See, e.g., M. Dondelinger et al., Front. Immunol. 9: 2278 (2018) for more information regarding antibody residue numbering formats.

[0007] Additionally, the disclosure includes anti-hVISTA Abs comprising a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1X2AMH (wherein X1 is H or S and X2 is H or Y) (SEQ ID NO: 693), a VH CDR2 comprising IIWYDGSNX3X4YADSVKG (wherein X3 is D or K and X4 is D or Y) (SEQ ID NO: 694), and / or a VH CDR3 comprising DSGFYX5SYYFDY (wherein X5 is D or S) (SEQ ID NO: 695). (See also Figure 29C). In Ab P1-061015, these positions are S, Y, K, Y, and S, respectively, while in P1-068748, these positions are H, H, D, D, and D. In such cases, the antibody may comprise a heavy chain variable region (VH) comprising the VH CDR1 of P1-068748. and P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H3 VH CDR1, CDR2 and / or CDR3 of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S or P1-068748_D57K_D100S. Note that the nomenclature herein is based on Kabat numbering. The above substitutions at X1, X2, X3, X4 and X5 are located at positions 31, 32, 58, 59 and 104 of the heavy chain sequences of SEQ ID NOs: 95 and 99, as can be seen, for example, from reviewing Tables 12 and 28 and SEQ ID NOs: 95 and 99.

[0008] The priority U.S. provisional application to which this application claims benefit contains at least one drawing executed in color. If the provisional application is later made publicly available, copies of the color drawing(s) will be available from the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0009] [Figure 1A] Figures 1A-C show that the extracellular domain of VISTA contains an exceptionally high frequency of histidine residues, many of which are conserved, and that at least some of these histidine residues may be involved in receptor-ligand binding. Figure 1A shows a graph of immunoglobulin domain-containing proteins, with the number of amino acid residues in the extracellular domain of each protein plotted on the x-axis and the frequency of histidine residues within the extracellular domain of each protein plotted on the y-axis. The magnitude of each data point corresponds to the total number of histidine residues in the extracellular domain of each protein. Figure 1B shows the aligned amino acid sequences of the extracellular domains of human, cynomolgus macaque, and mouse VISTA. The signal peptide (Sig) and transmembrane domain (TMD) sequence positions are indicated. Histidine residues conserved across all three species are shown in bold and underlined; histidine residues conserved across human and cynomolgus macaque are only shown in bold. Figure 1C shows a model of the three-dimensional structure of the human VISTA immunoglobulin domain, with histidine residues represented as ball-and-stick traces. [Figure 1B]Figures 1A-C show that the extracellular domain of VISTA contains an exceptionally high frequency of histidine residues, many of which are conserved, and that at least some of these histidine residues may be involved in receptor-ligand binding. Figure 1A shows a graph of immunoglobulin domain-containing proteins, with the number of amino acid residues in the extracellular domain of each protein plotted on the x-axis and the frequency of histidine residues within the extracellular domain of each protein plotted on the y-axis. The magnitude of each data point corresponds to the total number of histidine residues in the extracellular domain of each protein. Figure 1B shows the aligned amino acid sequences of the extracellular domains of human, cynomolgus macaque, and mouse VISTA. The signal peptide (Sig) and transmembrane domain (TMD) sequence positions are indicated. Histidine residues conserved across all three species are shown in bold and underlined; histidine residues conserved across human and cynomolgus macaque are only shown in bold. Figure 1C shows a model of the three-dimensional structure of the human VISTA immunoglobulin domain, with histidine residues represented as ball-and-stick traces. [Figure 1C]Figures 1A-C show that the extracellular domain of VISTA contains an exceptionally high frequency of histidine residues, many of which are conserved, and that at least some of these histidine residues may be involved in receptor-ligand binding. Figure 1A shows a graph of immunoglobulin domain-containing proteins, with the number of amino acid residues in the extracellular domain of each protein plotted on the x-axis and the frequency of histidine residues within the extracellular domain of each protein plotted on the y-axis. The magnitude of each data point corresponds to the total number of histidine residues in the extracellular domain of each protein. Figure 1B shows the aligned amino acid sequences of the extracellular domains of human, cynomolgus macaque, and mouse VISTA. The signal peptide (Sig) and transmembrane domain (TMD) sequence positions are indicated. Histidine residues conserved across all three species are shown in bold and underlined; histidine residues conserved across human and cynomolgus macaque are only shown in bold. Figure 1C shows a model of the three-dimensional structure of the human VISTA immunoglobulin domain, with histidine residues represented as ball-and-stick traces. [Figure 2A] Figures 2A-B show a model in which histidine residues in the extracellular domain of VISTA confer counterreceptor selectivity at acidic pH rather than physiological pH. Figure 2A shows the equilibrium between the absence and presence of protonation of the pyrrole ammonium group (NH) on histidine residues. The pKa of histidine in solution is 6.5, indicating that histidine residues are more likely to be protonated, and therefore positively charged, at pH 6.5 or below than at higher pHs. Figure 2B shows a model in which VISTA selectively associates with P-selectin glycoprotein ligand 1 (PSGL-1) or other counterreceptors and ligands ("VISTA-R") at acidic pH. Therefore, antibodies that bind to the extracellular domain of VISTA at acidic pH but not physiological pH may be crucial for inhibiting or modulating VISTA activity. [Figure 2B]Figures 2A-B show a model in which histidine residues in the extracellular domain of VISTA confer counterreceptor selectivity at acidic pH rather than physiological pH. Figure 2A shows the equilibrium between the absence and presence of protonation of the pyrrole ammonium group (NH) on histidine residues. The pKa of histidine in solution is 6.5, indicating that histidine residues are more likely to be protonated, and therefore positively charged, at pH 6.5 or below than at higher pHs. Figure 2B shows a model in which VISTA selectively associates with P-selectin glycoprotein ligand 1 (PSGL-1) or other counterreceptors and ligands ("VISTA-R") at acidic pH. Therefore, antibodies that bind to the extracellular domain of VISTA at acidic pH but not physiological pH may be crucial for inhibiting or modulating VISTA activity. [Figure 3] Figure 3 shows the levels of VISTA surface expression (mean fluorescent intensity (MFI) of anti-VISTA antibody staining) on tumor-infiltrating macrophages, dendritic cells, neutrophils, CD4+ effector T cells, CD4+ regulatory T cells, CD8+ T cells, natural killer (NK) cells, and B cells. VISTA is expressed on numerous tumor-infiltrating leukocytes, particularly myeloid cells. The tumor microenvironment is often acidic, allowing VISTA to associate with counterreceptors and ligands. [Figure 4A]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 4B]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 4C]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 4D]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 4E]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 4F-4G]Figures 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH, with little or no binding at neutral pH, and that this binding can be blocked by anti-VISTA antibodies. Figure 4A on the left shows a representative histogram of fluorescently conjugated recombinant VISTA multimers binding to activated human CD4+ T cells. Filled histograms, ranging from dark gray to light gray, represent binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH. Non-VISTA control multimer binding at pH 6.0 is shown as an unfilled histogram. On the right, the average MFI of VISTA (circles) and control (triangles) multimers binding to activated human CD4+ T cells from two donors at different pHs is graphed. Figure 4B shows representative histograms of recombinant VISTA multimer binding to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Unfilled, solid-bounded, and dotted-bounded histograms represent binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C shows representative recombinant VISTA multimer binding to activated human CD4+ T cells in the presence of an anti-VISTA blocking antibody (squares) or a non-VISTA-specific, isotype-matched control antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regression is also shown. Triangles represent background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. Figure 4D shows a representative two-dimensional flow cytometry plot of recombinant VISTA multimer binding at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (strain pGSD-677, American Type Culture Collection) transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibodies shown in Figure 4C.Cells left unstained by recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E shows representative histograms of recombinant mouse VISTA-Fc fusion protein binding to mouse splenocytes at pH 6.0 and pH 7.4. Filled histograms, ranging from dark gray to light gray, represent binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms represent binding to total splenocytes at pH 7.4. Figures 4F and 4G show that VISTA multimers bind to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4. [Figure 5A] Figures 5A–D show that VISTA preferentially mediates T cell suppression and cell:cell adhesion at acidic pH, and both effects can be reversed using anti-VISTA blocking antibodies. Figure 5A shows representative cell:cell conjugate formation at pH 6.0 and 7.0 between 293T cells expressing hVISTA or a vector control (plotted on the y-axis) and CHO cells endogenously expressing cell surface heparan sulfate on the x-axis. Figure 5B is a graph of the frequency of cell conjugates formed at pH 6.0 between the same cells in the presence of an anti-VISTA blocking antibody, an anti-VISTA non-blocking antibody, or an isotype-matched non-VISTA-specific control antibody. Figure 5C shows representative plots of luciferase activity produced by Jurkat (human T cell line) cells expressing an NFkB luciferase reporter after coculture at various pHs with 293T cells expressing hVISTA and the single-chain variable fragment of the anti-human T cell receptor agonist antibody OKT3 ("artificial antigen-presenting cells"). Co-cultured cells were treated with an anti-VISTA blocking antibody (squares) or an isotype-matched non-VISTA-specific control antibody (circles). In Figure 5D, the data shown in Figure 5A are plotted as the fold increase in luciferase signal ("effect size") with anti-VISTA antibody treatment compared to control. [Figure 5B]Figures 5A–D show that VISTA preferentially mediates T cell suppression and cell:cell adhesion at acidic pH, and both effects can be reversed using anti-VISTA blocking antibodies. Figure 5A shows representative cell:cell conjugate formation at pH 6.0 and 7.0 between 293T cells expressing hVISTA or a vector control (plotted on the y-axis) and CHO cells endogenously expressing cell surface heparan sulfate on the x-axis. Figure 5B is a graph of the frequency of cell conjugates formed at pH 6.0 between the same cells in the presence of an anti-VISTA blocking antibody, an anti-VISTA non-blocking antibody, or an isotype-matched non-VISTA-specific control antibody. Figure 5C shows representative plots of luciferase activity produced by Jurkat (human T cell line) cells expressing an NFkB luciferase reporter after coculture at various pHs with 293T cells expressing hVISTA and the single-chain variable fragment of the anti-human T cell receptor agonist antibody OKT3 ("artificial antigen-presenting cells"). Co-cultured cells were treated with an anti-VISTA blocking antibody (squares) or an isotype-matched non-VISTA-specific control antibody (circles). In Figure 5D, the data shown in Figure 5A are plotted as the fold increase in luciferase signal ("effect size") with anti-VISTA antibody treatment compared to control. [Figure 5C]Figures 5A–D show that VISTA preferentially mediates T cell suppression and cell:cell adhesion at acidic pH, and both effects can be reversed using anti-VISTA blocking antibodies. Figure 5A shows representative cell:cell conjugate formation at pH 6.0 and 7.0 between 293T cells expressing hVISTA or a vector control (plotted on the y-axis) and CHO cells endogenously expressing cell surface heparan sulfate on the x-axis. Figure 5B is a graph of the frequency of cell conjugates formed at pH 6.0 between the same cells in the presence of an anti-VISTA blocking antibody, an anti-VISTA non-blocking antibody, or an isotype-matched non-VISTA-specific control antibody. Figure 5C shows representative plots of luciferase activity produced by Jurkat (human T cell line) cells expressing an NFkB luciferase reporter after coculture at various pHs with 293T cells expressing hVISTA and the single-chain variable fragment of the anti-human T cell receptor agonist antibody OKT3 ("artificial antigen-presenting cells"). Co-cultured cells were treated with an anti-VISTA blocking antibody (squares) or an isotype-matched non-VISTA-specific control antibody (circles). In Figure 5D, the data shown in Figure 5A are plotted as the fold increase in luciferase signal ("effect size") with anti-VISTA antibody treatment compared to control. [Figure 5D]Figures 5A–D show that VISTA preferentially mediates T cell suppression and cell:cell adhesion at acidic pH, and both effects can be reversed using anti-VISTA blocking antibodies. Figure 5A shows representative cell:cell conjugate formation at pH 6.0 and 7.0 between 293T cells expressing hVISTA or a vector control (plotted on the y-axis) and CHO cells endogenously expressing cell surface heparan sulfate on the x-axis. Figure 5B is a graph of the frequency of cell conjugates formed at pH 6.0 between the same cells in the presence of an anti-VISTA blocking antibody, an anti-VISTA non-blocking antibody, or an isotype-matched non-VISTA-specific control antibody. Figure 5C shows representative plots of luciferase activity produced by Jurkat (human T cell line) cells expressing an NFkB luciferase reporter after coculture at various pHs with 293T cells expressing hVISTA and the single-chain variable fragment of the anti-human T cell receptor agonist antibody OKT3 ("artificial antigen-presenting cells"). Co-cultured cells were treated with an anti-VISTA blocking antibody (squares) or an isotype-matched non-VISTA-specific control antibody (circles). In Figure 5D, the data shown in Figure 5A are plotted as the fold increase in luciferase signal ("effect size") with anti-VISTA antibody treatment compared to control. [Figure 6A]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6B]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6C]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6D]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6E]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6F]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 6G]Figures 6A-G show that VISTA can be found in intracellular endosomes, particularly Rab11+ recycling endosomes, and can be recycled to and from the cell surface by endosomal trafficking. Figure 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. Figure 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A non-VISTA-binding control antibody of the same isotype as the VISTA antibody ("cAb") does not detectably bind to monocytes. Figure 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6.7 (light gray). Figure 6D shows the sensitivity of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), and 3 (squares) with cathepsin B-sensitive linkers and cytotoxic payloads, or a non-VISTA-specific control antibody (triangles). Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E compares hVISTA binding of anti-VISTA antibody 3 with that of an engineered variant ("VISTA mAb 3c") that does not exhibit improved binding at acidic pH. Figure 6F shows an antibody-drug conjugate assay comparing the potency of anti-VISTA antibodies 3 (squares) and 3c (diamonds). Figure 6G shows a schematic of endosomal trafficking, in which VISTA recycles to and from the cell surface via early and recycling endosomes. [Figure 7A]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 7B]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 7C]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 7D]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 7E]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 7F]Figures 7A-F show how an anti-VISTA antibody variant library was designed and screened to obtain an acidic pH-selective antibody. Figure 7A shows the amino acid substitutions made in the VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to generate the '029 library for screening. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the library tolerated the substitution of the negatively charged amino acids aspartic acid and glutamic acid and pH-sensitive histidine. X = H, D, or E. Sequences in parentheses were removed from the synthesis to avoid introducing disadvantages. A total of 647 unique sequences of P1-061029 HCDR3 with one or two mutations were synthesized. Figure 7B shows the procedure by which the '029 library was iteratively screened and selected for acidic pH-selective antibody variants. R represents the selection round. Figure 7C shows representative two-dimensional flow cytometry plot data showing the variant pool after nine rounds of selection. VISTA binding is plotted on the y-axis, and mutant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pHs are shown. Figure 7D shows a diagram of P1-061029 and its progeny clones binding to human VISTA at pH 6.0 and 7.4. Figure 7E shows a diagram of the off-rates of P1-061029 and its progeny clones for human VISTA at pH 6.0. Figure 7F shows SPR binding data for antibodies P1-068761, P1-068767, and P1-061029 with human VISTA at pH 6.0 and pH 7.4. [Figure 8A-8B]Figures 8A-F show the acidic pH-selective cell binding, blocking, and effector activity of VISTA antibodies P1-068761 and P1-068767. Figures 8A and 8B show the mean fluorescence intensity of acidic pH-selective antibodies P1-068761 (Figure 8A) and P1-068767 (Figure 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; highest curve in Figure 8A), 6.1 (squares; third-highest curve), 6.2 (triangles; second-highest curve), 6.4 (inverted triangles; fourth-highest curve near the pH 6.1 curve), 6.6 (diamonds; fourth-lowest curve), 7.0 (circles; third-lowest curve), 7.2 (squares; second-lowest curve), and 8.1 (open triangles; bottom-most curve in Figure 8A). Binding was detected using a fluorescently conjugated anti-human IgG secondary antibody. Figure 8C shows P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) binding to Raji cells ectopically expressing human VISTA at 3125 ng / mL at various pH levels. "pH50," the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D shows the mean fluorescence intensity (MFI) of isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 ("control"; see Figure 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes. Binding was detected by a fluorescently conjugated anti-human IgG secondary antibody. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), while a non-VISTA-specific control antibody (circles) did not block VISTA binding.Figure 8F shows the reduced efficacy of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. P1-061029 (squares), a non-VISTA-specific positive control antibody (circles), and a non-VISTA-specific negative control antibody (diamonds) are also shown. NK cell-specific lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown. [Figure 8C]Figures 8A-F show the acidic pH-selective cell binding, blocking, and effector activity of VISTA antibodies P1-068761 and P1-068767. Figures 8A and 8B show the mean fluorescence intensity of acidic pH-selective antibodies P1-068761 (Figure 8A) and P1-068767 (Figure 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; highest curve in Figure 8A), 6.1 (squares; third-highest curve), 6.2 (triangles; second-highest curve), 6.4 (inverted triangles; fourth-highest curve near the pH 6.1 curve), 6.6 (diamonds; fourth-lowest curve), 7.0 (circles; third-lowest curve), 7.2 (squares; second-lowest curve), and 8.1 (open triangles; bottom-most curve in Figure 8A). Binding was detected using a fluorescently conjugated anti-human IgG secondary antibody. Figure 8C shows P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) binding to Raji cells ectopically expressing human VISTA at 3125 ng / mL at various pH levels. "pH50," the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D shows the mean fluorescence intensity (MFI) of isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 ("control"; see Figure 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes. Binding was detected by a fluorescently conjugated anti-human IgG secondary antibody. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), while a non-VISTA-specific control antibody (circles) did not block VISTA binding.Figure 8F shows the reduced efficacy of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. P1-061029 (squares), a non-VISTA-specific positive control antibody (circles), and a non-VISTA-specific negative control antibody (diamonds) are also shown. NK cell-specific lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown. [Figure 8D]Figures 8A-F show the acidic pH-selective cell binding, blocking, and effector activity of VISTA antibodies P1-068761 and P1-068767. Figures 8A and 8B show the mean fluorescence intensity of acidic pH-selective antibodies P1-068761 (Figure 8A) and P1-068767 (Figure 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; highest curve in Figure 8A), 6.1 (squares; third-highest curve), 6.2 (triangles; second-highest curve), 6.4 (inverted triangles; fourth-highest curve near the pH 6.1 curve), 6.6 (diamonds; fourth-lowest curve), 7.0 (circles; third-lowest curve), 7.2 (squares; second-lowest curve), and 8.1 (open triangles; bottom-most curve in Figure 8A). Binding was detected using a fluorescently conjugated anti-human IgG secondary antibody. Figure 8C shows P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) binding to Raji cells ectopically expressing human VISTA at 3125 ng / mL at various pH levels. "pH50," the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D shows the mean fluorescence intensity (MFI) of isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 ("control"; see Figure 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes. Binding was detected by a fluorescently conjugated anti-human IgG secondary antibody. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), while a non-VISTA-specific control antibody (circles) did not block VISTA binding.Figure 8F shows the reduced efficacy of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. P1-061029 (squares), a non-VISTA-specific positive control antibody (circles), and a non-VISTA-specific negative control antibody (diamonds) are also shown. NK cell-specific lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown. [Figure 8E]Figures 8A-F show the acidic pH-selective cell binding, blocking, and effector activity of VISTA antibodies P1-068761 and P1-068767. Figures 8A and 8B show the mean fluorescence intensity of acidic pH-selective antibodies P1-068761 (Figure 8A) and P1-068767 (Figure 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; highest curve in Figure 8A), 6.1 (squares; third-highest curve), 6.2 (triangles; second-highest curve), 6.4 (inverted triangles; fourth-highest curve near the pH 6.1 curve), 6.6 (diamonds; fourth-lowest curve), 7.0 (circles; third-lowest curve), 7.2 (squares; second-lowest curve), and 8.1 (open triangles; bottom-most curve in Figure 8A). Binding was detected using a fluorescently conjugated anti-human IgG secondary antibody. Figure 8C shows P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) binding to Raji cells ectopically expressing human VISTA at 3125 ng / mL at various pH levels. "pH50," the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D shows the mean fluorescence intensity (MFI) of isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 ("control"; see Figure 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes. Binding was detected by a fluorescently conjugated anti-human IgG secondary antibody. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), while a non-VISTA-specific control antibody (circles) did not block VISTA binding.Figure 8F shows the reduced efficacy of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. P1-061029 (squares), a non-VISTA-specific positive control antibody (circles), and a non-VISTA-specific negative control antibody (diamonds) are also shown. NK cell-specific lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown. [Figure 8F]Figures 8A-F show the acidic pH-selective cell binding, blocking, and effector activity of VISTA antibodies P1-068761 and P1-068767. Figures 8A and 8B show the mean fluorescence intensity of acidic pH-selective antibodies P1-068761 (Figure 8A) and P1-068767 (Figure 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; highest curve in Figure 8A), 6.1 (squares; third-highest curve), 6.2 (triangles; second-highest curve), 6.4 (inverted triangles; fourth-highest curve near the pH 6.1 curve), 6.6 (diamonds; fourth-lowest curve), 7.0 (circles; third-lowest curve), 7.2 (squares; second-lowest curve), and 8.1 (open triangles; bottom-most curve in Figure 8A). Binding was detected using a fluorescently conjugated anti-human IgG secondary antibody. Figure 8C shows P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) binding to Raji cells ectopically expressing human VISTA at 3125 ng / mL at various pH levels. "pH50," the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D shows the mean fluorescence intensity (MFI) of isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 ("control"; see Figure 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes. Binding was detected by a fluorescently conjugated anti-human IgG secondary antibody. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), while a non-VISTA-specific control antibody (circles) did not block VISTA binding.Figure 8F shows the reduced efficacy of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. P1-061029 (squares), a non-VISTA-specific positive control antibody (circles), and a non-VISTA-specific negative control antibody (diamonds) are also shown. NK cell-specific lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown. [Figure 9] Figure 9 shows the enhanced pharmacokinetics (PK) of acidic pH-selective anti-VISTA antibodies in cynomolgus monkeys. The figure shows serum antibody concentrations over time in cynomolgus monkeys treated with VISTA antibody 2 ("control," circles, see Figure 6C), VISTA antibody 3 ("acidic pH-sensitive," squares, see Figure 6C), or P1-068767 (triangles). [Figure 10A] Figures 10A and 10B show the binding effects of mutations in acidic pH-selective anti-VISTA antibodies '761 and '767. Figure 10A shows the kinetic binding data for the P1-068761 revertant at pH 7.4, pH 6.7, and pH 6.0, and the position of the revertant relative to P1-068761. Figure 10B shows the kinetic binding data for the P1-068767 revertant at pH 7.4, pH 6.7, and pH 6.0, and the position of the revertant relative to P1-068767. [Figure 10B] Figures 10A and 10B show the binding effects of mutations in acidic pH-selective anti-VISTA antibodies '761 and '767. Figure 10A shows the kinetic binding data for the P1-068761 revertant at pH 7.4, pH 6.7, and pH 6.0, and the position of the revertant relative to P1-068761. Figure 10B shows the kinetic binding data for the P1-068767 revertant at pH 7.4, pH 6.7, and pH 6.0, and the position of the revertant relative to P1-068767. [Figure 11A]Figures 11A-C show epitope binning and mapping of various anti-VISTA antibodies. Figure 11A shows VISTA epitope competition for P1-068761 and P1-068767 compared to P1-061029 and a VISTA antibody control. Figures 11B and 11C show epitope representation across all residues of a blocking hVISTA antibody (Figure 11B) as listed in Table 14 compared to a non-blocking hVISTA antibody (mAb1; Figure 11C). Amino acid residues 66 (H) and 162 (A) are shown to indicate molecular orientation. Histidine residues are in gray, and epitope residues are in black. [Figure 11B] Figures 11A-C show epitope binning and mapping of various anti-VISTA antibodies. Figure 11A shows VISTA epitope competition for P1-068761 and P1-068767 compared to P1-061029 and a VISTA antibody control. Figures 11B and 11C show epitope representation across all residues of a blocking hVISTA antibody (Figure 11B) as listed in Table 14 compared to a non-blocking hVISTA antibody (mAb1; Figure 11C). Amino acid residues 66 (H) and 162 (A) are shown to indicate molecular orientation. Histidine residues are in gray, and epitope residues are in black. [Figure 11C] Figures 11A-C show epitope binning and mapping of various anti-VISTA antibodies. Figure 11A shows VISTA epitope competition for P1-068761 and P1-068767 compared to P1-061029 and a VISTA antibody control. Figures 11B and 11C show epitope representation across all residues of a blocking hVISTA antibody (Figure 11B) as listed in Table 14 compared to a non-blocking hVISTA antibody (mAb1; Figure 11C). Amino acid residues 66 (H) and 162 (A) are shown to indicate molecular orientation. Histidine residues are in gray, and epitope residues are in black. [Figure 12A]Figures 12A-C show imaged capillary isoelectric focusing (icIEF) data for the following: Figure 12A: P1-061029, Figure 12B: P1-068761, and Figure 12C: P1-068767. The isoelectric points of the major species (pI major) as well as pI markers are shown. [Figure 12B] Figures 12A-C show imaged capillary isoelectric focusing (icIEF) data for the following: Figure 12A: P1-061029, Figure 12B: P1-068761, and Figure 12C: P1-068767. The isoelectric points of the major species (pI major) as well as pI markers are shown. [Figure 12C] Figures 12A-C show imaged capillary isoelectric focusing (icIEF) data for the following: Figure 12A: P1-061029, Figure 12B: P1-068761, and Figure 12C: P1-068767. The isoelectric points of the major species (pI major) as well as pI markers are shown. [Figure 13A-1] Figures 13A and B show alignments of the variable regions of '029 and '015 progeny clones. Figure 13A shows alignments of the amino acid sequences of the variable regions of '029 and its progeny clones. Figure 13B shows alignments of the amino acid sequences of the variable regions of '015 and its progeny clones. [Figure 13A-2] Figures 13A and B show alignments of the variable regions of '029 and '015 progeny clones. Figure 13A shows alignments of the amino acid sequences of the variable regions of '029 and its progeny clones. Figure 13B shows alignments of the amino acid sequences of the variable regions of '015 and its progeny clones. [Figure 13B] Figures 13A and B show alignments of the variable regions of '029 and '015 progeny clones. Figure 13A shows alignments of the amino acid sequences of the variable regions of '029 and its progeny clones. Figure 13B shows alignments of the amino acid sequences of the variable regions of '015 and its progeny clones. [Figure 14]Figure 14 shows an alignment of the VH sequences of P1-068761 with and without the K16R and T84A substitutions. Double underlined residues indicate framework regions positions 16 and 84, and shaded areas indicate the CDRs. [Figures 15A-15D]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15E]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15F]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15G]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15H]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15I]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figures 15J-15K]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15L]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15M]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15N]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figure 15O]Figures 15A-O show wild-type C57BL6 mice implanted with MC38 tumors and treated with a nonbinding isotype-matched control antibody (black squares), the murine VISTA-blocking antibody VISTA.10 (upward-pointing triangles), the murine PD-1-blocking antibody (squares), or a combination of VISTA and PD-1-blocking antibodies (downward-pointing triangles). All antibodies were of the murine IgG1-D265A (Fc-inactive) isotype (see Figures 15A-D). These data are representative of three independent experiments. Figures 15A-D show tumor volume over time. n = 10 per group. "TF" indicates mice that rejected the tumor. Figures 15E and F show the frequency of intratumoral CD8+ and CD4+ T cells 7 days after the start of treatment. n = 5 per group. One-way ANOVA with Dunnett's multiple comparisons, P = 0.0001. Figures 15G and H show the results for individual mice shown in Figures 15A-D. Figure 15I: VISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (top two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward-pointing triangles) or with a murine PD-1-blocking antibody (bottom two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward-pointing triangles). Median tumor growth and the number of tumor-free (TF) mice at the end of the study relative to the total number of mice are shown adjacent to each curve (e.g., 0 / 7 TF). These data are representative of two independent experiments. Error bars represent the interquartile range. Figures 15J-M show tumor volumes in human VISTA knock-in (KI) mice implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (Figure 15J), a murine PD-1 blocking antibody (Figure 15K), a combination of a murine PD-1 blocking antibody and the non-pH-selective human VISTA blocking antibody P1-061029 (Figure 15L), or a combination of a murine PD-1 blocking antibody and the acidic pH-selective human VISTA blocking antibody P1-068767 (Figure 15M). All antibodies were of the murine IgG1-D265A isotype. Tumor volumes are shown over time. n = 5-8 per group. These data are representative of one independent experiment.Figure 15N shows human VISTA KI and wild-type littermate (WT) mouse serum antibody concentrations after intravenous injection of 5 mg / kg P1-061029 (WT, downward triangle; KI, square) or P1-068767 (WT, upward triangle; KI, diamond). The calculated serum mean residence times (MRT) of P1-061029 and P1-068767 in KI mice are estimated to be 4.1 and 71 h, respectively. n = 4 KI mice and 1–2 WT mice per antibody. These data are representative of a single experiment. Figure 15O shows cynomolgus monkey serum antibody concentrations after intravenous injection of 5 mg / kg VISTA.4 (circle) or P1-068767 (square). The calculated serum mean residence times (MRT) of VISTA.4 and P1-061029 are estimated to be 7.6 h and 717 h, respectively. n=1 macaque per antibody. Data are representative of a single experiment. Error bars represent standard error of the mean unless otherwise indicated. [Figures 16A-16C] Figures 16A-C show representative histograms of intratumoral CD8+ T cell expression of PD-1 (Figure 16A), LAG-3 (Figure 16B), and TIM-3 (Figure 16C) 7 days after treatment initiation. Error bars represent the standard error of the mean. [Figure 17A] Figures 17A-C show that VISTA binds to PSGL-1 at acidic pH and that this interaction is blocked by the VISTA antibodies P1-061029, P1-068761, P1-068767, and VISTA.4. Figure 17A shows BLI binding sensorgrams of P-selectin-Fc and VISTA-Fc binding to captured PSGL1 at pH 6.0 and pH 7.4. Figure 17B is a histogram showing that antibodies P1-061029, P1-068761, P1-068767, and VISTA.4 inhibit PSGL-1 binding to hVISTA. Figure 17C shows antibody blockade of VISTA-Fc binding to CHO-PSGL-1 cells by VISTA.4 (upward triangles) and by the anti-PSGL-1 antibody KPL-1 (circles). These data are representative of two independent experiments. Error bars represent the standard error of the mean. [Figure 17B]Figures 17A-C show that VISTA binds to PSGL-1 at acidic pH and that this interaction is blocked by the VISTA antibodies P1-061029, P1-068761, P1-068767, and VISTA.4. Figure 17A shows BLI binding sensorgrams of P-selectin-Fc and VISTA-Fc binding to captured PSGL1 at pH 6.0 and pH 7.4. Figure 17B is a histogram showing that antibodies P1-061029, P1-068761, P1-068767, and VISTA.4 inhibit PSGL-1 binding to hVISTA. Figure 17C shows antibody blockade of VISTA-Fc binding to CHO-PSGL-1 cells by VISTA.4 (upward triangles) and by the anti-PSGL-1 antibody KPL-1 (circles). These data are representative of two independent experiments. Error bars represent the standard error of the mean. [Figure 17C] Figures 17A-C show that VISTA binds to PSGL-1 at acidic pH and that this interaction is blocked by the VISTA antibodies P1-061029, P1-068761, P1-068767, and VISTA.4. Figure 17A shows BLI binding sensorgrams of P-selectin-Fc and VISTA-Fc binding to captured PSGL1 at pH 6.0 and pH 7.4. Figure 17B is a histogram showing that antibodies P1-061029, P1-068761, P1-068767, and VISTA.4 inhibit PSGL-1 binding to hVISTA. Figure 17C shows antibody blockade of VISTA-Fc binding to CHO-PSGL-1 cells by VISTA.4 (upward triangles) and by the anti-PSGL-1 antibody KPL-1 (circles). These data are representative of two independent experiments. Error bars represent the standard error of the mean. [Figure 18A]Figures 18A-E show representations of the co-crystal structure of P1-068767 Fab and hVISTA, or (in Figure 18E) the non-blocking antibody VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusual, histidine-rich extension of its central β-sheet. The VISTA IgV domain was co-crystallized with the P1-068767 fragment antigen-binding (Fab). The crystal structure of the VISTA + P1-068767 complex was determined at 1.6 Å resolution. Figure 18A shows the VISTA IgV domain:P1-068767 Fab co-crystal structure. Figure 18A shows the overall structure of the VISTA IgV domain in complex with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B shows a superposition of the VISTA and PD-L1 IgV domains. VISTA histidine residues are shown in stick representation. Figure 18B shows that the IgV domain of VISTA has an unusual, histidine-rich β-sheet extension. Figure 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) as revealed by the VISTA + P1-068767 crystal structure. Figure 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. Figure 18D shows a close-up of the interface between VISTA (gray ribbon diagram with epitope residues H121, H122, and H123 shown in stick representation) and P1-068767 (shown as an electrostatic surface with its residues E100 and D102 in stick representation). Figure 18D shows that the acidic pH-selective P1-068767 associates with VISTA histidines containing acidic residues. Figure 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA derived from P1-068767. [Figure 18B]Figures 18A-E show representations of the co-crystal structure of P1-068767 Fab and hVISTA, or (in Figure 18E) the non-blocking antibody VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusual, histidine-rich extension of its central β-sheet. The VISTA IgV domain was co-crystallized with the P1-068767 fragment antigen-binding (Fab). The crystal structure of the VISTA + P1-068767 complex was determined at 1.6 Å resolution. Figure 18A shows the VISTA IgV domain:P1-068767 Fab co-crystal structure. Figure 18A shows the overall structure of the VISTA IgV domain in complex with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B shows a superposition of the VISTA and PD-L1 IgV domains. VISTA histidine residues are shown in stick representation. Figure 18B shows that the IgV domain of VISTA has an unusual, histidine-rich β-sheet extension. Figure 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) as revealed by the VISTA + P1-068767 crystal structure. Figure 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. Figure 18D shows a close-up of the interface between VISTA (gray ribbon diagram with epitope residues H121, H122, and H123 shown in stick representation) and P1-068767 (shown as an electrostatic surface with its residues E100 and D102 in stick representation). Figure 18D shows that the acidic pH-selective P1-068767 associates with VISTA histidines containing acidic residues. Figure 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA derived from P1-068767. [Figure 18C]Figures 18A-E show representations of the co-crystal structure of P1-068767 Fab and hVISTA, or (in Figure 18E) the non-blocking antibody VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusual, histidine-rich extension of its central β-sheet. The VISTA IgV domain was co-crystallized with the P1-068767 fragment antigen-binding (Fab). The crystal structure of the VISTA + P1-068767 complex was determined at 1.6 Å resolution. Figure 18A shows the VISTA IgV domain:P1-068767 Fab co-crystal structure. Figure 18A shows the overall structure of the VISTA IgV domain in complex with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B shows a superposition of the VISTA and PD-L1 IgV domains. VISTA histidine residues are shown in stick representation. Figure 18B shows that the IgV domain of VISTA has an unusual, histidine-rich β-sheet extension. Figure 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) as revealed by the VISTA + P1-068767 crystal structure. Figure 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. Figure 18D shows a close-up of the interface between VISTA (gray ribbon diagram with epitope residues H121, H122, and H123 shown in stick representation) and P1-068767 (shown as an electrostatic surface with its residues E100 and D102 in stick representation). Figure 18D shows that the acidic pH-selective P1-068767 associates with VISTA histidines containing acidic residues. Figure 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA derived from P1-068767. [Figure 18D]Figures 18A-E show representations of the co-crystal structure of P1-068767 Fab and hVISTA, or (in Figure 18E) the non-blocking antibody VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusual, histidine-rich extension of its central β-sheet. The VISTA IgV domain was co-crystallized with the P1-068767 fragment antigen-binding (Fab). The crystal structure of the VISTA + P1-068767 complex was determined at 1.6 Å resolution. Figure 18A shows the VISTA IgV domain:P1-068767 Fab co-crystal structure. Figure 18A shows the overall structure of the VISTA IgV domain in complex with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B shows a superposition of the VISTA and PD-L1 IgV domains. VISTA histidine residues are shown in stick representation. Figure 18B shows that the IgV domain of VISTA has an unusual, histidine-rich β-sheet extension. Figure 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) as revealed by the VISTA + P1-068767 crystal structure. Figure 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. Figure 18D shows a close-up of the interface between VISTA (gray ribbon diagram with epitope residues H121, H122, and H123 shown in stick representation) and P1-068767 (shown as an electrostatic surface with its residues E100 and D102 in stick representation). Figure 18D shows that the acidic pH-selective P1-068767 associates with VISTA histidines containing acidic residues. Figure 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA derived from P1-068767. [Figure 18E]Figures 18A-E show representations of the co-crystal structure of P1-068767 Fab and hVISTA, or (in Figure 18E) the non-blocking antibody VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusual, histidine-rich extension of its central β-sheet. The VISTA IgV domain was co-crystallized with the P1-068767 fragment antigen-binding (Fab). The crystal structure of the VISTA + P1-068767 complex was determined at 1.6 Å resolution. Figure 18A shows the VISTA IgV domain:P1-068767 Fab co-crystal structure. Figure 18A shows the overall structure of the VISTA IgV domain in complex with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B shows a superposition of the VISTA and PD-L1 IgV domains. VISTA histidine residues are shown in stick representation. Figure 18B shows that the IgV domain of VISTA has an unusual, histidine-rich β-sheet extension. Figure 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) as revealed by the VISTA + P1-068767 crystal structure. Figure 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. Figure 18D shows a close-up of the interface between VISTA (gray ribbon diagram with epitope residues H121, H122, and H123 shown in stick representation) and P1-068767 (shown as an electrostatic surface with its residues E100 and D102 in stick representation). Figure 18D shows that the acidic pH-selective P1-068767 associates with VISTA histidines containing acidic residues. Figure 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA derived from P1-068767. [Figure 19] FIG. 19 shows the epitopes of VISTA.4 as determined by MS-HDX (MS trace). [Figure 20]Figure 20 shows the location of the VISTA.4 epitope in the amino acid sequence of hVISTA based on the data in Figure 19. Residues 57-68, 86-97, and 148-165, highlighted in Figure 19, are also represented in lighter grey text and underlined in Figure 20. [Figure 21A] Figures 21A and 21B show VISTA multimers binding to activated human CD4+ T cells at pH 6.0 in the presence of antibodies VISTA.4 (triangles), VISTA.5 (squares), and non-VISTA binding (control, circles). Figure 21B shows the blocking efficiency of each antibody compared to non-blocking T cells. One-way ANOVA with Dunnett's multiple comparisons, ***, P<0.001. These data are representative of more than four independent experiments. Error bars represent the standard error of the mean. [Figure 21B] Figures 21A and 21B show VISTA multimers binding to activated human CD4+ T cells at pH 6.0 in the presence of antibodies VISTA.4 (triangles), VISTA.5 (squares), and non-VISTA binding (control, circles). Figure 21B shows the blocking efficiency of each antibody compared to non-blocking T cells. One-way ANOVA with Dunnett's multiple comparisons, ***, P<0.001. These data are representative of more than four independent experiments. Error bars represent the standard error of the mean. [Figure 22A] Figure 22 shows that antibodies that block VISTA binding at acidic pH are functional. Effect of blocking antibody VISTA.4 (squares), non-blocking antibody VISTA.5 (triangles), and non-VISTA-binding (control, circles) antibodies on proliferation (Figure 22A) and interferon gamma production (Figure 22B) of human CD4+ T cells cocultured with 293T cells engineered to express VISTA and a TCR agonist (293T-OKT3-VISTA). One-way ANOVA with Dunnett's multiple comparisons, *, P<0.05. These data are representative of more than four independent experiments. [Figure 22B]Figure 22 shows that antibodies that block VISTA binding at acidic pH are functional. Effect of blocking antibody VISTA.4 (squares), non-blocking antibody VISTA.5 (triangles), and non-VISTA-binding (control, circles) antibodies on proliferation (Figure 22A) and interferon gamma production (Figure 22B) of human CD4+ T cells cocultured with 293T cells engineered to express VISTA and a TCR agonist (293T-OKT3-VISTA). One-way ANOVA with Dunnett's multiple comparisons, *, P<0.05. These data are representative of more than four independent experiments. [Figure 23] Figure 23 shows the effect of VISTA.4 blockade on Jurkat T cell activation (as measured by NF-kB inhibition) after co-culture with 293T-OKT3-VISTA cells at various pHs. These data are representative of a composite of three independent experiments. [Figure 24] Figure 24 shows the effect of pH on VISTA suppression of human CD4+ T cells. Cells were stimulated with plate-coated OKT3 and VISTA-Fc at the indicated pH in the presence of VISTA.4 (upward triangles), VISTA.5 (downward triangles), or a non-VISTA-binding antibody (antibody control, squares). Cells stimulated with plate-coated OKT3 and control IgG (VISTA control, filled circles) or without OKT3 (no OKT3, gray diamonds) are also shown. These data are representative of one independent experiment. [Figure 25A]Figures 25A–E show that VISTA:PSGL-1 binding specificity is determined by histidine and sulfotyrosine residues. As shown in Figure 25A, human PSGL-1 19-mer Fc recombinant protein was produced in cells with or without sialyl Lewis X decoration (SLX+ and SLX−, respectively). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. Data are representative of single independent experiments. As shown in Figure 25B, human PSGL-1 19-mer Fc glycopeptides produced with sialyl Lewis X decoration separated into fractions with greater than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. These data are representative of a single independent experiment. As presented in Figures 25C-25D, human VISTA-Fc recombinant proteins were produced in which the histidine residues at positions 153-155 were either left unchanged (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C shows the BLI binding magnitude of wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data are representative of a single experiment. Figure 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamond). These data are representative of two independent experiments. Figure 25E shows a computational model of the PSGL-1 19-mer glycopeptide (top) in complex with the histidine-rich ligand interface of VISTA (gray ribbon, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled. [Figure 25B]Figures 25A–E show that VISTA:PSGL-1 binding specificity is determined by histidine and sulfotyrosine residues. As shown in Figure 25A, human PSGL-1 19-mer Fc recombinant protein was produced in cells with or without sialyl Lewis X decoration (SLX+ and SLX−, respectively). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. Data are representative of single independent experiments. As shown in Figure 25B, human PSGL-1 19-mer Fc glycopeptides produced with sialyl Lewis X decoration separated into fractions with greater than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. These data are representative of a single independent experiment. As presented in Figures 25C-25D, human VISTA-Fc recombinant proteins were produced in which the histidine residues at positions 153-155 were either left unchanged (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C shows the BLI binding magnitude of wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data are representative of a single experiment. Figure 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamond). These data are representative of two independent experiments. Figure 25E shows a computational model of the PSGL-1 19-mer glycopeptide (top) in complex with the histidine-rich ligand interface of VISTA (gray ribbon, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled. [Figure 25C]Figures 25A–E show that VISTA:PSGL-1 binding specificity is determined by histidine and sulfotyrosine residues. As shown in Figure 25A, human PSGL-1 19-mer Fc recombinant protein was produced in cells with or without sialyl Lewis X decoration (SLX+ and SLX−, respectively). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. Data are representative of single independent experiments. As shown in Figure 25B, human PSGL-1 19-mer Fc glycopeptides produced with sialyl Lewis X decoration separated into fractions with greater than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. These data are representative of a single independent experiment. As presented in Figures 25C-25D, human VISTA-Fc recombinant proteins were produced in which the histidine residues at positions 153-155 were either left unchanged (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C shows the BLI binding magnitude of wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data are representative of a single experiment. Figure 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamond). These data are representative of two independent experiments. Figure 25E shows a computational model of the PSGL-1 19-mer glycopeptide (top) in complex with the histidine-rich ligand interface of VISTA (gray ribbon, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled. [Figure 25D]Figures 25A–E show that VISTA:PSGL-1 binding specificity is determined by histidine and sulfotyrosine residues. As shown in Figure 25A, human PSGL-1 19-mer Fc recombinant protein was produced in cells with or without sialyl Lewis X decoration (SLX+ and SLX−, respectively). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. Data are representative of single independent experiments. As shown in Figure 25B, human PSGL-1 19-mer Fc glycopeptides produced with sialyl Lewis X decoration separated into fractions with greater than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. These data are representative of a single independent experiment. As presented in Figures 25C-25D, human VISTA-Fc recombinant proteins were produced in which the histidine residues at positions 153-155 were either left unchanged (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C shows the BLI binding magnitude of wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data are representative of a single experiment. Figure 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamond). These data are representative of two independent experiments. Figure 25E shows a computational model of the PSGL-1 19-mer glycopeptide (top) in complex with the histidine-rich ligand interface of VISTA (gray ribbon, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled. [Figure 25E]Figures 25A–E show that VISTA:PSGL-1 binding specificity is determined by histidine and sulfotyrosine residues. As shown in Figure 25A, human PSGL-1 19-mer Fc recombinant protein was produced in cells with or without sialyl Lewis X decoration (SLX+ and SLX−, respectively). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. Data are representative of single independent experiments. As shown in Figure 25B, human PSGL-1 19-mer Fc glycopeptides produced with sialyl Lewis X decoration separated into fractions with greater than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). BLI binding magnitudes at pH 6.0 (white) and 7.4 (black) are shown for VISTA-Fc and P-selectin-Fc, as indicated. These data are representative of a single independent experiment. As presented in Figures 25C-25D, human VISTA-Fc recombinant proteins were produced in which the histidine residues at positions 153-155 were either left unchanged (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C shows the BLI binding magnitude of wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data are representative of a single experiment. Figure 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamond). These data are representative of two independent experiments. Figure 25E shows a computational model of the PSGL-1 19-mer glycopeptide (top) in complex with the histidine-rich ligand interface of VISTA (gray ribbon, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled. [Figure 26A] Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 26B]Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 26C]Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 26D]Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 26E]Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 26F]Figures 26A-F: Figure 26A shows the BLI binding magnitude of GP1BA-his (squares) and PSGL-1 19-mer-Fc (circles) to captured VISTA-Fc at the indicated pH. These data are representative of one experiment. Figure 26B shows a histogram of VISTA multimer binding to human platelets. Binding was performed in the presence of a non-VISTA-binding control antibody at pH 7.4 and pH 6.0, or in the presence of a VISTA.4 blocking antibody at pH 6.0. Unstained platelets (gray filled histograms) are also shown. These data are representative of two independent experiments. Figure 26C shows the BLI binding magnitude of VSIG-3-Fc binding to captured VISTA-Fc at the indicated pH. These data are representative of two independent experiments. Figure 26D shows the BLI binding magnitude of the binding of the indicated concentrations of VSIG-3-Fc to captured VISTA-Fc at pH 6.0. Competition was provided by buffer alone (leftmost bar), non-binding isotype-matched control antibody, human PSGL-1 19mer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data are representative of one independent experiment. Figure 26E shows VSIG-3-Fc binding to activated human PBMCT cells at pH 6.0 (circles) or pH 7.4 (squares). Binding of isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (gray triangles) is also shown. These data are representative of two independent experiments. Error bars represent the standard error of the mean. Figure 26F shows BLI binding magnitudes of VISTA-Fc (left) and PSGL-1 19mer-Fc (right) binding to captured VISTA-Fc at the indicated pH. These data are representative of one independent experiment. [Figure 27A]Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 27B] Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 27C]Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 27D] Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 27E]Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 27F] Figures 27A-F: Figures 27A-D show human and cynomolgus monkey sensorgrams of antibodies VISTA.4 and P1-068767 at pH 7.4 (left) and pH 6.0 (right). These data are representative of two independent experiments. Figure 27A: Human VISTA sensorgram of VISTA.4. Figure 27B: Human VISTA sensorgram of P1-068767. Figure 27C: Cynomolgus monkey VISTA sensorgram of VISTA.4. Figure 27D: Cynomolgus monkey VISTA sensorgram of P1-068767. Figure 27E shows the levels of labeled P1-061029 or P1-068767 antibody in various organs, blood, or tumors in human VISTA knock-in mice implanted with MC38 tumors and treated with the respective antibodies. Figure 27F shows tumor growth in mice treated with P1-061029 alone (left, downward triangles) or with P1-068767 alone (right, upward triangles). n=16 per group. These data are composites of two independent experiments. Error bars represent the standard error of the mean. [Figure 28]Figure 28 shows the results of an experiment in which human VISTA knock-in mice were implanted with MC38 tumors and treated with fluorescently labeled P1-061029 (left) or P1-068767 (right). Radiation efficiencies (x10) in the indicated organs 51 hours after injection are shown. These data are representative of a single experiment. [Figure 29A] Figures 29A-C show the results of SPR assays of the binding of anti-hVISTA antibodies to hVISTA. Figure 29A shows the relative hVISTA binding (%Rmax) by SPR of the P1-068744 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29B shows the relative hVISTA binding (%Rmax) by SPR of the P1-068748 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29C shows a summary of the results as an alignment of the antibody heavy chain CDRs. The delineated amino acid residues of P1-068744 and P1-068748 that are in bold are those whose reversion to the corresponding residue in P1-061015 maintained pH selectivity and improved kd at pH 6.0 compared to the P1-068744 or P1-068748 antibodies. The double-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 caused loss of pH selectivity of P1-068744 or P1-068748 and are therefore critical residues in these antibodies for pH-selective binding to hVISTA. The jagged-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 was detrimental to the kinetics and / or binding of P1-068744 or P1-068748 to hVISTA and are therefore critical residues in these antibodies for binding to hVISTA. Alternative names for the depicted amino acid residues in the P1-068744 and P1-068748 antibodies are shown. [Figure 29B]Figures 29A-C show the results of SPR assays of the binding of anti-hVISTA antibodies to hVISTA. Figure 29A shows the relative hVISTA binding (%Rmax) by SPR of the P1-068744 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29B shows the relative hVISTA binding (%Rmax) by SPR of the P1-068748 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29C shows a summary of the results as an alignment of the antibody heavy chain CDRs. The delineated amino acid residues of P1-068744 and P1-068748 that are in bold are those whose reversion to the corresponding residue in P1-061015 maintained pH selectivity and improved kd at pH 6.0 compared to the P1-068744 or P1-068748 antibodies. The double-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 caused loss of pH selectivity of P1-068744 or P1-068748 and are therefore critical residues in these antibodies for pH-selective binding to hVISTA. The jagged-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 was detrimental to the kinetics and / or binding of P1-068744 or P1-068748 to hVISTA and are therefore critical residues in these antibodies for binding to hVISTA. Alternative names for the depicted amino acid residues in the P1-068744 and P1-068748 antibodies are shown. [Figure 29C]Figures 29A-C show the results of SPR assays of the binding of anti-hVISTA antibodies to hVISTA. Figure 29A shows the relative hVISTA binding (%Rmax) by SPR of the P1-068744 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29B shows the relative hVISTA binding (%Rmax) by SPR of the P1-068748 revertants compared to the binding of the parent P1-061015 antibody at pH 6.0 (black bars) and pH 7.4 (gray bars). Figure 29C shows a summary of the results as an alignment of the antibody heavy chain CDRs. The delineated amino acid residues of P1-068744 and P1-068748 that are in bold are those whose reversion to the corresponding residue in P1-061015 maintained pH selectivity and improved kd at pH 6.0 compared to the P1-068744 or P1-068748 antibodies. The double-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 caused loss of pH selectivity of P1-068744 or P1-068748 and are therefore critical residues in these antibodies for pH-selective binding to hVISTA. The jagged-underlined amino acid residues are those whose reversion to the corresponding residue in P1-061015 was detrimental to the kinetics and / or binding of P1-068744 or P1-068748 to hVISTA and are therefore critical residues in these antibodies for binding to hVISTA. Alternative names for the depicted amino acid residues in the P1-068744 and P1-068748 antibodies are shown. [Figure 30A] Figures 30A-D show results from testing the binding of anti-hVISTA antibodies (10 μg / ml) to 293T cells engineered to express the hVISTA extracellular domain in HBSS / MES buffer at pH 6.0 (black bars) and pH 7.4 (white bars). Results are shown as percent binding normalized to the binding of P1-061029, which is set at 100%. Figures 30A, 30B, 30C, and 30D each show the results for a particular set of antibodies as indicated in each bar graph below. [Figure 30B] Figures 30A-D show results from testing the binding of anti-hVISTA antibodies (10 μg / ml) to 293T cells engineered to express the hVISTA extracellular domain in HBSS / MES buffer at pH 6.0 (black bars) and pH 7.4 (white bars). Results are shown as percent binding normalized to the binding of P1-061029, which is set at 100%. Figures 30A, 30B, 30C, and 30D each show the results for a particular set of antibodies as indicated in each bar graph below. [Figure 30C] Figures 30A-D show results from testing the binding of anti-hVISTA antibodies (10 μg / ml) to 293T cells engineered to express the hVISTA extracellular domain in HBSS / MES buffer at pH 6.0 (black bars) and pH 7.4 (white bars). Results are shown as percent binding normalized to the binding of P1-061029, which is set at 100%. Figures 30A, 30B, 30C, and 30D each show the results for a particular set of antibodies as indicated in each bar graph below. [Figure 30D] Figures 30A-D show results from testing the binding of anti-hVISTA antibodies (10 μg / ml) to 293T cells engineered to express the hVISTA extracellular domain in HBSS / MES buffer at pH 6.0 (black bars) and pH 7.4 (white bars). Results are shown as percent binding normalized to the binding of P1-061029, which is set at 100%. Figures 30A, 30B, 30C, and 30D each show the results for a particular set of antibodies as indicated in each bar graph below. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition In this application, the use of "or" means "and / or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers back to more than one said independent or dependent claim in the alternative only. The terms "comprising," "including," and "having" may be used interchangeably herein. In accordance with the present invention, an "isolated" molecule is one that has been removed from its natural environment. As such, the term "isolated" does not necessarily reflect the extent to which the molecule has been purified.

[0011] The term "polypeptide" refers to a polymer of amino acid residues and is not limited to a minimum length. A "protein" can include one or more polypeptides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" or "protein" refers to a polypeptide or protein, respectively, containing modifications (generally conservative in nature) to the native sequence, such as deletions, additions, and substitutions, so long as the protein maintains the desired activity. These modifications can be deliberate, as by site-directed mutagenesis, or accidental, such as through mutations of hosts producing the protein or errors due to PCR amplification. A protein can comprise two or more polypeptides.

[0012] "VISTA" is an abbreviation for V-domain immunoglobulin-containing suppressor of T-cell activation, a member of the B7 family of immune checkpoint regulators. VISTA is also known as PD-1 homolog (PD1H), B7-H5, C10orf54, differentiation of ESC-1 (Dies-1), platelet receptor Gi24 precursor, and death domain 1 alpha (DD1α). The terms "hVISTA" or "huVISTA" refer to the human VISTA protein. The amino acid sequence of hVISTA, including its signal peptide, is provided by SEQ ID NO: 1, while the sequence without the signal peptide is provided by SEQ ID NO: 2 (see Sequence Listing below). The extracellular domain or "ECD" of VISTA or "VISTA-ECD" refers to the portion of the VISTA protein located in the extracellular space, which in the case of hVISTA, includes amino acids 1-162 of SEQ ID NO: 2 (see also Figure 1B). A portion of the "IgV domain" of hVISTA comprises residues 5-135 of SEQ ID NO:2.

[0013] The term "leader peptide" or "leader sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from mammalian cells. Leader sequences may be cleaved upon export of the polypeptide from mammalian cells to form the mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached.

[0014] The term "antibody" or "Ab" is used broadly herein to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. As used herein, this term refers to a molecule comprising at least complementarity-determining regions (CDRs) 1, 2, and 3 of a heavy chain and at least CDRs 1, 2, and 3 of a light chain, which molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, such as mouse, cynomolgus monkey, etc.

[0015] The term "heavy chain" or "HC" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine (K).

[0016] The term "heavy chain variable region" or "VH" refers to the region of a heavy chain comprising heavy chain complementarity-determining region (CDR) 1, framework region (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the heavy chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4. As detailed below, in some embodiments, heavy chain CDR1 comprises residues 26-35 of a VH SEQ ID NO: herein, heavy chain CDR2 comprises residues 50-66 of a VH SEQ ID NO: herein, and heavy chain CDR3 comprises residues 99-110 of a VH SEQ ID NO: herein. In other embodiments, where specified, heavy chain CDR1 corresponds to Kabat residues 31-35, heavy chain CDR2 corresponds to Kabat residues 50-65, and heavy chain CDR3 corresponds to Kabat residues 95-102. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.). In some embodiments, the heavy chain CDRs are as specified herein, e.g., in the sequence listing below, or in Table 2.

[0017] The term "light chain" or "LC" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0018] The term "light chain variable region" or "VL" refers to a region comprising light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also comprises FR1 and / or FR4. As specified below, in some embodiments, light chain CDR1 comprises residues 24-35 of a VL SEQ ID NO: herein, light chain CDR2 comprises residues 51-57 of a VL SEQ ID NO: herein, and light chain CDR3 comprises residues 90-98 of a VL SEQ ID NO: herein. In other embodiments, where specified, light chain CDR1 corresponds to Kabat residues 24-34, light chain CDR2 corresponds to Kabat residues 50-56, and light chain CDR3 corresponds to Kabat residues 89-97. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.). In some embodiments, the light chain CDRs are as specified herein, eg, in the sequence listing.

[0019] A "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region derived from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region derived from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are derived from a first species, and all of the constant regions of the chimeric antibody are derived from a second species.

[0020] A "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is a Fab, scFv, (Fab')2, etc.

[0021] "Human antibody," as used herein, refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, e.g., XenoMouse®, and antibodies selected using in vitro methods, e.g., phage display, in which the antibody repertoire is based on human immunoglobulin sequences.

[0022] "VISTA antibody" or "anti-VISTA antibody," as used herein, refers to an antibody that specifically binds to VISTA under at least some conditions, such as acidic pH. In some embodiments, the antibody may be a "huVISTA antibody" or "anti-huVISTA antibody," indicating that it specifically binds to human VISTA protein under at least some conditions, such as at acidic pH. A VISTA antibody that specifically binds to the extracellular domain (ECD) of VISTA may be referred to, for example, as a "VISTA-ECD antibody."

[0023] In some embodiments, the antibodies herein may contain one or more "conservative substitutions" compared to a particular sequence. As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an antibody herein is replaced with another amino acid residue from the same side chain family (e.g., basic, acidic, beta-branched, aromatic, uncharged polar). Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are described, for example, in Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0024] In some embodiments, the antibody may bind to VISTA with higher affinity at acidic pH than at neutral and / or physiological pH, hi some embodiments, the antibody may bind to VISTA with higher affinity at acidic pH and may bind only negligibly or nonspecifically at neutral and / or physiological pH.

[0025] The "K" of the antibody binding to a protein, e.g., the VISTA-ECD protein D" or "dissociation constant" is a measure of the affinity or specific binding of an antibody to a protein, e.g., a VISTA-ECD protein. D is high K D exhibiting improved binding or affinity over K D is the "off-rate" or k for antibodies and polypeptides. off or k d and "on-rate" or k on or k a The off-rate and on-rate are the rates at which two binding partners associate and dissociate in the system. Thus, a slower off-rate, where the on-rate remains approximately constant, indicates a higher overall affinity and, therefore, a lower K D As used herein, k "less than or equal to" a particular value off is k off or "off-rate" is as specified or slower than the rate specified.

[0026] The term "specific binding" or "specifically binds" or similar terms refers to the K of binding of two polypeptides, such as an antibody and its polypeptide target. D is smaller than would be the case between two random polypeptides present under the same conditions. D is smaller than that due to non-specific aggregation of polypeptides in the system.

[0027] In some embodiments, an antibody specifically binds to a VISTA-ECD protein at a particular pH or pH range. As used herein, an "acidic" pH generally refers to a pH below 7.0, a "basic" pH generally refers to a pH above 7.0, and a "neutral" pH generally refers to a pH of about 7.0. As used herein, "physiological pH" refers to a pH under normal (i.e., non-cancerous) physiological conditions, e.g., 7.35 to 7.45, or 7.3 to 7.4, e.g., about 7.4. As used herein, phrases such as "binding under acidic conditions" or "binding under physiological conditions," when used in connection with binding between two molecules, such as VISTA and a VISTA binding partner or VISTA and a T cell, refer to binding at acidic pH and binding at physiological pH, respectively.

[0028] When referring to an antibody that "blocks the binding of" or "inhibits the binding of" a ligand (or receptor) or a competing antibody to a receptor (or ligand) alone or on a cell, binding is blocked if there is a statistically significant overall reduction compared to a control, e.g., a 50% or greater overall reduction, e.g., a 75%, 80%, 85%, 90%, 95% or greater overall reduction. An "anti-VISTA blocking antibody" is one that can block the binding of VISTA to PSGL-1 or another VISTA ligand or receptor or heparan sulfate proteoglycan, for example, at least under some conditions, e.g., at acidic pH.

[0029] "Preferentially accumulate" or "preferably accumulate," when referring to the accumulation of an antibody in a tumor in a patient administered the antibody compared to that in an organ or tissue, e.g., blood, of the patient, refers to at least 50%, 75%, 100% (i.e., 2-fold), 5-fold or more accumulation in the tumor compared to that in the organ or tissue. For example, an acidic pH-selective binding antibody accumulates to at least 2-fold higher levels in the tumor of a subject compared to its accumulation in the blood.

[0030] "Tumor model," as used herein, refers to an in vivo preclinical assay that can be used to study the biological activity of a VISTA-ECD antibody, including xenograft or native mouse tumor assay systems. In some cases, the tumor model allows for tracking the size or growth of the tumor upon treatment with the antibody and / or the presence of immune cells, e.g., specific types of T cells or NK cells, in the tumor to determine whether the antibody has elicited or enhanced an immune response.

[0031] The term "immunostimulatory agent," as used herein, refers to a molecule that stimulates the immune system by either acting as an agonist of immunostimulatory molecules, including costimulatory molecules, or as an antagonist of immunoinhibitory molecules, including co-inhibitory molecules. An immunostimulatory or immunoinhibitory molecule can be an immune checkpoint regulator, such as VISTA or another B7 family member, or another molecule as described further below. An immunostimulatory agent can be a biologic, such as an antibody or antibody fragment, other protein, or vaccine, or can be a small molecule drug. "Immunostimulatory molecules" include receptors or ligands that act to enhance, stimulate, induce, or otherwise "turn on" an immune response. Immunostimulatory molecules, as defined herein, include costimulatory molecules. "Immuno-inhibitory molecules" include receptors or ligands that act to inhibit, suppress, or otherwise "turn off" an immune response. Immuno-inhibitory molecules, as defined herein, include co-inhibitory molecules. Such immunostimulatory and immunoinhibitory molecules can be, for example, receptors or ligands found on immune cells such as T cells, or on cells involved in innate immunity such as NK cells.

[0032] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the sequences being compared.

[0033] The terms "causing" or "enhancing" refer to the initiation or increase of any event (e.g., protein-ligand binding) or the initiation or increase of any biological activity (e.g., immune response) or phenotypic characteristic, or the initiation or increase of the prevalence, extent, or likelihood of that activity or characteristic. "Causing" or "enhancing" is the initiation or increase of an activity, function, and / or amount compared to a reference. The causing or enhancing need not be complete. For example, in certain embodiments, "enhancing" refers to the ability to cause an overall increase of 20% or more. In another embodiment, "enhancing" refers to the ability to cause an overall increase of 50% or more. In yet other embodiments, "enhancing" refers to the ability to cause an overall increase of 75%, 85%, 90%, 95%, or more.

[0034] The term "inhibition" or "inhibiting" more generally refers to the reduction or interruption of any event (e.g., protein-ligand binding) or the reduction or interruption of any phenotypic characteristic, or the reduction or interruption of the prevalence, degree, or likelihood of that characteristic. "Reducing" or "inhibiting" is the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. Inhibition or reduction need not be complete. For example, in certain embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 50% or more. In yet other embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0035] "Treatment," as used herein, covers any administration or application of a therapeutic agent for a disease in a human, and includes inhibiting the progression of the disease or one or more symptoms of the disease, inhibiting or slowing the progression of the disease or one or more symptoms thereof, partially or completely alleviating the disease or one or more symptoms thereof, or preventing the recurrence of one or more symptoms of the disease.

[0036] The terms "subject" and "patient" are used interchangeably herein to refer to a human.

[0037] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective in treating a disease or disorder in a subject to partially or completely alleviate one or more symptoms. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0038] The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also referred to as benign tumors), pre-malignant, or malignant. Cancer cells can be solid cancer cells or leukemia cancer cells. The term "tumor growth" is used herein to refer to proliferation or growth by a cell or cells comprising a cancer, leading to a corresponding increase in the size or extent of the cancer.

[0039] Examples of cancer that can be used in the treatment methods herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia.More specifically, non-limiting examples of such cancer include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder carcinoma, gastric cancer, melanoma and various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

[0040] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive (sequential) administration in any order.

[0041] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with a therapeutic agent that together comprises a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritating to the skin and does not cause injection site reactions.

[0042] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents that may be administered in the methods herein include, but are not limited to, alkylating agents such as thiotepa and Cytoxan® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its analogs adozelesin, carzelesin, and bizene); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxychloride, amide, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycins, including dynemycin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin , puromycin, quelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioglucan anine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilon;Etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; Schizophyllan; spirogermanium; tenuazonic acid; triazicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane® cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, IL), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO);Retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)), and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0043] Further non-limiting exemplary chemotherapeutic agents that may be administered in the methods herein include antihormonal agents that act to regulate or inhibit hormone action on cancer, e.g., antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fairston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, e.g., 4(5)-imidazole, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and alimidazole. Dex® anastrozole and the like; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abherent cell proliferation, such as PKC-alpha, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., Angiozyme® ribozyme) and HER2 expression inhibitors; gene therapy vaccines, such as Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin® rIL-2; lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0044] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a low molecular weight substance, polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof, that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesired vascular permeability. It should be understood that anti-angiogenic agents include substances that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents that can be administered in the methods herein include antibodies against angiogenic agents or other antagonists, such as antibodies against VEGF-A [e.g., bevacizumab (Avastin®)] or antibodies against VEGF-A receptors (e.g., KDR receptors or Flt-1 receptors), anti-PDGFR inhibitors, such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or, for example, those described in International Patent Application WO2004 / 113304).Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3, listing antiangiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2, listing known antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1, listing antiangiogenic agents used in clinical trials).

[0045] As used herein, the term "growth inhibitor" refers to a compound or composition that inhibits the growth of cells (e.g., cells expressing VEGF) in vitro or in vivo. Thus, the growth inhibitor that can be administered in the methods herein can be one that significantly reduces the percentage of cells in S phase (e.g., cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, substances that block cell cycle progression (at a location other than S phase), such as substances that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Substances that arrest G1 also lead to S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found, for example, on page 13, in "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al., Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1 (WB Saunders, Philadelphia, 1995). Taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analog of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to the inhibition of mitosis in cells.

[0046] The term "antineoplastic composition" refers to a composition useful for treating cancer that comprises at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapeutic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA antibodies), and the like. These include ipilimumab (YERVOY®), PD-1 or PD-L1 inhibitors (e.g., Opdivo®, Keytruda®, Tecentriq®, Bavencio®, IMFINZI®), TIM3 inhibitors (e.g., anti-TIM3 antibodies), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, CTLA4, TIM3, or VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemicals. Combinations thereof are also encompassed by this disclosure.

[0047] Antibody that specifically binds to VISTA-ECD at acidic pH VISTA has many histidine residues in its extracellular domain (ECD), so its folding and overall structure, as well as the surface available for binding of ligands such as antibodies, are significantly different at acidic pH than at neutral pH, particularly the pK of histidine. a The pH may differ from that of around pH 6.5, which is generally acidic. Because tumor microenvironments are generally acidic, binding to VISTA in those microenvironments may require antibodies to bind with specificity to VISTA at an acidic pH, where at least some of the surface histidine residues are more likely to be protonated.

[0048] The following sequence listing provides the amino acid sequence of human VISTA (hVISTA) with and without the signal peptide (SEQ ID NO:1 and SEQ ID NO:2 (mature hVISTA)), respectively. The signal peptide comprises amino acid residues 1-32 of SEQ ID NO:1. The extracellular domain (ECD) consists of amino acid residues 1-162 of SEQ ID NO:2. The IgV domain comprises amino acid residues 37-167 of SEQ ID NO:1 and amino acid residues 5-135 of SEQ ID NO:2. The stalk region is located at amino acid residues 172-194 of SEQ ID NO:1 and amino acid residues 136-162 of SEQ ID NO:2, and the transmembrane domain is located at amino acid residues 195-216 of SEQ ID NO:1 and amino acid residues 163-184 of SEQ ID NO:2. Amino acid residues 187 of SEQ ID NO:1 and 155 of SEQ ID NO:2 (bold and underlined) can be either D or E, which corresponds to a polymorphism in hVISTA. The residues are shown in bold and underlined. Thus, SEQ ID NO: 1 and SEQ ID NO: 2 encompass both human polymorphisms at that residue. Histidine residues in the ECD of VISTA are shaded grey.

[0049] An anti-VISTA antibody (Ab) may specifically bind to VISTA-ECD or a fragment thereof at an acidic pH, for example, comprising the IgV domain of VISTA or a region derived from hVISTA, for example, comprising amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein at a pH less than 7.0. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein at a pH less than 6.8. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein at a pH less than 6.5. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein at a pH less than 6.3. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein at a pH less than 6.0. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.8. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.5. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.3. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.0.

[0050] Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 5.0 to pH 7.0. Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 5.0 to pH 6.5. Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 5.0 to pH 6.0. Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 5.5 to pH 7.0. Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 5.5 to pH 6.5. Certain Abs specifically bind to the VISTA-ECD protein at a pH within the range of pH 6.0 to pH 6.5.

[0051] VISTA-ECD protein, e.g., hVISTA-ECD or a fragment thereof, comprising a region derived from hVISTA, e.g., the IgV domain of VISTA or the IgV domain of hVISTA, e.g., amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2, at a pH of 6.5 or less, for 10 -6 K below M D Also provided herein are Abs that bind at 10 -7 K below M D In some embodiments, the Ab binds at 10 -8 K below M D In some embodiments, the Ab binds at 10 -9 K below M D In some embodiments, the Ab binds at 10 -10 K below M D For example, Ab binds to the VISTA-ECD protein at a pH of 6.5 or less and at a concentration of 10 -8 K below M D can be bonded with

[0052] VISTA-ECD protein and 10 -6 K below M D Also provided herein are Abs that bind at 10 -7 K below M D In some embodiments, the Ab binds at 10 -8 K below M D In some embodiments, the Ab binds at 10 -9 K below M D In some embodiments, the Ab binds at 10 -10 K below M D For example, the Ab binds to the VISTA-ECD protein at a pH of 6.5 or less, e.g., within the pH range of 6.0 to 6.5, at a pH of 10 -7 K below M D Furthermore, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, for example, within the pH range of 6.0 to 6.5, at a pH of 10.-8 K below M D The Ab can bind to hVISTA-ECD at a pH of 6.5 or less, for example, within the pH range of 6.0 to 6.5, at 10 -9 K below M D can be bonded with

[0053] VISTA-ECD protein, e.g., hVISTA-ECD or a fragment thereof, comprising a region derived from hVISTA, e.g., the IgV domain of VISTA or e.g., amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2, is incubated for 10 min at a pH of 6.5 or less, at 25°C, or at 37°C. -5 s -1 The following k off Also provided herein are Abs that specifically bind at 10° C. or 10° C. at 25° C. or 37° C. In some embodiments, the Abs specifically bind at 10° C. or 10° C. at 25° C. or 37° C. -4 s -1 The following k off In some embodiments, the Ab has a 2:10 at 25°C or 37°C. -4 s -1 The following k off In some embodiments, the Ab has a 5 to 10 at 25°C or at 37°C. -4 s -1 The following k off In some embodiments, the Ab has a serotype of 7-10 at 25°C or 37°C. -4 s -1 The following k off In some embodiments, the Ab has a 10 -3 s -1 The following k off In some embodiments, the Ab has a 2:10 at 25°C or 37°C. -3 s -1 The following k off In some embodiments, the Ab has a 5 to 10 at 25°C or at 37°C. -3 s -1 The following k off In some embodiments, the Ab has a serotype of 7-10 at 25°C or 37°C. -3 s -1 The following koff In some embodiments, the Ab has a 10 -2 s -1 The following k off In some embodiments, the solubility is 10% at 25° C. or 10% at 37° C. -1 s -1 The following k off For example, the Ab binds to the VISTA-ECD protein at a pH of 6.5 or less, at 25°C, or at 37°C for 10 min. -3 s -1 The following k off The Ab can specifically bind to the hVISTA-ECD protein at a pH of 6.5 or less, at 25°C, or at 37°C for 10 min. -3 s -1 The following k off Furthermore, the Ab can specifically bind to the VISTA-ECD protein at a pH of 6.5 or less, at 25°C, or at 37°C for 10 min. -2 s -1 The following k off can specifically bind to

[0054] VISTA-ECD protein, e.g., hVISTA-ECD or a fragment thereof, comprising a region derived from hVISTA, e.g., the IgV domain of VISTA, or e.g., amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2, and (i) 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less or 10 -10 K below M D and (ii) 10 as measured, for example, at 25°C or at 37°C. -5 s -‘1 Below, 10 -4 (or 2, 5 or 7 10 -4 ) below, 10 -3 (or 2, 5 or 7 10 -4 )s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1The following k off Provided herein are Abs that bind to VISTA-ECD proteins at a pH of 6.5 or less and at a rate of 10 -7 K below M D and 10 as measured at, for example, 25°C or at 37°C. -3 s -1 The following k off The Ab binds to the VISTA-ECD protein at a pH of 6.5 or less and binds at a rate of 10 -8 K below M D and 10 as measured at, for example, 25°C or at 37°C. -3 s -1 The following k off The Ab binds to the VISTA-ECD protein at a pH of 6.5 or less and binds at a rate of 10 -8 K below M D and 10 as measured at, for example, 25°C or at 37°C. -2 s -1 The following k off For example, the Ab can bind to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -7 K below M D and 10 as measured at, for example, 25°C or at 37°C. -3 s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -9 K below M D and 10 as measured at, for example, 25°C or at 37°C. -3 s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -9 K below M D and 10 as measured at, for example, 25°C or at 37°C. -2 s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -8 K below M Dand 10 as measured at, for example, 25°C or at 37°C. -4 (or 2, 5 or 7 10 -4 )s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -8 K below M D and 10 as measured at, for example, 25°C or at 37°C. -5 (or 2, 5 or 7 10 -5 )s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -9 K below M D and 10 as measured at, for example, 25°C or at 37°C. -4 (or 2, 5 or 7 10 -4 )s -1 The following k off The Ab binds to the hVISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -9 K below M D and 10 as measured at, for example, 25°C or at 37°C. -5 (or 2, 5 or 7 10 -5 )s -1 The following k off It can be combined at speed.

[0055] VISTA-ECD protein and, for example, at a pH of 6.5 or less, at 25°C, or at 37°C for 10 minutes. 4 M -1 s -1 Higher than on Provided herein are Abs that specifically bind at 10 5 M -1 s -1 More than k on In some such embodiments, the Ab may bind at 10 6 M -1 s -1 More than k on In some such embodiments, the Ab may bind at 107 M -1 s -1 More than k on For example, the Ab may bind to the VISTA-ECD protein at a pH of 6.5 or less, e.g., 10 as measured at 25°C, or 37°C. 6 M -1 s -1 More than k on For example, an Ab may bind to the ECD of hVISTA at a pH of 6.5 or less, e.g., 10 as measured at 25°C, or 37°C. 6 M -1 s -1 More than k on can be bonded with

[0056] VISTA-ECD protein and, for example, at a pH of 6.5 or less, (i) 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less or 10 -10 K below M D and (ii) 10 as measured, for example, at 25°C or at 37°C. 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on For example, the Ab binds to the VISTA-ECD protein at a pH of 6.5 or less and at a concentration of 10 -7 K below M D and 10 as measured at, for example, 25°C or at 37°C. 6 M -1 s -1 More than k on For example, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less and at a rate of 10 -8 K below M Dand 10 as measured at, for example, 25°C or at 37°C. 6 M -1 s -1 More than k on For example, Abs can bind to hVISTA-ECD at pH 6.5 or below at a rate of 10 -7 K below M D and 10 as measured at, for example, 25°C or at 37°C. 6 M -1 s -1 Higher than on For example, Abs can bind to hVISTA-ECD at pH 6.5 or below at a rate of 10 -8 K below M D and 10 as measured at, for example, 25°C or at 37°C. 6 M -1 s -1 More than k on It can be combined at speed.

[0057] In some embodiments, the Ab is co-cultured with the VISTA-ECD protein at a pH of 6.5 or less, for 10 -7 K below M D as measured at 10°C, e.g., 25°C, or 37°C. -5 s -1 Below, 2 10 -5 s -1 Below, 5 10 -5 s -1 Below, 7 10 -5 s -1 Below, 10 -4 s -1 Below, 2 10 -4 s -1 Below, 5 10 -4 s -1 Below, 7 10 -4 s -1 Below, 10 -3 s -1 Below, 2 10 -3 s -1 Below, 5 10 -3 s -1 Below, 7 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10-1 s -1 The following k off In some embodiments, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -9 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off In some such embodiments, the Ab may bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -10 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off can be bonded with

[0058] In some embodiments, the Ab is co-cultured with the VISTA-ECD protein at a pH of 6.5 or less, for 10 -7 K below M D 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k onIn some embodiments, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -8 K below M D 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on In some embodiments, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -9 K below M D 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on In some such embodiments, the Ab may bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -10 K below M D 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on can be bonded with

[0059] In some embodiments, the Ab is co-cultured with the VISTA-ECD protein at a pH of 6.5 or less, for 10 -7 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on In some embodiments, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -8 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s-1 More than k on In some embodiments, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -9 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1 That's it, 10 7 M -1 s -1 More than k on In some such embodiments, the Ab may bind to the VISTA-ECD protein at a pH of 6.5 or less, at 10 -10 K below M D 10 as measured at, for example, 25°C or at 37°C -5 s -1 Below, 10 -4 s -1 Below, 10 -3 s -1 Below, 10 -2 s -1 Less than or equal to 10 -1 s -1 The following k off 10 as measured at, for example, 25°C or at 37°C 4 M -1 s -1 That's it, 10 5 M -1 s -1 That's it, 10 6 M -1 s -1That's it, 10 7 M -1 s -1 More than k on can be bonded with

[0060] As noted above, in some of the above embodiments, the VISTA-ECD protein is hVISTA-ECD or a portion of hVISTA-ECD, such as the IgV domain. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 20-95 of SEQ ID NO:2. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 20-70 of SEQ ID NO:2. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 35-95 of SEQ ID NO:2. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 35-70 of SEQ ID NO:2. In some of the above embodiments, the epitope is a three-dimensional epitope that includes not only one of the above portions of SEQ ID NO:2 derived from residues 20-95, 20-70, 35-95, or 35-70, but also another portion of SEQ ID NO:2, such as residues 95-105 of SEQ ID NO:2. In certain embodiments, the Ab binds to an epitope of hVISTA bound by the Abs described in WO2015 / 097536. For example, the Ab may compete or cross-compete with the Abs disclosed in WO2015 / 097536 for binding to hVISTA. In certain embodiments, the Ab binds to a conformational epitope of human VISTA. In certain embodiments, the Ab binds to a conformational epitope comprising or residing within residues 103-111 of SEQ ID NO:2 and 136-146 of SEQ ID NO:2 of human VISTA. In certain embodiments, the Ab binds to a conformational epitope comprising or residing within residues 24-36, 54-65, and 100-102 of SEQ ID NO:2 of human VISTA. In certain embodiments, the Ab binds to a conformational epitope comprising amino acid residues in the FG loop of human VISTA. In some embodiments, the Ab binds to a polypeptide comprising amino acid residues 35-127 and / or 37-125 of SEQ ID NO:2.In some embodiments, the Ab binds to a VISTA ECD polypeptide comprising amino acid residues 350-127 of SEQ ID NO:2, or a portion thereof, but the antibody does not bind, or binds with reduced affinity, to a VISTA ECD polypeptide or portion thereof comprising an amino acid substitution (1) at one of the following amino acid residues: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127, and SEQ ID NO:2, or (2) at one of the following amino acid residues: Y37, T39, R54, F62, Q63, H66, L115, V117, I119, S124, or E125. In some embodiments, the anti-VISTA antibody has the same binding characteristics (or substantially the same binding characteristics) as the antibodies described herein, e.g., as shown in the Examples and / or in the claims.

[0061] Some of the above-described antibodies may exhibit differential binding affinity to the VISTA-ECD protein depending on pH. Certain Abs that specifically bind to the VISTA-ECD protein under acidic conditions, e.g., at pH 6.5 or below, also specifically bind to the VISTA-ECD protein with similar affinity at neutral and / or alkaline pH (i.e., they are "pan-binders"). For example, some such Abs exhibit a K D However, at pH 7.0, D 10 at both pH 6.5 and pH 7.0 (e.g., at a constant temperature of 25°C or 37°C) to be within 1.5 times of -7 K below M D Some such Abs can bind to the VISTA-ECD protein at a K D However, at pH 7.0, D 10 at both pH 6.5 and pH 7.0 (e.g., at a constant temperature of 25°C or 37°C) to be within 1.5 times of -8 K below M DSome such Abs can bind to the hVISTA-ECD protein at a K D However, at pH 7.0, D 10 at both pH 6.5 and pH 7.0 (e.g., at a constant temperature of 25°C or 37°C) to be within 1.5 times of -8 K below M D can be bonded with

[0062] Certain Abs that specifically bind to VISTA-ECD protein under acidic conditions, e.g., at pH 6.5 or below, may bind to VISTA-ECD protein with lower affinity under neutral, physiological, and / or alkaline conditions ("pH-sensitive binders" or "pH-sensitive Abs"). Certain Abs that specifically bind to VISTA-ECD protein under acidic conditions, e.g., at pH 6.5 or below, may have insignificant, e.g., nearly undetectable, binding to VISTA-ECD protein under neutral, physiological, and / or alkaline conditions. For example, in some embodiments, an Ab binds to VISTA-ECD protein with a 10 affinity at pH 6.5. -8 K below M D and 10 at pH 7.0 and / or pH 7.4 -8 K is larger than M D In some such embodiments, the Ab may bind to the VISTA-ECD protein at 10 -8 K below M D and at pH 7.0 and / or pH 7.4, a K that is more than 1.5 times greater than that at pH 6.5 D In certain embodiments, the VISTA-ECD protein may bind to a VISTA-ECD protein at a K that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, or 5000-fold lower at pH 6.5 than at pH 7.0. DpH-sensitive Abs are provided that specifically bind to VISTA-ECD proteins at a constant temperature (e.g., 25° C. or 37° C.). For example, in some cases, the Ab binds to the VISTA-ECD protein with a K that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, or 5000-fold less at pH 6.0 than at pH 7.0 and / or pH 7.4. D The mixture is then allowed to bind at a constant temperature (e.g., 25°C or 37°C).

[0063] In certain embodiments, the Ab binds to the VISTA-ECD protein and has a lower kA under acidic conditions than under neutral, physiological, or alkaline conditions. off In certain embodiments, the VISTA-ECD protein specifically binds to the VISTA-ECD protein at acidic conditions, e.g., at pH 6.5, pH 7.0, and / or pH 7.4, as measured at 25°C or at 37°C. off at least 1.5x, 2x, 5x, 10x, 20x, 50x, 100x or 1000x lower than off In other words, the off-rate is slower at acidic pH than at neutral pH. For example, in some embodiments, the Ab binds to the VISTA-ECD protein with a k that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6.0 relative to pH 7.0 and / or pH 7.4, e.g., as measured at 25°C or 37°C. off In certain embodiments, the VISTA-ECD protein specifically binds to the VISTA-ECD protein at a rate of, for example, a K at pH 6.5, a K at pH 7.4, as measured at 25°C, or at 37°C. off at least 1.5x, 2x, 5x, 10x, 20x, 50x, 100x or 1000x lower than off In some embodiments, the Ab binds to the VISTA-ECD protein with a k that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6.0 versus pH 7.4, e.g., as measured at 25°C or at 37°C. offIn certain embodiments, the VISTA-ECD protein specifically binds to the VISTA-ECD protein at a kPa (kcal) of 100 kJ / mL at pH 6.0-6.5, pH 7.0-7.4, as measured, for example, at 25°C or at 37°C. off at least 1.5x, 2x, 5x, 10x, 20x, 50x, 100x or 1000x lower than off Abs that bind to

[0064] In certain embodiments, the VISTA-ECD protein and the VISTA-ECD protein have a higher k in acidic conditions relative to neutral, physiological, or alkaline conditions. on In certain embodiments, the VISTA-ECD protein specifically binds to the Ab at a pH of 6.5, 7.0, and / or 7.4 as measured at 25° C. or 37° C. on At least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold higher than on For example, in some embodiments, the Ab binds to the aVISTA-ECD protein with a k that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold higher at pH 6.0 than at pH 7.0 and / or pH 7.4, e.g., as measured at 25°C or at 37°C. on It specifically binds to

[0065] In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH at which at least one histidine residue in SEQ ID NO: 1 is protonated, e.g., His98. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH below 6.5, e.g., a pH at which most histidine residues in the ECD are protonated, which is predicted to be between pH 6.0 and pH 6.5.

[0066] Also encompassed herein are Abs that specifically bind to VISTA-ECD proteins at neutral, physiological, or alkaline pH with high affinity relative to acidic pH, so long as the binding affinity at acidic pH remains high. For example, an Ab may have a K at pH 7.0 that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500-fold, or 1000-fold lower than at pH 6.5. D Although the Ab binds to the VISTA-ECD protein at pH 6.5 and pH 7.0, the Ab binds to the VISTA-ECD protein at pH 6.5 and pH 7.0 at 10 -8 K below M D can be bonded with

[0067] Also encompassed herein are Abs that share one or more of the above properties in this section. D , k off , k on Therefore, the specific epitope should not be treated in isolation. Thus, an Ab can bind to an epitope containing one of the regions of SEQ ID NO:2 above, and its K D , k off or k on The antibody may have general binding or pH-sensitive or pH-selective binding properties as described above, as exhibited by one or more of the following behaviors:

[0068] In any of the above embodiments, the Ab can be, for example, a full-length antibody (i.e., comprising a full-length heavy chain (with or without a C-terminal lysine) and a full-length light chain), or an antigen-binding fragment, such as a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an scFv fragment, an Fv fragment, or the Ab can be a chimeric, humanized, or human antibody, or the Ab can be a bispecific or multispecific antibody.

[0069] Determining how well an Ab binds to a VISTA-ECD protein at a given pH can be performed using several different methods, such as by surface plasmon resonance (SPR), e.g., by a BIACORE® assay. An exemplary SPR assay involves capturing one or several antibodies on a CM4 sensor chip using an immobilized capture reagent (e.g., using a Biacore® anti-human Fc capture kit, GE Healthcare catalog number BR-1008-39 or a Biacore® anti-mouse capture kit, GE Healthcare catalog number BR-1008-39) and running a concentration series of VISTA antigen as the analyte to determine binding kinetics and affinity in a running buffer with the desired pH. In one embodiment, VISTA is injected at a flow rate of 30 μL / min at two to five concentrations ranging from 0.1 nM to 500 nM (e.g., 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM) with an association time of up to 4 minutes and a dissociation time of up to 10 minutes. Between binding cycles, the capture surface is regenerated according to the manufacturer's instructions for each capture kit. All data are double-referenced using a reference flow cell and a blank injection. Data with simple 1:1 kinetics are fitted to a Langmuir binding model with mass transfer using Biacore® T200 evaluation software. SPR methods, as described in the Examples, may also be used.

[0070] The affinity of Abs for VISTA ECD polypeptides can be determined using cells expressing VISTA ECD polypeptides, PSGL-1, or heparan sulfate on their surface; this method involves flow cytometry, which can determine the binding of Abs to VISTA-ECD bound to cells at a given pH, e.g., pH 6.5 or lower. An exemplary flow cytometry assay involves resuspending 293T cells or other cells ectopically expressing hVISTA ECD in a buffer consisting of HBSS + 1% BSA adjusted to a desired pH, e.g., pH 6.0 with MES or pH 7.4 with HEPES. Serial dilutions of Abs (e.g., human IgG) against hVISTA are made starting at approximately 20 μg / mL and incubated with the resuspended cells at 4° C. for 30 minutes. The cells are then washed twice with the same buffer, maintaining the desired pH, for example, 6.0 or 7.4, and incubated with a fluorophore-conjugated secondary antibody that recognizes the primary antibody (e.g., human IgG) and is stable at reduced pH. The cells are then washed as before and immediately acquired without fixation on a BD Fortessa or other flow cytometer. The affinity of the Ab for the VISTA ECD polypeptide can be determined as described in the Examples.

[0071] In certain embodiments, an Ab that binds to hVISTA ECD blocks the binding of hVISTA to its binding partner (e.g., a VISTA receptor), for example, on a cell. Inhibition or blocking can be 100%, or at least 99%, 95%, 90%, 85%, 80%, 75%, or 50%. In certain embodiments, an Ab binds to a VISTA-ECD protein at an acidic pH, e.g., pH 6.5 or below, and inhibits the binding of VISTA to its binding partner by at least 50%, e.g., at least 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH below pH 7.0. In certain embodiments, an Ab specifically binds to a VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH below pH 6.8. In certain embodiments, an Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.5. In certain embodiments, an Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.3. In certain embodiments, an Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.0. In certain embodiments, an Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.8. In certain embodiments, an Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.5. In certain embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits binding of VISTA to its binding partner by at least 50% at a pH that is less than pH 5.3.In certain embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits binding of VISTA to its binding partner by at least 50% at a pH that is less than pH 5.0.

[0072] Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of pH 5.0 to pH 7.0. Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of pH 5.0 to pH 6.5. Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of pH 5.0 to pH 6.0. Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of pH 5.5 to pH 7.0. Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of pH 5.5 to pH 6.5. Certain Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the range of 6.0 to 6.5. Inhibition of binding can be determined as described in the Examples.

[0073] The VISTA binding partner can be PSGL-1, e.g., human PSGL-1. The sequences of human PSGL-1 isoforms are provided herein as SEQ ID NOS: 3-10. VISTA binds to PSGL-1 with or without sialylated Lewis X. The binding partner can also be, for example, a heparan sulfate proteoglycan present on certain cells.

[0074] Inhibition of VISTA binding to a binding partner can be determined by measuring the inhibition of binding of VISTA (or VISTA ECD or VISTA IgV domain or VISTA-positive cells) to cells to which VISTA binds, such as T cells (e.g., CD4+ T cells, CD8+ T cells, either activated or not), NK cells, or other cells to which VISTA binds, in the presence and absence of the antibody. An exemplary experiment that can be used to determine whether an antibody inhibits binding of VISTA to its binding partner or T cells expressing the binding partner is a flow cytometry assay, for example, an assay comprising the following: human peripheral blood mononuclear cells, buffy coat, or leukopak obtained from donor blood are resuspended in a buffer consisting of HBSS+1% BSA adjusted to the desired pH, e.g., pH 6.0 with MES and pH 7.4 with HEPES. The cells are then incubated with 20 μg / mL of a recombinant chimeric protein consisting of hVISTA ECD fused to human IgG1 Fc (VISTA-Fc) and varying concentrations of a candidate VISTA blocking antibody or control antibody for 30 minutes at 4° C. The cells are then washed twice with the same buffer while maintaining the desired pH, e.g., pH 6.0 or 7.4, and incubated for an additional 30 minutes at 4° C. with a fluorophore-conjugated secondary antibody that recognizes VISTA-Fc but not the candidate blocking antibody or control antibody, and that is stable at reduced pH. The cells are then washed as before, not fixed, and immediately acquired on a BD Fortessa or other flow cytometer. Inhibition of binding can be determined, for example, as described in the Examples.

[0075] In certain embodiments, the Abs described herein can induce or enhance an immune response, e.g., an antigen-specific immune response. In certain embodiments, the Abs stimulate T cell activity, particularly at the acidic pH found, for example, in the tumor microenvironment. Stimulation of T cell activity can be measured, for example, in a mixed lymphocyte reaction (MLR) or in an in vitro assay using antigen-presenting cells (natural or artificial) and T cells. Stimulation of T cell activity can also be measured, for example, using the Jurkat assay described in the Examples. Stimulation of T cell activity can also be measured by determining IFN-γ secretion from T cells, with enhanced IFN-γ secretion indicating T cell stimulation. Secretion of other cytokines from activated T cells can also be measured. In certain embodiments, activated T cell signaling, such as NF-kB levels, is measured. In certain embodiments, the Abs described herein inhibit cell adhesion, which can be measured as described in the Examples.

[0076] The activity of anti-VISTA Abs can also be demonstrated in monocyte, ADCC and ADCP assays, particularly at the acidic pH found, for example, in the tumor microenvironment.

[0077] In certain embodiments, the anti-VISTA Abs inhibit tumor growth in a tumor model, for example, a human VISTA knock-in tumor model.

[0078] As shown in the Examples herein, recycling of anti-VISTA Abs in endosomes to enhance the pharmacokinetic (PK) properties of the antibody, i.e., half-life, etc., requires that the anti-VISTA antibody bind to VISTA under acidic conditions. Thus, anti-VISTA Abs that bind to VISTA at low pH, e.g., at a pH of 6.5 or less as further described herein, are also predicted to have a longer, acceptable half-life compared to VISTA antibodies that do not bind to VISTA at acidic pH.

[0079] In some embodiments, VISTA antibodies preferentially bind to tumors over bone marrow cells in vivo. Certain anti-VISTA Abs herein have been shown to preferentially localize to tumors over blood in VISTA knock-in mice. For example, in some embodiments, antibodies herein accumulate at least two-fold higher levels in tumors relative to blood 24-51 hours after administration of the Ab to a subject. In some embodiments, anti-VISTA Abs do not accumulate significantly more in the liver or lungs relative to the blood of a subject. In some embodiments, anti-VISTA Abs in tumors can persist over time, for example, up to at least 51 hours after administration of the VISTA Ab to a subject. In some embodiments, the localization of anti-VISTA Abs in a subject is determined by administering Abs labeled with a PET tracer to the subject.

[0080] Exemplary hVISTA-ECD Binding Abs Provided herein are Abs that preferentially bind to hVISTA(ECD) at acidic pH (eg, in acidic conditions) relative to physiological or neutral pH.

[0081] This disclosure relates to the following: P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E 102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32 Described are anti-hVISTA Abs including P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, and P1-068748_D57K_D100S. The antibody may contain the heavy chain CDRs of either the P1-068744 or P1-068748 Ab modified to restore at least one H, D, or E residue to the residue found at the same position in the P1-061015 parent antibody. The antibody may, in some embodiments, comprise the light chain CDRs of antibody P1-061015.

[0082] More generally, the disclosure includes anti-hVISTA Abs comprising a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1YAMH (wherein X1 is E or S) (SEQ ID NO: 690), a VH CDR2 comprising X2IWYDGSNKYX3ADSVKG (wherein X2 is H or I and X3 is E or Y) (SEQ ID NO: 691), and / or a VH CDR3 comprising DSGFYX4SYYFDX5 (wherein X4 is E or S and X5 is E or Y) (SEQ ID NO: 692). In Ab P1-061015, these positions are S, I, Y, S, and Y, respectively, while in P1-068744, these positions are E, H, E, E, and E (see, e.g., Figure 29C). In such cases, the antibody may have a VH CDR1 of P1-068744. and P1-68744_E31S (i.e., containing the CDRs of P1-068744 except when X1 is S), P1-68744_H50I (i.e., X2 is I), P1-68744_E59Y (i.e., X3 is Y), P1-068744_E100S (i.e. X4 is S), P1-068744_E102Y (i.e. X5 is Y), P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, or P1-068744_E59Y_E102Y. Note that the nomenclature for these antibodies used above is somewhat based on the Kabat numbering scheme, which numbers residues by their structural position rather than by their sequence position. Because antibodies may have CDRs of different lengths, the numbering scheme may not necessarily correspond to the number of residue positions in the antibody sequence.The above substitutions at X1, X2, X3, X4, and X5 are located at positions 31, 50, 60, 104, and 110 of the heavy chain sequences of SEQ ID NOs: 95 and 103 herein, as can be readily seen from reviewing Tables 11 and 27. See, e.g., M. Dondelinger et al., Front. Immunol. 9: 2278 (2018) for more information regarding antibody residue numbering formats.

[0083] In some embodiments, the antibody may comprise the heavy chain CDRs of P1-068744 or one of the above mutants where X2 is H. In some embodiments, the antibody may comprise the heavy chain CDRs of P1-068744 or one of the above mutants where X2 is H and X4 is E (i.e., does not contain an H50I or E100S mutation). Examples include antibodies that comprise the heavy chain CDRs of P1-068744, P1-068744_E31S, P1-068744_E31S_E102Y, P1-068744_E102Y, P1-068744_E31S_E59Y, P1-068744_E59Y, and P1-068744_E59Y_E102Y. In some embodiments, these antibodies have a CDR of (a) < 5 x 10 -8 M, <1 x 10 -8 M, <5 x 10 -9 M, or <3 x 10 -9 Affinity for hVISTA (e.g., by surface plasmon resonance (SPR)) represented by Kd in M; (b) 1 × 10 at pH 7.4 -8 and 1 × 10 -6 and / or (c) the antibody further has a Kd (e.g., as measured by SPR) of <5×10 -3 (1 / s), <4×10 -3 (1 / s) or <3×10 -3and further has a koff at pH 6.0 of (1 / s) (e.g., as measured by SPR). In other embodiments, the antibody retains an H at position X2 and an E at position X3. In some embodiments, the antibody retains an E at both positions X3 and X4. Also, in some embodiments, the antibody retains an H at X2, an E at X3, and an E at X4 compared to P1-068744. Examples include antibodies comprising the heavy chain CDRs of P1-068744, P1-068744_E31S, P1-068744_E31S_E102Y, and P1-068744_E102Y. In the above embodiments, the antibody may comprise the heavy chain CDRs of one of these antibodies and the light chain CDRs of the P061015 antibody. In some embodiments, the anti-hVISTA antibody is one described herein, e.g., one described above, with no other amino acid substitutions in the CDRs or variable regions.

[0084] Additionally, the disclosure includes anti-hVISTA Abs comprising a heavy chain variable region (VH) comprising a VH CDR1 comprising GFTFSX1X2AMH (wherein X1 is H or S and X2 is H or Y) (SEQ ID NO: 693), a VH CDR2 comprising IIWYDGSNX3X4YADSVKG (wherein X3 is D or K and X4 is D or Y) (SEQ ID NO: 694), and / or a VH CDR3 comprising DSGFYX5SYYFDY (wherein X5 is D or S) (SEQ ID NO: 695). In Ab P1-061015, these positions are S, Y, K, Y, and S, respectively, while in P1-068748, these positions are H, H, D, D, and D (see Figure 29C). In such cases, the antibody may comprise a heavy chain variable region (VH) comprising the VH CDR1 of P1-068748. and P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H VH CDR1, CDR2 and / or CDR3 of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S or P1-068748_D57K_D100S. Note that the nomenclature herein is based on Kabat numbering. The above substitutions at X1, X2, X3, X4 and X5 are located at positions 31, 32, 58, 59 and 104 of the heavy chain sequences of SEQ ID NOs: 95 and 99, as can be seen, for example, from reviewing Tables 12 and 28 and SEQ ID NOs: 95 and 99.

[0085] In some such embodiments, the heavy chain CDRs may retain a D at position X5 (i.e., amino acid position 104; i.e., they do not contain a D100S substitution as described in the nomenclature above). In some cases, the antibody retains a D at sequence position X5 and also exhibits pH-selective binding to hVISTA. In some cases, the antibody retains a D at position X4. In some cases, the antibody retains a D at both positions X4 and X5 (i.e., does not contain either a D58Y or a D100S substitution relative to P1-068744). In some cases, the antibody comprises the heavy chain CDRs of P1-068748, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, or P1-068748_H31S_D57K. In some such cases, the antibody may comprise the heavy chain CDRs of one of those antibodies and the light chain CDRs of the P061015 antibody. In some embodiments, the antibody has (a) < 1 x 10 -8 M, <7 x 10 -9 M, or <5 x 10 -9 Affinity for hVISTA (e.g., by surface plasmon resonance (SPR)) represented by Kd in M; (b) 5 × 10 at pH 7.4 -8 and 1 × 10 -6 and / or (c) the antibody further has a Kd (e.g., as measured by SPR) of <5×10 -3 (1 / s), <4×10 -3 (1 / s) or <3×10 -3 (1 / s) (e.g., as measured by SPR). In some embodiments, the anti-hVISTA antibody is one described herein, e.g., one described above, with no other amino acid substitutions in the CDRs or variable regions.

[0086] In certain embodiments, an anti-hVISTA antibody comprises the VH and VL CDRs of P1-068744 or P1-068748, or comprises the VH and VL of P1-068744 or P1-068748 with amino acid reversions to those in P1-061015, where these amino acid reversions do not affect the desired characteristics of the antibody. For example, an anti-hVISTA antibody may comprise the VH and VL or VH and VL CDRs of P1-068744 or P1-068748 with back mutations that do not affect pH selectivity and / or binding kinetics, as further described herein.

[0087] In certain embodiments, an anti-hVISTA Ab comprises a heavy chain variable region ("VH") comprising the VH CDR1, CDR2 and / or CDR3 of any of the anti-hVISTA Abs provided herein. In certain embodiments, an anti-hVISTA Ab comprises a VH comprising the VH CDR1, CDR2 and CDR3 of any of the anti-hVISTA Abs provided herein. In certain embodiments, an anti-hVISTA Ab comprises a VH comprising the VH CDR1, CDR2 and CDR3 of any of the anti-hVISTA Abs provided herein. The Ab can be P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y _E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S and / or a VH comprising a VH CDR1, CDR2 and / or CDR3 of P1-068748_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. The VH CDR1, CDR2, and CDR3 of each of these species comprise amino acid positions 26-35 (VH CDR1), 50-66 (VH CDR2), and 99-110 (VH CDR3) of the VH sequences of each of the above antibody species provided in the sequence listing below. The CDRs are also underlined and in bold in each of the VH sequences of the above antibody species provided in the sequence listing below.

[0088] In certain embodiments, the anti-hVISTA Ab comprises a VL comprising the VL CDR1, CDR2, and CDR3 of any of the anti-hVISTA Abs provided herein. In certain embodiments, the anti-hVISTA Ab comprises any of the following: P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1 -069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_ E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H3 and a VL comprising a VL CDR1, CDR2 and CDR3 of one of P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. The VL CDR1, CDR2, and CDR3 of each of these species comprise amino acid positions 24-35 (VL CDR1), 51-57 (VL CDR2), and 90-98 (VL CDR3) of the VL sequences of each of the above antibody species provided in the sequence listing below. The CDRs are also underlined and in bold in each of these sequences.

[0089] In certain embodiments, an anti-hVISTA Ab comprises a VH that comprises the VH CDR1, CDR2, and / or CDR3 of any of the anti-hVISTA Abs provided herein, and a VL that comprises the CDR1, CDR2, and / or CDR3 of any of the anti-hVISTA Abs provided herein. In certain embodiments, an anti-hVISTA Ab comprises a VH that comprises the VH CDR1, CDR2, and CDR3 of any of the anti-hVISTA Abs provided herein, and a VL that comprises the CDR1, CDR2, and CDR3 of any of the anti-hVISTA Abs provided herein. In certain embodiments, an anti-hVISTA Ab comprises a VH that comprises the VH CDR1, CDR2, and CDR3 of any of the anti-hVISTA Abs provided herein. The Ab can be P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-0687 61_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068 761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E1 00fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-0687 67_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E or P1-061029_Y32E_F100fE VHA VH comprising CDR1, CDR2 and / or CDR3 and P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1- 069061, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061 015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y _E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S and a VL comprising a VL CDR1, CDR2 and CDR3 of P1-068748_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

[0090] In some embodiments, the anti-hVISTA Ab (a) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-061029 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-061029; (b) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-061015 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-061015; (c) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068757 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068757; (d) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068759 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068759; (e) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068761 and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068761; (f) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068763 and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068763; (g) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068765 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068765; (h) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068767; (i) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068769 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068769; (j) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068771 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068771; (k) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068773 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068773; (l) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068775 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068775; (m) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069059 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069059; (n) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069061 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069061; (o) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069063 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069063; (p) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-069065 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069065; (q) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069067 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069067; (r) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069069 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069069; (s) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-069071 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069071; (t) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-069073 and VL comprising VL CDR1, CDR2 and CDR3 of P1-069073; (u) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069075 and VL comprising VL CDR1, CDR2 and CDR3 of P1-069075; (v) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-069077 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-069077; (w) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068736 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068736; (x) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068738 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068738; (y) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068740 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068740; (z) a VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068742 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068742; (aa) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068744; (bb) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068746 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068746; (cc) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748 and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748; (dd) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068750 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068750; (ee) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068752 and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068752; (ff) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068754 and VL comprising VL CDR1, CDR2 and CDR3 of P1-068754; (gg) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068761_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E55A; (hh) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068761_H100G and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068761_H100G; (ii) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of P1-068761_E56N and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068761_E56N; (jj) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068761_E55A_E56N and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E55A_E56N; (kk) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068761_E30D and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D; (ll) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E30D_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D_E55A; (mm) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E56N_H100G and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E56N_H100G; (nn) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E30D_H100G and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D_H100G; (oo) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068761_E30D_E56N and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D_E56N; (pp) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068761_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E100fF; (qq) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068761_E55A_E100fF and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068761_E55A_E100fF; (rr) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_H100G_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_H100G_E100fF; (ss) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E30D_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D_E100fF; (tt) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E56N_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E56N_E100fF; (uu) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E32Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E32Y; (vv) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E32Y_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E32Y_E55A; (ww) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E32Y_E56N and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E32Y_E56N; (xx) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068761_E30D_E32Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068761_E30D_E32Y; (yy) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068761_E32Y_H100G and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068761_E32Y_H100G; (zz) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068761_E32Y_E100fF and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of P1-068761_E32Y_E100fF; (aaa) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_D52N_D102V and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_D52N_D102V; (bbb) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_D52N and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_D52N; (ccc) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_D52N_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_D52N_E55A; (ddd) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_E55A_D102V and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E55A_D102V; (eee) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of P1-068767_D102V and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068767_D102V; (fff) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E55A; (ggg) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068767_E30D_D52N and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E30D_D52N; (hhh) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_E30D_D102V and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E30D_D102V; (iii) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of P1-068767_E30D and a VL comprising VL CDR1, CDR2, and CDR3 of P1-068767_E30D; (jjj) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068767_E30D_E55A and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E30D_E55A; (kkk) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_E100fF_D102V and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E100fF_D102V; (lll) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_E55A_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E55A_E100fF; (mmm) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068767_D52N_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_D52N_E100fF; (nnn) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068767_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E100fF; (ooo) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068767_E30D_E100fF and VL comprising VL CDR1, CDR2 and CDR3 of P1-068767_E30D_E100fF; (ppp) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-061029_F100fE_V102D and VL comprising VL CDR1, CDR2 and CDR3 of P1-061029_F100fE_V102D; (qqq) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-61029_F100fE and VL comprising VL CDR1, CDR2 and CDR3 of P1-61029_F100fE; (rrr) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-61029_V102D and VL comprising VL CDR1, CDR2 and CDR3 of P1-61029_V102D; (sss) a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-061029_Y32E and a VL comprising VL CDR1, CDR2, and CDR3 of P1-061029_Y32E; (ttt) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-061029_Y32E_F100fE and VL comprising VL CDR1, CDR2 and CDR3 of P1-061029_Y32E_F100fE; (uuu) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068744_E31S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E31S; (vvv) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_H50I and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_H50I; (www) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E59Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E59Y; (xxx) VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of P1-068744_E100S and VL comprising VL CDR1, CDR2, and CDR3 of P1-068744_E100S; (yyy) VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068744_E102Y and VL comprising VL CDR1, CDR2, and CDR3 of P1-068744_E102Y; (zzz) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E31S_H50I and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E31S_H50I; (aaaa) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_H50I_E59Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_H50I_E59Y; (bbbb) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E5;9Y_E100S and VL comprising the amino acid sequence of VL CDR1, CDR2 and CDR3 of P1-068744_E5;9Y_E100S; (cccc) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E100S_E102Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E100S_E102Y; (dddd) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E31S_E102Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E31S_E102Y; (eeee) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E31S_E59Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E31S_E59Y; (ffff) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068744_E31S_E100S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E31S_E100S; (gggg) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_H50I_E100S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_H50I_E100S; (hhhh) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_H50I_E102Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_H50I_E102Y; (iiii) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068744_E59Y_E102Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068744_E59Y_E102Y; (jjjj) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of P1-068748_H31S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H31S; (kkkk) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H32Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H32Y; (llll) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_D57K and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_D57K; (mmmm) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_D58Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_D58Y; (nnnn) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_D100S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_D100S; (oooo) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H31S_H32Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H31S_H32Y; (pppp) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H32Y_D57K and a VL comprising the VL CDR1, CDR2 and CDR3 of P1-068748_H32Y_D57K; (qqqq) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_D57K_D58Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_D57K_D58Y; (rrrr) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_D58Y_D100S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_D58Y_D100S; (ssss) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H31S_D57K and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H31S_D57K; (tttt) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H31S_D58Y and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H31S_D58Y; (uuuu) VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H31S_D100S and VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H31S_D100S, (vvvv) a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068748_H32Y_D58Y and a VL comprising VL CDR1, CDR2 and CDR3 of P1-068748_H32Y_D58Y; (www) A VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068748_H32Y_D100S and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of P1-068748_H32Y_D100S; or (xxxx) VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of P1-068748_D57K_D100S and VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of P1-068748_D57K_D100S may include:

[0091] Again, the following sequence listing provides the heavy and light chain variable region sequences and full-length heavy and light chain sequences of the antibodies listed above having an IgG1.3 heavy chain constant region (except where a different Hc constant region is noted in the table), noting the positions of their VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 by amino acid residue, with the CDRs in each VH and VL sequence being bolded and underlined. Thus, for example, as noted by the bolded, underlined amino acids of SEQ ID NO:67 shown in the sequence listing, VH CDR1 of P1-061029 comprises amino acids 26-35 of SEQ ID NO:67, while VH CDR2 comprises amino acids 50-66 of SEQ ID NO:67, VH CDR3 comprises amino acids 99-110 of SEQ ID NO:67, etc.

[0092] In certain embodiments, the anti-hVISTA Ab comprises a VH comprising the amino acid sequence of the VH of any of the anti-hVISTA Abs provided herein. The individual VH sequences of the specific antibody species provided herein are listed in the Sequence Listing. In certain embodiments, the anti-hVISTA Ab comprises P1-061029 or P1-061015 or its progeny, such as P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E10 2Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1 and a VH comprising the amino acid sequence of P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

[0093] In some embodiments, the anti-hVISTA Ab is selected from the group consisting of antibodies P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P 1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H 31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748 _D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D and P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, but with one, two, three, four or five amino acid substitutions, for example one, two, three, four or five conservative substitutions, in the framework regions of the VH sequence. P1-061029 or its progeny may contain K16R and / or T84A substitutions in the VH framework regions (P1-061015 already has an R and an A at those positions).

[0094] In certain embodiments, an anti-hVISTA Ab comprises a VH CDR1, CDR2, and CDR3 that comprise the amino acid sequences of the VH CDRs of any of the anti-hVISTA Abs provided herein, and that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH of any of the anti-hVISTA Abs provided herein. Ab P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-0 68769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1 -069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P 1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-06874 8_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H 31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_ and a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the VH of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. In certain embodiments, the VH of the antibody differs from that of the VH sequence shown in the Sequence Listing by one, two, three, four, or five amino acid substitutions in the framework regions of the VH sequence, e.g., one, two, three, four, or five conservative substitutions, or, e.g., one or both of K16R and / or T84A substitutions in P1-061029 or its progeny.

[0095] In certain embodiments, the anti-hVISTA Ab comprises a VH consisting of the amino acid sequence of the VH of any of the anti-hVISTA Abs provided herein. In certain embodiments, the anti-hVISTA Ab comprises P1-061029 or P1-061015 or its progeny, such as P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H3 1S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_ D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D5 or P1-068748_D57K_D100S, and in the case of P1-061029 or its progeny, optionally have one or both of a K16R and / or T84A substitution.

[0096] In certain embodiments, the anti-hVISTA Ab comprises a VL comprising the amino acid sequence of the VL of any of the anti-hVISTA Abs provided herein. In certain embodiments, the anti-hVISTA Ab comprises P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59 Y_E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31 S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. In certain embodiments, an anti-hVISTA Ab comprises a VL CDR1, CDR2, and CDR3 that comprise the amino acid sequences of the VL CDRs of any of the anti-hVISTA Abs provided herein, and that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL of any of the anti-hVISTA Abs provided herein. Ab P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-0 68769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1 -069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1 -068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748 _H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H3 1S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D 100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. In certain embodiments, the VL of the antibody differs from that of the VL sequence shown in the Sequence Listing by one, two, three, four, or five amino acid substitutions, e.g., one, two, three, four, or five conservative substitutions, in the framework regions of the VL sequence. For example, P1-061015 or its progeny may have a T85V substitution in the VL framework region.

[0097] In certain embodiments, the anti-hVISTA Ab comprises a VL consisting of the amino acid sequence of the VL of any of the anti-hVISTA Abs provided herein. In certain embodiments, the anti-hVISTA Ab comprises P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E10 2Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1- The VL comprises a VL consisting of the amino acid sequence of 068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

[0098] In certain embodiments, the anti-hVISTA Ab comprises a VH comprising the amino acid sequence of the VH of any of the anti-hVISTA Abs provided herein, and a VL comprising the amino acid sequence of the VL of any of the anti-hVISTA Abs provided herein. In certain of these embodiments, the anti-hVISTA Ab is P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100 S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P 1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, and a VL comprising the amino acid sequence of the VL of P1-061029 (SEQ ID NO: 68) or P1-061015 (SEQ ID NO: 96) or P1-061015 (SEQ ID NO: 569; see P1-061015 T85V) comprising a T85V substitution.

[0099] However, in certain embodiments, the VH of the antibody is selected from the group consisting of P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1- 069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068 736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-06 8748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068 748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748 _D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, but with one, two, three, four or five amino acid substitutions, for example one, two, three, four or five conservative substitutions, in the framework regions of the VH sequence, and the VL is of P1-061029 or P1-061015 or P1-061015 T85V.

[0100] In certain embodiments, the anti-hVISTA Ab is P1-061029 or P1-061015 or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069 063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1 -068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P 1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-06874 8_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H 31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_ and a VH and VL comprising the VH and VL amino acid sequences of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, wherein the VH of P1-061029 or its descendants may comprise one or both of a K16R and / or T84A substitution, and further, the VL of P1-061015 or its descendants may comprise a T85V substitution.

[0101] In certain embodiments, the anti-hVISTA Abs comprise a VH CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VH CDR of any of the anti-hVISTA Abs provided herein, and a VL CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VL CDR of any of the anti-hVISTA Abs provided herein, and also comprise a VH and VL that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, respectively, to the corresponding VH and VL of any of the anti-hVISTA Abs provided herein. In certain embodiments, the VH and VL of the antibody differ from the VH and VL sequences set forth in the Sequence Listing by one, two, three, four or five amino acid substitutions in the framework regions of the sequences, e.g., one, two, three, four or five conservative substitutions, or, e.g., one or both of a K16R and / or T84A substitution in the VH sequence in the case of P1-061029 or its progeny, or a T85V substitution in the case of P1-061015 or its progeny.

[0102] In certain embodiments, the anti-hVISTA Ab comprises a VH and a VL consisting of the amino acid sequences of the VH and VL of any of the anti-hVISTA Abs provided herein. In certain embodiments, the anti-hVISTA Ab is P1-061029 or P1-061015, or its progeny, e.g., P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069062, P1-069073, P1-069081, P1-069091, P1-069092, P1-069093, P1-069094, P1-069095, P1-069096, P1-069097, P1-069098, P1-069099, P1-069010, P1-069011, P1-069012, P1-069013, P1-069014, P1-069015, P1-069016, P1-069017, P1-069018, P1-069019, P1-069020, P1-069021, P1-069022, P1-069023, P1-069024, P1-069025, P1-069026, P1 69063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P 1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-0 68744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748_H32Y , P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y , P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-06 and a VH and a VL consisting of the amino acid sequences of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, wherein the VH or P1-061029 or its descendants may be modified by having one or both of a K16R and / or a T84A substitution, and the VL of P1-061015 or its descendants may be modified by having a T85V substitution.

[0103] Anti-hVISTA Ab (a) VH comprising the amino acid sequence of VH of P1-061029 and VL comprising the amino acid sequence of VL of P1-061029; (b) VH comprising the amino acid sequence of VH of P1-061015 and VL comprising the amino acid sequence of VL of P1-061015; (c) VH comprising the amino acid sequence of VH of P1-068757 and VL comprising the amino acid sequence of VL of P1-068757; (d) VH comprising the amino acid sequence of VH of P1-068759 and VL comprising the amino acid sequence of VL of P1-068759; (e) VH comprising the amino acid sequence of VH of P1-068761 and VL comprising the amino acid sequence of VL of P1-068761; (f) VH comprising the amino acid sequence of VH of P1-068763 and VL comprising the amino acid sequence of VL of P1-068763; (g) VH comprising the amino acid sequence of VH of P1-068765 and VL comprising the amino acid sequence of VL of P1-068765; (h) VH comprising the amino acid sequence of VH of P1-068767 and VL comprising the amino acid sequence of VL of P1-068767; (i) a VH comprising the amino acid sequence of the VH of P1-068769 and a VL comprising the amino acid sequence of the VL of P1-068769; (j) VH comprising the amino acid sequence of VH of P1-068771 and VL comprising the amino acid sequence of VL of P1-068771; (k) VH comprising the amino acid sequence of VH of P1-068773 and VL comprising the amino acid sequence of VL of P1-068773; (l) VH comprising the amino acid sequence of VH of P1-068775 and VL comprising the amino acid sequence of VL of P1-068775; (m) a VH comprising the amino acid sequence of the VH of P1-069059 and a VL comprising the amino acid sequence of the VL of P1-069059; (n) a VH comprising the amino acid sequence of the VH of P1-069061 and a VL comprising the amino acid sequence of the VL of P1-069061; (o) VH comprising the amino acid sequence of VH of P1-069063 and VL comprising the amino acid sequence of VL of P1-069063; (p) VH comprising the amino acid sequence of VH of P1-069065 and VL comprising the amino acid sequence of VL of P1-069065; (q) a VH comprising the amino acid sequence of the VH of P1-069067 and a VL comprising the amino acid sequence of the VL of P1-069067; (r) VH comprising the amino acid sequence of VH of P1-069069 and VL comprising the amino acid sequence of VL of P1-069069; (s) a VH comprising the amino acid sequence of the VH of P1-069071 and a VL comprising the amino acid sequence of the VL of P1-069071; (t) VH comprising the amino acid sequence of VH of P1-069073 and VL comprising the amino acid sequence of VL of P1-069073; (u) VH comprising the amino acid sequence of VH of P1-069075 and VL comprising the amino acid sequence of VL of P1-069075; (v) VH comprising the amino acid sequence of VH of P1-069077 and VL comprising the amino acid sequence of VL of P1-069077; (w) VH comprising the amino acid sequence of VH of P1-068736 and VL comprising the amino acid sequence of VL of P1-068736; (x) VH comprising the amino acid sequence of VH of P1-068738 and VL comprising the amino acid sequence of VL of P1-068738; (y) VH comprising the amino acid sequence of VH of P1-068740 and VL comprising the amino acid sequence of VL of P1-068740; (z) VH comprising the amino acid sequence of VH of P1-068742 and VL comprising the amino acid sequence of VL of P1-068742; (aa) VH comprising the amino acid sequence of VH of P1-068744 and VL comprising the amino acid sequence of VL of P1-068744; (bb) VH comprising the amino acid sequence of VH of P1-068746 and VL comprising the amino acid sequence of VL of P1-068746; (cc) VH comprising the amino acid sequence of VH of P1-068748 and VL comprising the amino acid sequence of VL of P1-068748; (dd) VH comprising the amino acid sequence of VH of P1-068750 and VL comprising the amino acid sequence of VL of P1-068750; (ee) VH comprising the amino acid sequence of VH of P1-068752 and VL comprising the amino acid sequence of VL of P1-068752; (ff) VH comprising the amino acid sequence of VH of P1-068754 and VL comprising the amino acid sequence of VL of P1-068754; (gg) VH comprising the amino acid sequence of VH of P1-068761_E55A and VL comprising the amino acid sequence of VL of P1-068761_E55A; (hh) VH comprising the amino acid sequence of VH of P1-068761_H100G and VL comprising the amino acid sequence of VL of P1-068761_H100G; (ii) VH comprising the amino acid sequence of VH of P1-068761_E56N and VL comprising the amino acid sequence of VL of P1-068761_E56N; (jj) VH comprising the amino acid sequence of VH of P1-068761_E55A_E56N and VL comprising the amino acid sequence of VL of P1-068761_E55A_E56N; (kk) VH comprising the amino acid sequence of VH of P1-068761_E30D and VL comprising the amino acid sequence of VL of P1-068761_E30D; (ll) VH comprising the amino acid sequence of VH of P1-068761_E30D_E55A and VL comprising the amino acid sequence of VL of P1-068761_E30D_E55A; (mm) VH comprising the amino acid sequence of VH of P1-068761_E56N_H100G and VL comprising the amino acid sequence of VL of P1-068761_E56N_H100G; (nn) VH comprising the amino acid sequence of VH of P1-068761_E30D_H100G and VL comprising the amino acid sequence of VL of P1-068761_E30D_H100G; (oo) VH comprising the amino acid sequence of VH of P1-068761_E30D_E56N and VL comprising the amino acid sequence of VL of P1-068761_E30D_E56N; (pp) VH comprising the amino acid sequence of VH of P1-068761_E100fF and VL comprising the amino acid sequence of VL of P1-068761_E100fF; (qq) VH comprising the amino acid sequence of VH of P1-068761_E55A_E100fF and VL comprising the amino acid sequence of VL of P1-068761_E55A_E100fF; (rr) VH comprising the amino acid sequence of VH of P1-068761_H100G_E100fF and VL comprising the amino acid sequence of VL of P1-068761_H100G_E100fF; (ss) VH comprising the amino acid sequence of VH of P1-068761_E30D_E100fF and VL comprising the amino acid sequence of VL of P1-068761_E30D_E100fF; (tt) VH comprising the amino acid sequence of VH of P1-068761_E56N_E100fF and VL comprising the amino acid sequence of VL of P1-068761_E56N_E100fF; (uu) VH comprising the amino acid sequence of VH of P1-068761_E32Y and VL comprising the amino acid sequence of VL of P1-068761_E32Y; (vv) VH comprising the amino acid sequence of VH of P1-068761_E32Y_E55A and VL comprising the amino acid sequence of VL of P1-068761_E32Y_E55A; (ww) VH comprising the amino acid sequence of VH of P1-068761_E32Y_E56N and VL comprising the amino acid sequence of VL of P1-068761_E32Y_E56N; (xx) VH comprising the amino acid sequence of VH of P1-068761_E30D_E32Y and VL comprising the amino acid sequence of VL of P1-068761_E30D_E32Y; (yy) VH comprising the amino acid sequence of VH of P1-068761_E32Y_H100G and VL comprising the amino acid sequence of VL of P1-068761_E32Y_H100G; (zz) VH comprising the amino acid sequence of VH of P1-068761_E32Y_E100fF and VL comprising the amino acid sequence of VL of P1-068761_E32Y_E100fF; (aaa) VH comprising the amino acid sequence of VH of P1-068767_D52N_D102V and VL comprising the amino acid sequence of VL of P1-068767_D52N_D102V; (bbb) VH comprising the amino acid sequence of VH of P1-068767_D52N and VL comprising the amino acid sequence of VL of P1-068767_D52N; (ccc) VH comprising the amino acid sequence of VH of P1-068767_D52N_E55A and VL comprising the amino acid sequence of VL of P1-068767_D52N_E55A; (ddd) VH comprising the amino acid sequence of VH of P1-068767_E55A_D102V and VL comprising the amino acid sequence of VL of P1-068767_E55A_D102V; (eee) VH comprising the amino acid sequence of VH of P1-068767_D102V and VL comprising the amino acid sequence of VL of P1-068767_D102V; (fff) VH comprising the amino acid sequence of VH of P1-068767_E55A and VL comprising the amino acid sequence of VL of P1-068767_E55A; (ggg) VH comprising the amino acid sequence of VH of P1-068767_E30D_D52N and VL comprising the amino acid sequence of VL of P1-068767_E30D_D52N; (hhh) VH comprising the amino acid sequence of VH of P1-068767_E30D_D102V and VL comprising the amino acid sequence of VL of P1-068767_E30D_D102V; (iii) VH comprising the amino acid sequence of VH of P1-068767_E30D and VL comprising the amino acid sequence of VL of P1-068767_E30D; (jjj) VH comprising the amino acid sequence of VH of P1-068767_E30D_E55A and VL comprising the amino acid sequence of VL of P1-068767_E30D_E55A; (kkk) VH comprising the amino acid sequence of VH of P1-068767_E100fF_D102V and VL comprising the amino acid sequence of VL of P1-068767_E100fF_D102V; (lll) VH comprising the amino acid sequence of VH of P1-068767_E55A_E100fF and VL comprising the amino acid sequence of VL of P1-068767_E55A_E100fF; (mmm) VH comprising the amino acid sequence of VH of P1-068767_D52N_E100fF and VL comprising the amino acid sequence of VL of P1-068767_D52N_E100fF; (nnn) VH comprising the amino acid sequence of VH of P1-068767_E100fF and VL comprising the amino acid sequence of VL of P1-068767_E100fF; (ooo) VH comprising the amino acid sequence of VH of P1-068767_E30D_E100fF and VL comprising the amino acid sequence of VL of P1-068767_E30D_E100fF; (ppp) VH comprising the amino acid sequence of VH of P1-061029_F100fE_V102D and VL comprising the amino acid sequence of VL of P1-061029_F100fE_V102D; (qqq) VH comprising the amino acid sequence of VH of P1-061029_F100fE and VL comprising the amino acid sequence of VL of P1-061029_F100fE; (rrr) VH comprising the amino acid sequence of VH of P1-061029_V102D and VL comprising the amino acid sequence of VL of P1-061029_V102D; (sss) VH comprising the amino acid sequence of VH of P1-061029_Y32E and VL comprising the amino acid sequence of VL of P1-061029_Y32E; (ttt) VH comprising the amino acid sequence of VH of P1-061029_Y32E_F100fE and VL comprising the amino acid sequence of VL of P1-061029_Y32E_F100fE; (uuu) VH comprising the amino acid sequence of VH of P1-068744_E31S and VL comprising VL of P1-068744_E31S; (vvv) VH comprising the amino acid sequence of VH of P1-068744_H50I and VL comprising VL of P1-068744_H50I; (www) VH comprising the amino acid sequence of VH of P1-068744_E59Y and VL comprising VL of P1-068744_E59Y; (xxx) VH comprising the amino acid sequence of VH of P1-068744_E100S and VL comprising VL of P1-068744_E100S; (yyy) VH comprising the amino acid sequence of VH of P1-068744_E102Y and VL comprising VL of P1-068744_E102Y; (zzz) VH comprising the amino acid sequence of VH of P1-068744_E31S_H50I and VL comprising VL of P1-068744_E31S_H50I; (aaaa) VH comprising the amino acid sequence of VH of P1-068744_H50I_E59Y and VL comprising VL of P1-068744_H50I_E59Y; (bbbb) VH comprising the amino acid sequence of VH of P1-068744_E59Y_E100S and VL comprising VL of P1-068744_E5;9Y_E100S; (cccc) VH comprising the amino acid sequence of VH of P1-068744_E100S_E102Y and VL comprising VL of P1-068744_E100S_E102Y; (dddd) VH comprising the amino acid sequence of VH of P1-068744_E31S_E102Y and VL comprising VL of P1-068744_E31S_E102Y; (eeee) VH comprising the amino acid sequence of VH of P1-068744_E31S_E59Y and VL comprising VL of P1-068744_E31S_E59Y; (ffff) VH comprising the amino acid sequence of VH of P1-068744_E31S_E100S and VL comprising VL of P1-068744_E31S_E100S; (gggg) VH comprising the amino acid sequence of VH of P1-068744_H50I_E100S and VL comprising VL of P1-068744_H50I_E100S; (hhhh) VH comprising the amino acid sequence of VH of P1-068744_H50I_E102Y and VL comprising VL of P1-068744_H50I_E102Y; (iiii) VH comprising the amino acid sequence of VH of P1-068744_E59Y_E102Y and VL comprising VL of P1-068744_E59Y_E102Y; (jjjj) VH comprising the amino acid sequence of VH of P1-068748_H31S and VL comprising VL of P1-068748_H31S; (kkkk) VH comprising the amino acid sequence of VH of P1-068748_H32Y and VL comprising VL of P1-068748_H32Y; (llll) VH comprising the amino acid sequence of VH of P1-068748_D57K and VL comprising VL of P1-068748_D57K; (mmmm) VH comprising the amino acid sequence of VH of P1-068748_D58Y and VL comprising VL of P1-068748_D58Y; (nnnn) VH comprising the amino acid sequence of VH of P1-068748_D100S and VL comprising VL of P1-068748_D100S; (oooo) VH comprising the amino acid sequence of VH of P1-068748_H31S_H32Y and VL comprising VL of P1-068748_H31S_H32Y; (pppp) VH comprising the amino acid sequence of VH of P1-068748_H32Y_D57K and VL comprising VL of P1-068748_H32Y_D57K; (qqqq) VH comprising the amino acid sequence of VH of P1-068748_D57K_D58Y and VL comprising VL of P1-068748_D57K_D58Y; (rrrr) VH comprising the amino acid sequence of VH of P1-068748_D58Y_D100S and VL comprising VL of P1-068748_D58Y_D100S; (ssss) VH comprising the amino acid sequence of VH of P1-068748_H31S_D57K and VL comprising VL of P1-068748_H31S_D57K; (tttt) VH comprising the amino acid sequence of VH of P1-068748_H31S_D58Y and VL comprising VL of P1-068748_H31S_D58Y; (uuuu) VH comprising the amino acid sequence of VH of P1-068748_H31S_D100S and VL comprising VL of P1-068748_H31S_D100S, (vvvv) VH comprising the amino acid sequence of VH of P1-068748_H32Y_D58Y and VL comprising VL of P1-068748_H32Y_D58Y; (www) VH comprising the amino acid sequence of VH of P1-068748_H32Y_D100S and VL comprising VL of P1-068748_H32Y_D100S, or (xxxx) may comprise a VH comprising the amino acid sequence of the VH of P1-068748_D57K_D100S and a VL comprising the VL of P1-068748_D57K_D100S, wherein the VH of (a) or (c) to (ttt) may comprise one or both of a K16R and a T84A substitution, and the VL of (b) or (uuu) to (xxxx) may comprise a T85V substitution.

[0104] Anti-hVISTA Ab (a) a VH comprising the amino acid sequence of the VH of P1-061029 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-061029; (b) a VH comprising the amino acid sequence of the VH of P1-068757 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068757; (c) a VH comprising the amino acid sequence of the VH of P1-068759 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068759; (d) a VH comprising the amino acid sequence of the VH of P1-068761 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761; (e) a VH comprising the amino acid sequence of the VH of P1-068763 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068763; (f) a VH comprising the amino acid sequence of the VH of P1-068765 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068765; (g) a VH comprising the amino acid sequence of the VH of P1-068767 modified by a K16R and / or T84A substitution and a VL comprising the amino acid sequence of the VL of P1-068767; (h) a VH comprising the amino acid sequence of the VH of P1-068769 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068769; (i) a VH comprising the amino acid sequence of the VH of P1-068771 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068771; (j) a VH comprising the amino acid sequence of the VH of P1-068773 modified by K16R and / or T84A substitutions and a VL comprising the amino acid sequence of the VL of -068773; (k) a VH comprising the amino acid sequence of the VH of P1-068775 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068775; (l) a VH comprising the amino acid sequence of the VH of P1-069059 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069059; (m) a VH comprising the amino acid sequence of the VH of P1-069061 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069061; (n) a VH comprising the amino acid sequence of the VH of P1-069063 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069063; (o) a VH comprising the amino acid sequence of the VH of P1-069065 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069065; (p) a VH comprising the amino acid sequence of the VH of P1-069067 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069067; (q) a VH comprising the amino acid sequence of the VH of P1-069069 modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069069; (r) a VH comprising the amino acid sequence of the VH of P1-069071 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069071; (s) a VH comprising the amino acid sequence of the VH of P1-069073 modified by K16R and / or T84A substitutions; and a VL comprising the amino acid sequence of the VL of P1-069073; (t) a VH comprising the amino acid sequence of the VH of P1-069075 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069075; (u) a VH comprising the amino acid sequence of the VH of P1-069077 modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-069077; (v) a VH comprising the amino acid sequence of the VH of P1-068761_E55A modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E55A; (w) a VH comprising the amino acid sequence of the VH of P1-068761_H100G modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_H100G; (x) a VH comprising the amino acid sequence of the VH of P1-068761_E56N modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E56N; (y) a VH comprising the amino acid sequence of the VH of P1-068761_E55A_E56N modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E55A_E56N; (z) a VH comprising the amino acid sequence of the VH of P1-068761_E30D modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E30D; (aa) a VH comprising the amino acid sequence of the VH of P1-068761_E30D_E55A modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E30D_E55A; (bb) a VH comprising the amino acid sequence of the VH of P1-068761_E56N_H100G modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E56N_H100G; (cc) a VH comprising the amino acid sequence of the VH of P1-068761_E30D_H100G modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E30D_H100G; (dd) a VH comprising the amino acid sequence of the VH of P1-068761_E30D_E56N modified by a K16R and / or a T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E30D_E56N; (ee) a VH comprising the amino acid sequence of the VH of P1-068761_E100fF modified by K16R and / or T84A substitutions, and a VL comprising the amino acid sequence of the VL of P1-068761_E100fF; (ff) a VH comprising the amino acid sequence of the VH of P1-068761_E55A_E100fF modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_E55A_E100fF; (gg) a VH comprising the amino acid sequence of the VH of P1-068761_H100G_E100fF modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of P1-068761_H100G_E100fF; (hh) a VH comprising the amino acid sequence of the VH of P1-068761_E30D_E100fF modified by a K16R and / or T84A substitution, and a VL comprising the amino acid sequence of the VL of...

Claims

1. 1. An isolated antibody that specifically binds to human VISTA (hVISTA), wherein the antibody has been engineered by replacing one to five glutamic acid (E), aspartic acid (D), or histidine (H) amino acids in the VH CDR of antibody P1-068744 or antibody P1-068748 with amino acid residues found at the same positions in antibody P1-061015, and wherein the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of the isolated antibody considered as a whole differ from those of antibody P1-061015.

2. 2. The isolated antibody of claim 1, comprising the VH CDR1, CDR2 and / or CDR3 of P1-061015 or a variant thereof comprising 1-3 or 1-2 amino acid differences in the VH CDR1, CDR2 and / or CDR3 compared to antibody P1-061015.

3. 3. The isolated antibody of claim 2, wherein no single VH CDR contains more than two amino acid differences compared to the corresponding VH CDR of P1-061015.

4. 4. The isolated antibody of claim 2 or 3, wherein the amino acid difference comprises a substitution of an amino acid residue in P1-061015 with a glutamic acid residue (E), an aspartic acid residue (D), or a histidine residue (H).

5. -GFTFSX １ X ２ AMH (where X １ is E, H or S, and X ２ is H or Y) -X ３ IWYDGSNX ４ X ５ X ６ ADSVKG (where X ３ is H or I, and X ４ is D or K, and X ５ is D or Y, and X ６ is E or Y) and / or -DSGFYX ７ SYYFDX ８ (where X ７ is D, E or S, and X ８ is E or Y) 5. The isolated antibody of claim 2, comprising:

6. -GFTFSX １ YAMH (where X １ is E or S; -X ２ IWYDGSNKYX ３ ADSVKG (where X ２ is H or I, and X ２ is E or Y) and / or -DSGFYX ４ SYYFDX ５ (where X ４ is E or S, and X ５ is E or Y) The antibody of any one of claims 2 to 4, comprising:

7. -GFTFSX １ X ２ AMH (where X １ is H or S, and X ２ is H or Y; -IIWYDGSNX ３ X ４ YADSVKG (where X ３ is D or K, and X ４ is D or Y), and / or -DSGFYX ５ SYYFDY (where X ５ is D or S) The antibody of any one of claims 2 to 4, comprising:

8. P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S _H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y _E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S 8. The isolated antibody of any one of claims 1 to 7, comprising a VH CDRl, CDR2 and / or CDR3 of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

9. P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S _H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y _E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S 8. The isolated antibody of any one of claims 1 to 7, comprising a VH CDR1, CDR2 and CDR3 of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

10. 10. The isolated antibody of claim 8, comprising VL CDR1, CDR2 and CDR3 of the VL of P1-061015.

11. P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S _H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068 748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S; or P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y modified by 1, 2, 3, 4 or 5 amino acid substitutions P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-06874 11. The isolated antibody of any one of claims 1 to 10, comprising a VH of P1-068748_H31S_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

12. P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S _H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E10 2Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1- P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S; or P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S_H50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y modified by 1, 2, 3, 4 or 5 amino acid substitutions in the framework regions P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-06874 11. The isolated antibody of any one of claims 1 to 10, comprising a VH of P1-068748_H31S_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

13. 13. The isolated antibody of any one of claims 1 to 12, comprising a VL comprising an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical to that of P1-061015.

14. The isolated antibody of claim 13, comprising the VL of P1-061015.

15. 14. The isolated antibody of claim 13, comprising a VL comprising the amino acid sequence of the VL of P1-061015 modified by a T85V substitution.

16. 16. The isolated antibody of any one of claims 1 to 15, comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 182, 183 or 184.

17. 16. The isolated antibody of any one of claims 1 to 15, comprising the LC of P1-061015 or the LC of P1-061015 modified by a T85V substitution.

18. 18. The isolated antibody of any one of claims 1 to 17, which binds to a histidine-rich region of hVISTA, e.g., at or near a histidine-rich β-sheet stretch.

19. 19. The isolated antibody of claim 18, which binds to a histidine-rich region of hVISTA, e.g., at or near a histidine-rich beta-sheet extension, in conditions having a pH of 6.0 to 6.

5.

20. For example, P1-061015, P1-068744, P1-068748, P1-068749 ... 68744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-068744_E31S_H 50I, P1-068744_H50I_E59Y, P1-068744_E59Y_E100S, P1-068744_E100S_E102Y, P1-068744_E31S_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H 31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748 _D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D 20. The isolated antibody of any one of claims 1 to 19, which competes or cross-competes with one or more antibodies described herein comprising a VH and VL of: P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S.

21. 21. The isolated antibody of any one of claims 1 to 20, which binds to epitope group A, for example as determined by a competitive biolayer interferometry (BLI) epitope binning assay described in the Examples.

22. 22. The isolated antibody of any one of claims 1 to 21, which does not significantly bind to hVISTA modified in that one or more of the following amino acid residues have been mutated: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127, as determined, for example, by yeast mutation analysis as described in the Examples.

23. 23. The isolated antibody (Ab) of any one of claims 1 to 22, which specifically binds to hVISTA under acidic conditions, e.g., at a pH of 6.5 (e.g., as measured by one of the assays described in the Examples), inhibits the interaction between hVISTA and (a) T cells and / or (b) PSGL-1 and / or (c) VSIG-3 (e.g., inhibits the interaction between H153 and H154 of hVISTA having SEQ ID NO: 1 and PSGL-1 tyrosines Y46 and Y48); - enhancing T cell activation, for example, by enhancing T cell proliferation, enhancing IFN-γ production from T cells and / or stimulating T cell receptor-mediated NF-kB signaling; contacts hVISTA by one or more (e.g., at least 1-3, 1-5, 1-10, 5-10, 5-15, or all) energetically important contact residues Y37, T39, R54, F62, H66, V117, I119, or S124, where the numbering is that of mature hVISTA (SEQ ID NO: 2), as determined, for example, using yeast surface display and NGS assays described in the Examples; binds to the histidine-rich β-sheet extension of hVISTA, e.g., as determined by crystallography, e.g., as described in the Examples; makes contacts (at a distance of 4.0 angstroms (Å) or less) with H121, H122 and / or H123 of mature hVISTA, e.g., by hydrogen bonds, as determined, e.g., by crystallography, e.g., as described in the Examples; Region 1 of hVISTA having SEQ ID NO: 1 may have the strongest binding to region 2 as determined by MS-HDX as described in the Examples: ５７ LGPVDKGHDVTF ６８ (SEQ ID NO: 566), Region 2: ８６ RRPIRNLTFQDL ９７ (SEQ ID NO:567) and region 3: １４８ VVEIRHHSEHRVHGAME １６５ (SEQ ID NO: 568), competes with one or more antibodies described herein for binding to hVISTA, e.g., P1-061015, P1-068744, P1-068748, P1-061029, P1-068761, P1-068767, and VISTA.4 (two-way competition); make contacts with hVISTA through at least one or more glutamic acid, aspartic acid, or histidine residues located in VH CDR1, CDR2, or CDR3; have low target-mediated drug disposition, e.g., leading to a mean residence time (MRT) of at least 100, 200, 300, 400, 500, 600, or 700 hours, as measured as described in the Examples; - in MC38 tumor-bearing hVISTA knock-in mice, preferentially accumulates in tumor tissue compared to lung, liver, and spleen; and / or - In subjects receiving the antibody, it preferentially accumulates in tumor tissue compared to the lung, liver, and spleen. Isolated antibody (Ab).

24. The K for binding to hVISTA under acidic conditions, e.g., at a pH of 6.5, is higher than that under neutral or physiological pH. Ｄ or a K that is at least 10, 100, or 1000 times lower than k Ｄ 24. The isolated antibody (Ab) of any one of claims 1 to 23, which specifically binds to hVISTA at (and / or koff) (e.g., as measured by one of the assays described in the Examples), specifically binds to hVISTA, e.g., a polypeptide comprising the histidine-rich region of the ECD or amino acid residues 35-127 of SEQ ID NO:2, at an acidic pH, e.g., pH 6.0 or pH 6.5; lacks significant binding to hVISTA, e.g., a polypeptide comprising the histidine-rich region of the ECD or amino acid residues 35-127 of SEQ ID NO:2, at physiological or neutral pH, e.g., pH 7.4 or pH 7.0; specifically binds to the histidine-rich region of cyno VISTA, e.g., the ECD, at an acidic pH, e.g., pH 6.0 or pH 6.5; lacks significant binding to cyno VISTA, e.g., the histidine-rich region of the ECD, at physiological or neutral pH, e.g., pH 7.4 or pH 7.0; has reduced binding to hVISTA ECD having a substitution at one or more of the following amino acids: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127 compared to hVISTA ECD having SEQ ID NO:2; cross-compete with P1-061015, P1-068744 and / or P1-068748 for binding to hVISTA; inhibits binding of hVISTA to VISTA-expressing human T cells (e.g., naive or activated T cells) at an acidic pH, e.g., pH 6.0 or pH 6.5; inhibits the binding of hVISTA to PSGL-1 at an acidic pH, e.g., pH 6.0 or pH 6.5 (e.g., inhibiting the interaction between H153 and H154 of hVISTA having SEQ ID NO: 1 and PSGL-1 tyrosines Y46 and Y48), wherein PSGL-1 has or does not have a sialyl Lewis X, and the tyrosine is preferably sulfotyrosine; - has a mean residence time (MRT) in cynomolgus monkeys of at least 100, 200, 300, 350, 400, 450, 500, 600, or 700 hours (e.g., at least 350 hours), measured, for example, as described in the Examples; stimulate T cell activation, for example, by enhancing T cell proliferation, enhancing IFN-γ production from T cells and / or stimulating T cell receptor-mediated NF-kB signaling; VISTA-mediated cell: inhibits cell adhesion; specifically binds to hVISTA in samples of human tumor cells or inflamed human tissues that express VISTA; contacts hVISTA through one or more (e.g., at least 1-3, 1-5, 1-10, 5-10, 5-15, or all) energetically important contact residues Y37, T39, R54, F62, H66, V117, I119, or S124, where the numbering is that of mature hVISTA (SEQ ID NO: 2), e.g., as determined using yeast surface display and NGS assays described in the Examples; Region 1 of hVISTA having SEQ ID NO: 1 may have the strongest binding to region 2 as determined by MS-HDX as described in the Examples: ５７ LGPVDKGHDVTF ６８ (SEQ ID NO: 566), Region 2: ８６ RRPIRNLTFQDL ９７ (SEQ ID NO:567) and region 3: １４８ VVEIRHHSEHRVHGAME １６５ (SEQ ID NO: 568), binds to the histidine-rich β-sheet extension of hVISTA, e.g., as determined by crystallography, e.g., as described in the Examples; makes contacts (at a distance of 4.0 angstroms (Å) or less) with H121, H122 and / or H123 of mature hVISTA, e.g., by hydrogen bonds, as determined, e.g., by crystallography, e.g., as described in the Examples; contacts hVISTA through at least one or more glutamic acid, aspartic acid, or histidine residues located in VH CDR1, CDR2, or CDR3 Isolated antibody (Ab).

25. 25. The isolated antibody of any one of claims 1 to 24, having an isoelectric point (pI) of between 6.5 and 6.8, for example as measured by icIEF.

26. 26. The isolated antibody of any one of claims 1 to 25, which is an IgG antibody.

27. 27. The isolated antibody of claim 26, which is an IgG1, IgG2 or IgG4 antibody (IgG4 optionally having S228P).

28. 28. The isolated antibody of any one of claims 1 to 27, which is an effector-less antibody, e.g., an antibody that lacks ADCC and / or CDC and / or does not significantly bind to one or more FcγRs, e.g., FcγRIII.

29. 29. The isolated antibody of claim 28, wherein the constant region comprises one to five mutations in the wild-type heavy chain constant region that reduce an effector function of the antibody and / or its ability to bind to one or more FcγRs, e.g., FcγRIII, compared to that of the corresponding wild-type heavy chain constant region.

30. 30. The isolated antibody of any one of claims 1 to 29, wherein the constant region of the antibody is IgG1.3, IgG1.1, or IgG1 with a P238K substitution (e.g., IgG1.P238K).

31. 31. The isolated antibody of any one of claims 1 to 30, which has effector function and / or binds to one or more FcγRs, e.g., FcγRIII.

32. 32. The isolated antibody of claim 31, which is defucosylated (e.g., a defucosylated IgG1 antibody).

33. 33. The isolated antibody of claim 31 or 32, wherein the constant region comprises one to five mutations that enhance the effector function of the antibody and / or its ability to bind to one or more FcγRs, e.g., FcγRIII, compared to the corresponding wild-type constant region.

34. 34. The isolated antibody of any one of claims 1 to 33, which is a full-length antibody or an antibody comprising a full-length heavy chain (with or without a C-terminal lysine) and a full-length light chain.

35. 34. The isolated antibody of any one of claims 1 to 33, which is an antigen-binding fragment of an antibody.

36. 36. The isolated antibody of any one of claims 1 to 35, which is a multimeric (e.g., dimeric or trimeric) antibody.

37. 37. The isolated antibody of any one of claims 1 to 36, which is linked (e.g., covalently) to another molecule.

38. 38. The isolated antibody of claim 37, wherein the other molecule is a label.

39. 39. The isolated antibody of claim 37 or 38, wherein the other molecule is a peptide.

40. 40. The isolated antibody of any one of claims 1 to 39, which is an antibody drug conjugate (ADC) or an activatable antibody.

41. 41. An isolated nucleic acid encoding the antibody of any one of claims 1 to 40.

42. 41. An isolated nucleic acid encoding the heavy and / or light chain of an antibody according to any one of claims 1 to 40.

43. 41. A composition comprising an isolated nucleic acid encoding a heavy chain of the antibody of any one of claims 1 to 40 and a nucleic acid encoding a light chain of the antibody.

44. 44. A cell comprising the isolated nucleic acid of any one of claims 41 to 43.

45. 45. A method for preparing an antibody, comprising culturing the cell of claim 44 under conditions in which the antibody is expressed.

46. 41. A composition comprising the isolated antibody, nucleic acid, composition or cell of any one of claims 1 to 40 and a pharmaceutically acceptable carrier.

47. 47. The composition of claim 46, comprising a second therapeutic agent.

48. 48. The composition of claim 47, wherein the second therapeutic agent is an immunostimulant or a chemotherapeutic agent.

49. 49. The composition of claim 48, wherein the second therapeutic agent is an immunostimulant that is an antagonist of an immunosuppressive molecule, e.g., PD-1 / PD-L1, CTLA-4, and LAG-3, or an agonist of an immunostimulatory molecule, e.g., GITR and OX40.

50. 41. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition or isolated antibody of any one of claims 1 to 40 that stimulates an immune response and / or is a VISTA antagonist antibody.

51. 51. The method of claim 50, wherein the subject has VISTA-positive cells, e.g., in a cancer tumor.

52. 52. The method of claim 51, wherein the VISTA-positive cells are VISTA-positive infiltrating lymphoid (e.g., T cells) or myelomonocytic cells.

53. 53. The method of any one of claims 50 to 52, wherein the subject is first tested for the presence of VISTA-positive cells in the tumor.

54. 54. The method of any one of claims 50 to 53, further comprising administering a second therapy.

55. 55. The method of claim 54, wherein the second therapy is chemotherapy, radiation therapy, surgery, or administration of a second agent.

56. 56. The method of claim 55, wherein the second therapy is a second agent, and the second agent is an immunostimulant or a chemotherapeutic agent.

57. 56. The method of claim 55, wherein the second therapeutic agent is an immunostimulant that is an antagonist of an immunosuppressive molecule, e.g., PD-1 / PD-L1, CTLA-4, and LAG-3, or an agonist of an immunostimulatory molecule, e.g., GITR and OX40.

58. 41. A method of treating an infectious disease (e.g., a viral disease) in a subject, comprising administering to the subject a therapeutically effective amount of an isolated antibody of any one of claims 1 to 40, wherein the antibody stimulates an immune response and / or is a VISTA antagonist.

59. 41. A method of treating inflammation, inflammatory conditions and autoimmune diseases, graft-versus-host disease or diseases that benefit from a reduced immune response, comprising administering to a subject a therapeutically effective amount of an isolated antibody of any one of claims 1 to 40, wherein the antibody inhibits an immune response, e.g., T cell activation, or is a VISTA agonist.

60. 60. The method of any one of claims 50 to 59, wherein the antibody that binds to hVISTA is an antibody described in any one of claims 1 to 40.

61. 1. A method for improving the anti-tumor efficacy of an antibody (Ab) that specifically binds to human VISTA extracellular domain (hVISTA-ECD), comprising: a. providing Ab P1-061015, P1-068744 or P1-068748; b. Substitution of 1 to 5 amino acid residues in the heavy or light chain of the Ab with different amino acid residues (e.g., with glutamic acid, aspartic acid, or histidine residues, or in the case of P1-06844 and P1-06848, with the corresponding residues in P1-061015), wherein the 1 to 5 amino acid residues are contact residues with hVISTA-ECD; c. Determining whether the Ab obtained in (b) has a higher affinity for hVISTA-ECD at pH 6.5 or less compared to the Ab in (a); and d. pH 6.5 or less, 10 －７ K below M Ｄ repeating steps (a)-(c) for several rounds sufficient to obtain an Ab that binds to hVISTA-ECD. A method comprising:

62. 1. A method for improving the anti-tumor efficacy of an antibody (Ab) that specifically binds to human VISTA extracellular domain (hVISTA-ECD), comprising: a. providing Ab P1-061015, P1-068744 or P1-068748; b. preparing a library of variants of the Ab of (a), each variant comprising substitution of 1 to 5 amino acid residues in the heavy or light chain of the Ab with a different amino acid residue (e.g., glutamic acid, aspartic acid, or histidine residues, or in the case of P1-06844 and P1-06848, the corresponding residues in P1-061015), wherein the 1 to 5 amino acid residues are contact residues with hVISTA-ECD; c. pH 6.5 or less, 10 －７ K below M Ｄ (b) selecting Abs from the library of mutants that bind to hVISTA-ECD; and d. Optionally, testing the antitumor efficacy of the Abs of (c) in a tumor model. A method comprising:

63. 1. A method of improving the efficacy of an antibody that binds human VISTA (hVISTA), comprising increasing the number of aspartic acid, glutamic acid and / or histidine residues in one or more VH CDRs of Ab P1-061015, P1-068744 or P1-068748 to enhance binding of the antibody to hVISTA at acidic pH.

64. 64. The method of any one of claims 61 to 63, further comprising counterselecting for antibodies that bind to VISTA at physiological pH.

65. 65. The method of any one of claims 61 to 64, further comprising selecting an antibody that is either a VISTA antagonist or a VISTA agonist.

66. 66. The method of any one of claims 61 to 65, further comprising selecting an antibody that inhibits the interaction between VISTA and a VISTA co-receptor (e.g., PSGL-1 and / or VSIG-3) and / or the interaction between VISTA and T cells or myelomonocytic cells.

67. 67. The method of any one of claims 61 to 66, further comprising selecting an antibody having one or more of the properties of antibody P1-068744 or P1-068748.

68. 1. A method for improving the anti-tumor efficacy of an antibody (Ab) that specifically binds to human VISTA extracellular domain (hVISTA-ECD), comprising: a. providing Ab P1-061015, P1-068744 or P1-068748; b. Substituting 1 to 5 amino acid residues in the heavy or light chain of the Ab, e.g., in the CDRs, with different amino acid residues, e.g., the 1 to 5 amino acid residues are contact residues with hVISTA-ECD, e.g., one or more of amino acid residues 31, 32, 50, 57, 58, 59, 100, or 102; c. determining whether the Ab obtained in (b) has a higher affinity for hVISTA-ECD at pH 6.5 than at pH 7.0 compared to the Ab in (a), and / or determining whether the Ab obtained in (b) has a similar or higher affinity at pH 6.5 compared to the antibody in (a), and / or whether it has a lower affinity at pH 7.0 compared to the antibody in (a); d. pH 6.5, 10 －７ K below M Ｄ Binds to hVISTA-ECD at pH 7.0 and 10 －６ K above M Ｄ repeating steps (a)-(c) for several rounds sufficient to obtain an Ab that binds to hVISTA-ECD. A method comprising:

69. 41. A method for detecting VISTA in a sample, comprising contacting the sample with a VISTA antibody of any one of claims 1 to 40.

70. 70. The antibody, composition or method of any one of claims 1 to 69, wherein the antibody inhibits the interaction between the hVISTA extracellular domain (ECD) and PSGL-1.

71. 71. The antibody, composition or method of any one of claims 1-70, wherein the antibody competes with an antibody described herein for binding to hVISTA ECD (e.g., using BLI as described herein).

72. The antibody, composition or method of any one of claims 1 to 71, wherein the antibody interacts with a histidine triad (H121, 122 and 123 of mature hVISTA) as determined by X-ray crystallography, e.g., as determined herein.

73. 1. A method for selectively targeting an anti-VISTA Ab to a tumor in a subject, the method comprising administering to a human having at least one tumor an Ab that specifically binds to human VISTA under acidic conditions.

74. 74. The method of claim 73, wherein the anti-VISTA Ab preferentially accumulates in tumor cells relative to bone marrow cells, relative to blood, and / or relative to liver, lung, and spleen cells.

75. 75. The method of claim 73 or 74, wherein the anti-VISTA Ab accumulates to at least a 2-fold higher level in the tumor compared to the blood 24 to 51 hours after administration of the Ab to the subject.

76. 76. The method of any one of claims 73-75, wherein the anti-VISTA Ab does not accumulate significantly more in the liver or lungs compared to the blood of the subject.

77. 77. The method of any one of claims 73-76, wherein anti-VISTA Abs have been shown to preferentially accumulate in tumors over blood in VISTA knock-in mice.

78. 78. The method of any one of claims 73-77, wherein accumulation of the anti-VISTA Ab in the tumor is sustained over time, for example, up to at least 51 hours after administration of the VISTA Ab to the subject.

79. 79. The method of any one of claims 73-78, wherein the accumulation of anti-VISTA Ab in the tumor is determined by administering to the subject Ab labeled with a PET tracer.

80. 80. The method of any one of claims 73-79, wherein the anti-VISTA Ab specifically binds to VISTA at pH 6.5 but does not significantly bind at pH 7.

0.

81. 81. The method of any one of claims 73 to 80, wherein the anti-VISTA Ab preferentially accumulates in tumor tissue of hVISTA knock-in mice bearing MC-38 tumors relative to blood and / or relative to lung, liver and spleen.

82. Anti-VISTA Ab was found in VISTA.4, P1-061015, P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1- 069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1 -068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748_ H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31 S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D10 82. The method of any one of claims 73 to 81, wherein the VH is any one of P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, and in the case of P1-061029 or its progeny, the VH may comprise one or both of a K16R and a T84A substitution, and in the case of P1-061015 or its progeny, the VL may comprise a T85V substitution.

83. The K for binding to hVISTA under acidic conditions, e.g., at a pH of 6.5, is higher than that under neutral or physiological pH, as determined by surface plasmon resonance (SPR) as described in Example 30 herein. Ｄ or a K that is at least 10, 100, or 1000 times lower than k Ｄ 40. An isolated antibody that specifically binds to human VISTA (hVISTA) at (and / or koff), wherein the anti-VISTA Ab may be (a) an antibody of any one of claims 1 to 40, or (b) an antibody selected from the group consisting of antibodies: VISTA.4, P1-061015, P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-06906 5, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-06873 6, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1 -068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748 _H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H3 1S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D 100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, wherein in the case of P1-061029 or its progeny, the VH may comprise one or both of a K16R and a T84A substitution, and in the case of P1-061015 or its progeny, the VL may comprise a T85V substitution.

84. An isolated antibody that specifically binds to human VISTA (hVISTA), wherein the antibody inhibits binding of hVISTA to cells to which hVISTA binds, e.g., cells expressing human PSGL-1, wherein binding is determined according to the protocol described in Example 31 herein, and wherein the anti-VISTA Ab can be (a) an antibody described in any one of claims 1 to 40, or (b) an antibody comprising the antibody: VISTA. 4, P1-061016, P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-06876 7, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-06906 5, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-06873 6, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1 -068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748 _H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H3 1S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58Y, P1-068748_D58Y_D 100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S, wherein in the case of P1-061029 or its progeny, the VH may comprise one or both of a K16R and a T84A substitution, and in the case of P1-061015 or its progeny, the VL may comprise a T85V substitution.

85. 85. The isolated antibody of claim 84, wherein the cells to which hVISTA binds are human leukocytes, human PBMCs, or human T cells, the cells are stimulated with anti-CD3 / CD28 bead stimulation for 72 to 96 hours, and the hVISTA in the binding protocol is a VISTA multimer or a VISTA-Fc chimeric protein.

86. 86. The isolated antibody of claim 84 or 85, which inhibits hVISTA binding to cells by at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

87. 87. The isolated antibody of any one of claims 84 to 86, which inhibits binding of hVISTA on a first cell to human VSIG-3 on a second cell, wherein the second cell is a cell that expresses human VSIG-3, and wherein binding of the first cell to the second cell is determined according to the protocol set forth in Example 31.

88. 88. The isolated antibody of claim 87, wherein the first cell is a HEK293 cell that ectopically expresses hVISTA, the second cell is a CHO cell that ectopically expresses human VSIG-3, and the hVISTA expressed by the first cell is a VISTA multimer or a VISTA-Fc chimeric protein.

89. 89. The isolated antibody of claim 87 or 88, which inhibits binding of a first cell to a second cell by at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

90. 1. An isolated antibody that specifically binds to human VISTA (hVISTA), as determined using competitive SPR epitope binning as described in Example 32 herein, comprising antibodies VISTA.4, P1-061015, P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-06906 5, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-06873 6, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754、P1-068761_E55A、P1-068761_H100G、P1-068761_E56N、P1-068761_E55A_E56N、P1-068761_E30D、P1-068761_E30D_E55A、P1-068761_E56N_H100G、P1-068761_E30D_H100G、P1-068761_E30D_E56N、P1-068761_E100fF、P1-068761_E55A_E100fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1-068761_E32Y_E56N、P1-068761_E30D_E32Y、P1-068761_E32Y_H100G、P1-068761_E32Y_E100fF、P1-068767_D52N_D102V、P1-068767_D52N、P1-068767_D52N_E55A、P1-068767_E55A_D102V、P1-068767_D102V、P1-068767_E55A、P1-068767_E30D_D52N、P1-068767_E30D_D102V、P1-068767_E30D、P1-068767_E30D_E55A、P1-068767_E100fF_D102V、P1-068767_E55A_E100fF、P1-068767_D52N_E100fF、P1-068767_E100fF、P1-068767_E30D_E100fF、P1-061029_F100fE_V102D、P1-061029_F100fE、P1-061029_V102D、P1-061029_Y32E、P1-061029_Y32E_F100fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100S、P1-068744_E102Y、P1-068744_E31S_H50I、P1-068744_H50I_E59Y、P1-068744_E59Y_E100S、P1-068744_E100S_E102Y、P1-068744_E31S_E102YP1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068 748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P an isolated antibody that blocks (i.e., unidirectional) or cross-blocks (i.e., bidirectional) the binding of any one of P1-068748_D57K_D58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S to hVISTA.

91. An isolated anti-PSGL-1 antibody that inhibits the binding of human VISTA (hVISTA) to cells, wherein the cells are cells to which hVISTA binds, e.g., cells that express human PSGL-1, and wherein binding is determined according to the protocol set forth in Example 31 herein.

92. 92. The isolated anti-PSGL-1 antibody of claim 91, wherein the cells to which hVISTA binds are human leukocytes, human PBMCs, or human T cells, the cells are stimulated with anti-CD3 / CD28 bead stimulation for 72 to 96 hours, and the hVISTA in the binding protocol is a VISTA multimer or a VISTA-Fc chimeric protein.

93. 93. The isolated anti-PSGL-1 antibody of claim 91 or 92, which inhibits hVISTA binding to cells by at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

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