Antibodies that bind to vista at acidic ph

Abstract

The present application relates to antibodies that specifically bind to V-domain immunoglobulin-containing T cell activation inhibitor (VISTA) at acidic pH and their use in cancer treatment. In some embodiments, the antibodies specifically bind to human VISTA at acidic pH, but do not significantly bind to human VISTA at neutral or physiological pH.

Description

Technical Field

[0001] This application relates to antibodies that specifically bind to T-cell activation inhibitory factor (VISTA) containing V-domain immunoglobulins at acidic pH and their use in cancer treatment.

[0002] Background Technology and Summary of the Invention

[0003] VISTA, a T-cell activation inhibitor containing the V domain Ig, is a co-inhibitory member of the B7 immune receptor family expressed by myeloid monocytes and other leukocytes. However, the mechanisms by which VISTA suppresses the immune response are poorly understood.

[0004] The inventors have discovered that, unlike other known immune receptors, VISTA selectively binds to its antireceptor and functions at acidic pH, exhibiting minimal activity at physiological pH (e.g., 7.3–7.4). Therefore, VISTA can suppress immune responses in acidic microenvironments such as tumor beds or sites of inflammation without interfering with cells circulating in the blood or residing in non-inflammatory, non-acidic tissues. Furthermore, the inventors have discovered that anti-VISTA antibodies can be engineered to selectively bind to VISTA at acidic pH, exhibiting minimal or no binding at physiological pH, thus reflecting VISTA's own acidic pH selectivity. These acidic pH-selective antibodies, compared to antibodies that bind VISTA at physiological pH, can provide properties suitable for treating diseases such as cancer.

[0005] This 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., under acidic conditions). This disclosure also relates to antibodies that specifically bind to the extracellular domain (ECD) of VISTA, such as hVISTA, at acidic pH, and have few or no binding at neutral or physiological pH. The inventors have indicated herein that the hVISTA-ECD amino acid sequence includes numerous conserved and non-conserved histidine residues, and that the frequency of histidine residues in the VISTA ECD is exceptionally high relative to other B7 family members and other immunoglobulin superfamily members. (See also...) Figure 1A and Figure 1BIn solution, the amino acid histidine has a pKa of approximately 6.5, meaning that at or below pH 6.5, histidine residues within proteins are often protonated and therefore positively charged, while at pH above 6.5, they gradually become unprotonated and neutral in charge. Tumor microenvironments and inflamed tissues are often acidic; therefore, VISTA proteins found in these microenvironments may have at least partially protonated histidine residues. As discussed herein, the inventors have hypothesized that histidine protonation can affect the conformation, surface structure, and / or charge density of VISTA, which in turn can create pH-specific or pH-selective epitopes for both receptor-ligand interactions and antibody binding. Targeting VISTA with antibodies that bind at acidic pH rather than neutral or physiological pH prevents target-mediated drug treatment via circulating and lymphoid organ-resident myeloid mononuclear cells, thereby improving antibody pKa, receptor occupancy, and activity in the tumor microenvironment. Acidic pH-selective antibodies can also improve the specificity of VISTA antibodies against intratumoral rather than circulating target cells in therapeutic modalities such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), complement-dependent cytotoxicity (CDC), and payload (antibody-drug conjugate) delivery.

[0006] This disclosure describes the following anti-hVISTA Abs: 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_E59 Y_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_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 modifications of P1-068744 or P1-068748Ab to restore at least one H, D, or E residue to the heavy chain CDR of the residue found at the same position in the parent antibody P1-061015.

[0007] More generally, this disclosure includes anti-hVISTA Ab containing a heavy chain variable region (VH), said heavy chain variable region comprising VH CDR1 containing GFTFSX1YAMH, wherein X1 is E or S (SEQ ID NO: 690); VH CDR2 containing X2IWYDGSNKYX3ADSVKG, wherein X2 is H or I, and X3 is E or Y (SEQ ID NO: 691); and / or VHCDR3 containing DSGFYX4SYYFDX5, wherein X4 is E or S, and X5 is E or Y (SEQ ID NO: 692). (See also) Figure 29CIn 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 contain VH CDR1, CDR2, and / or CDR3 of P1-068744, but with the following specified responses to the positions of P1-61015 (see Tables 11 and 27): P1-068744_E31S (i.e., containing the CDRs of P1-068744, with the exception that X1 is S), P1-68744_H50I (i.e., X2 is I), P1-68744_E59Y (i.e., X3 is Y), P1-068 744_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 naming of these antibodies used above is based on the Kabat numbering scheme, which numbers residues partly by their structural position rather than by their sequence position. Because antibodies can have CDRs of varying lengths, the numbering scheme may not necessarily correspond to the positional numbering of residues in the antibody sequence. The above substitutions at X1, X2, X3, X4, and X5 are for positions 31, 50, 60, 104, and 110 of the heavy chain sequence located in SEQ ID Nos: 95 and 103 herein, as can also be readily seen from reviewing Tables 11 and 27. For further information on antibody residue numbering formats, see, for example, M. Dondelinger et al., Front. Immunol. 9:2278 (2018).

[0008] Furthermore, this disclosure includes anti-hVISTA Ab containing a heavy chain variable region (VH), said heavy chain variable region comprising VH CDR1 containing GFTFSX1X2AMH, wherein X1 is H or S and X2 is H or Y (SEQ ID NO: 693); VH CDR2 containing IIWYDGSNX3X4YADSVKG, wherein X3 is D or K and X4 is D or Y (SEQ ID NO: 694); and / or VH CDR3 containing DSGFYX5SYYFDY, wherein X5 is D or S (SEQ ID NO: 695). (See also) Figure 29CIn Ab P1-061015, these positions are S, Y, K, Y, and S, while in P1-068748, these positions are H, H, D, D, and D. In such cases, the antibody may contain VH CDR1, CDR2, and / or CDR3 of P1-068748, but with the following specified responses to the positions of P1-61015 (see Tables 12 and 28): P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K VH CDR1, CDR2, and / or CDR3 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. Note that the naming in this document 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 sequence of SEQ ID No: 95 and 99, as can also be seen by reviewing Tables 12 and 28 and SEQ ID No: 95 and 99. Attached Figure Description

[0009] The priority claim of this application is made in a U.S. provisional application containing at least one color drawing. If the provisional application subsequently becomes publicly available, copies of the color drawing may be obtained from the United States Patent and Trademark Office upon request and payment of the necessary fees.

[0010] Figures 1A to 1C The extracellular domain of VISTA contains an exceptionally high frequency of histidine residues, many of which are conserved, and at least some of which are involved in receptor-ligand binding. Figure 1A A graph showing proteins containing immunoglobulin domains, 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 in the extracellular domain of each protein plotted on the y-axis. The size of each data point corresponds to the total number of histidine residues in the extracellular domain of each protein. Figure 1BThis displays the aligned amino acid sequences of the extracellular domains of VISTA from humans (SEQ ID NO: 165), cynomolgus monkeys (SEQ ID NO: 166), and mice (SEQ ID NO: 167). Signal peptide (Sig) and transmembrane domain (TMD) sequence positions are labeled. Conserved histidine residues across all three species are shown in bold and underlined; conserved histidine residues between humans and cynomolgus monkeys are shown only in bold. Figure 1C A model showing the three-dimensional structure of the human VISTA immunoglobulin domain. Histidine residues are depicted as sphere and stick traces.

[0011] Figures 2A to 2B This model demonstrates that histidine residues in the extracellular domain of VISTA confer reverse receptor selectivity to acidic pH rather than physiological pH. Figure 2A This shows the balance between the absence and presence of protonation of the pyrrolidone (NH) group in histidine residues. Histidine has a pKa of 6.5 in solution, indicating that histidine residues are more likely to be protonated and therefore positively charged at pH 6.5 and lower compared to higher pH levels. Figure 2B This model demonstrates that VISTA selectively binds to P-selectin glycoprotein ligand 1 (PSGL-1) or other anti-receptor and ligand (VISTA-R) at acidic pH. Therefore, antibodies that bind to the extracellular domain of VISTA at acidic pH, rather than at physiological pH, may be crucial for inhibiting or modulating VISTA activity.

[0012] Figure 3 This diagram shows the level of VISTA expression on the surface of tumor-infiltrating macrophages, dendritic cells, neutrophils, CD4+ effector T cells, CD4+ regulatory T cells, CD8+ T cells, natural killer (NK) cells, and B cells (mean fluorescence intensity (MFI) of anti-VISTA antibody staining). VISTA is expressed on many tumor-infiltrating leukocytes, particularly myeloid cells. The tumor microenvironment is often acidic, which allows VISTA to bind to its antireceptor and ligand.

[0013] Figures 4A to 4G The study showed that VISTA selectively binds to leukocytes and PSGL-1 at acidic pH, with little or no binding at neutral pH, and this binding can be blocked by anti-VISTA antibodies. Figure 4AOn the left, representative histograms of the binding of fluorescently conjugated recombinant VISTA multimers to activated human CD4+ T cells are shown. Filled histograms, from dark gray to light gray, depict binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms are labeled with their corresponding pH values. Binding of the non-VISTA control multimer at pH 6.0 is shown as an unfilled histogram. On the right, the mean MFI of binding of VISTA (circles) and control (triangles) multimers to activated human CD4+ T cells from two donors is illustrated at different pH values. Figure 4B Representative histograms showing the binding of recombinant VISTA multimer to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. Histograms filled from dark gray to light gray depict binding to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes at pH 6.0. Histograms with unfilled solid and dashed borders depict binding to total PBMC lymphocytes and monocytes, respectively, at pH 7.4. Figure 4C The diagram shows representative binding of recombinant VISTA multimers to activated human CD4+ T cells in the presence of 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 depict background signal from activated human CD4+ T cells not stained with recombinant VISTA multimers. Figure 4D Representative two-dimensional flow cytometry plots showing the binding of recombinant VISTA multimers to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (pGSD-677, American Type Culture Collection) transfected to express human PSGL-1 at pH 6.0. Multimer binding is shown in both presence and absence. Figure 4C The staining was performed using the anti-VISTA blocking antibody shown. Cells not stained with recombinant VISTA multimers are displayed as controls. PSGL-1 antibody staining is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis. Figure 4E Representative histograms showing the binding of recombinant mouse VISTA-Fc fusion protein to mouse spleen cells at pH 6.0 and pH 7.4. Filled histograms, from dark gray to light gray, depict binding to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells at pH 6.0. Unfilled histograms depict binding to total spleen cells at pH 7.4. Figure 4F and Figure 4GThe VISTA multimer was shown to bind to monocytes and neutrophils, and this binding was achieved more strongly at pH 6.0 than at pH 7.4.

[0014] Figures 5A to 5D The results showed that VISTA preferentially mediates T cell suppression and cell-cell adhesion at acidic pH, and both effects can be reversed by anti-VISTA blocking antibodies. Figure 5A Representative cell-cell junction formation is shown between 293T cells expressing hVISTA or vector control (plotted on the y-axis) and CHO cells expressing endogenous cell surface heparan sulfate on the x-axis at pH 6.0 and 7.0. Figure 5B This is a graph showing the frequency of cell conjugates formed between the aforementioned cells at pH 6.0 in the presence of anti-VISTA blocking antibodies, anti-VISTA non-blocking antibodies, or isotype-matched non-VISTA-specific control antibodies. Figure 5C A representative graph showing the luciferase activity produced by Jurkat (human T cell line) cells expressing the NFkB luciferase reporter after co-culturing 293T cells (“artificial antigen-presenting cells”) expressing hVISTA and the single-chain variable fragment of the anti-human T cell receptor agonist antibody OKT3 at various pH conditions. Anti-VISTA blocking antibody (squares) or isotype-matched non-VISTA-specific control antibody (circles) was added to the co-cultured cells. Figure 5D middle, Figure 5A The data shown are plotted as a fold increase in luciferase signal ("effect size") in the case of anti-VISTA antibody treatment relative to the control.

[0015] Figures 6A to 6G VISTA was found in intracellular endosomes, particularly Rab11+ recirculating endosomes, and could be transported and recirculated to and from the cell surface via endosome transport. Figure 6A This study shows the colocalization of VISTA, Rab5 (early endosomal marker), Rab7 (late endosomal marker), and Rab11 (recirculating endosomal marker) in 293T cells expressing human VISTA. Figure 6B This demonstrates the co-localization of VISTA and Rab11 within human monocytes. Intracellular VISTA co-localizes with Rab11+ circulating endosomes. Non-VISTA-binding control antibodies (“cAb”) of the same isotype as the VISTA antibody do not bind to monocytes in a detectable manner. Figure 6C The images show the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray), and 6. (light gray). Figure 6DThis diagram illustrates the susceptibility of VISTA-expressing acute myeloid leukemia (AML) cell lines to killing by the aforementioned anti-VISTA antibodies 1 (inverted triangle), 2 (circle), 3 (square), or non-VISTA-specific control antibodies (triangle) carrying cathepsin B-sensitive linkers and cytotoxic payloads. Cell viability (CellTiter-Glo LU) is plotted on the y-axis, and antibody concentration is plotted on the x-axis. Figure 6E Compare the hVISTA binding of anti-VISTA antibody 3 with the hVISTA binding of an engineered variant (“VISTA mAb 3c”) that does not show impaired binding at acidic pH. Figure 6F An antibody-drug conjugate assay showing the efficacy of anti-VISTA antibodies 3 (square) and 3c (rhombus). Figure 6G This diagram illustrates endosome transport, where VISTA recirculates to and from the cell surface via early endosomes and recirculating endosomes.

[0016] Figures 7A to 7F This demonstrates how to design and screen anti-VISTA antibody variant libraries to obtain acidic pH-selective antibodies. Figure 7A (SEQ ID NOs: 168-181 and 696-737, respectively) show amino acid substitutions performed in VH CDR3 of the anti-human VISTA antibody clone P1-061029 (abbreviated as '029) to create a '029 library for screening. To potentially improve binding to the histidine-rich regions of VISTA at acidic pH, the library allows substitutions with negatively charged amino acids aspartic acid and glutamic acid, as well as pH-responsive histidines. X = H, D, or E. The bracketed sequences were removed from the synthesis to avoid introducing adverse factors. A total of 647 unique sequences of P1-061029HCDR3 with 1-2 mutations were synthesized. Figure 7B This shows the procedure used for iterative screening and selection of the '029 library for acid pH-selective antibody variants. R indicates the selection round. Figure 7C Representative two-dimensional flow cytometry data are displayed, showing the variant pool after 9 rounds of selection. VISTA binding is plotted on the y-axis, and variant antibody expression is plotted on the x-axis. Binding data at various antibody concentrations and pH values ​​are shown. Figure 7D Graphs showing the binding of P1-061029 and its progeny clones to human VISTA at pH 6.0 and 7.4. Figure 7E A graph showing the dissociation rates of P1-061029 and its progeny clones with human VISTA at pH 6.0. Figure 7F Data on the SPR binding of antibodies P1-068761, P1-068767, and P1-061029 to human VISTA at pH 6.0 and pH 7.4 are shown.

[0017] Figures 8A to 8F The acid pH-selective cell binding, blocking, and effector activities of VISTA antibodies P1-068761 and P1-068767 were demonstrated. Figure 8A and Figure 8B The antibody P1-068761 (shows acidic pH-selective antibody) Figure 8A ) and P1-068767 ( Figure 8B The mean fluorescence intensity of the binding with Raji cells ectopically expressing human VISTA. Cells at approximately pH 6.0 (circles; Figure 8A The highest curve in the range, 6.1 (square; third highest curve), 6.2 (triangle; second highest curve), 6.4 (inverted triangle; fourth highest curve close to pH 6.1), 6.6 (diamond; fourth curve from the bottom), 7.0 (circle; third curve from the bottom), 7.2 (square; second curve from the bottom), and 8.1 (unfilled triangle; Figure 8A The lower part of the curve was stained. Binding was detected using fluorescently conjugated anti-human IgG secondary antibody. Figure 8C The binding of P1-068767 (circle) and isotype-matched nonspecific control antibody (triangle) to Raji cells ectopically expressing human VISTA is shown at various pH values ​​and 3125 ng / mL. The “pH50”, representing the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. Figure 8D Showing isotype-matched nonspecific control antibody (filled and unfilled circles for pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 (“Control”, see below). Figure 6C The mean fluorescence intensity (MFI) of P1-068761 (filled and unfilled squares at pH 7.0 and 6.0, respectively), P1-068767 (filled and unfilled triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and unfilled inverted triangles at pH 7.0 and 6.0, respectively) binding to human monocytes was detected by fluorescently conjugated anti-human IgG secondary antibodies. Figure 8E The results show that P1-061029 (square), P1-068761 (triangle), and P1-068767 (inverted triangle) similarly blocked the binding of recombinant VISTA multimers to activated human CD4+ T cells at pH 6.0, while the non-VISTA-specific control antibody (circle) did not block VISTA binding. Figure 8FThe efficacy of P1-068761 (triangle) and P1-068767 (inverted triangle) in mediating antibody-dependent cytotoxicity (ADCC) at physiological pH is shown. P1-061029 (square), the non-VISTA-specific positive control (antibody (circle)), and the non-VISTA-specific negative control antibody (diamond) are also shown. NK cell-specific lysis of target cells is plotted as a percentage of total target cells on the y-axis, and antibody concentration is plotted on the x-axis. Nonlinear regression is also shown.

[0018] Figure 9 This figure shows the enhanced pharmacokinetics (PK) of the acidic pH-selective anti-VISTA antibody in cynomolgus monkeys. The figure shows VISTA antibody 2 (“Control”, circled, see [reference]). Figure 6C VISTA antibody 3 (“acid pH sensitive”, square, see also) Figure 6C Serum antibody concentration over time in cynomolgus monkeys treated with P1-068767 (triangle) or P1-068767 (triangle).

[0019] Figure 10A and Figure 10B (SEQ ID NO: 67 P1-061029-pos 26-35, pos 50-66, pos 99-110) shows the binding effect of mutations in acidic pH selective anti-VISTA antibodies '761 and '767. Figure 10A This shows the kinetic binding data of the P1-068761 reversion mutant at pH 7.4, pH 6.7, and pH 6.0, as well as their positions relative to the reversion mutation of P1-068761. Figure 10B This shows the kinetic binding data of the P1-068767 reversion mutant at pH 7.4, pH 6.7, and pH 6.0, as well as their positions relative to the reversion mutation of P1-068767.

[0020] Figures 11A to 11C This shows the epitope division and localization of various anti-VISTA antibodies. Figure 11A The results show that P1-068761 and P1-068767 compete for the VISTA epitope compared to the P1-061029 and VISTA antibody controls. Figure 11B and Figure 11C Compared to the non-blocking hVISTA antibody (mAb1); Figure 11C ), as listed in Table 14 regarding the blocking hVISTA antibody ( Figure 11B Images of all epitopes of the amino acid residues. Residues 66(H) and 162(A) are indicated to represent the orientation of the molecule. Histidine residues are shown in gray, and epitope residues are shown in black.

[0021] Figures 12A to 12C The following is displayed as isoelectric focusing (icIEF) imaging data: Figure 12A P1-061029 Figure 12B P1-068761 and Figure 12C P1-068767 indicates the isoelectric point (pI main) and pI marking for the main types.

[0022] Figure 13A and Figure 13B Shows the alignment of the variable regions of the '029 and '015 offspring clones. Figure 13A (SEQ ID NO: 67) shows an alignment of the amino acid sequence of the variable region of '029 and its progeny clones. Figure 13B (SEQ ID NO: 95) shows an alignment of the amino acid sequence of the variable region of '015 and its progeny clones.

[0023] Figure 14 The alignment of the VH sequence of P1-068761 with and without K16R and T84A substitutions is shown. Double-underlined residues indicate positions 16 and 84 in the frame region, and shaded areas indicate CDRs.

[0024] Figures 15A to 15O Wild-type C57BL6 mice were implanted with MC38 tumors and treated with a non-binding isotype-matched control antibody (black square), mouse VISTA blocking antibody VISTA.10 (upward triangle), mouse PD-1 blocking antibody (square), or a combination of VISTA blocking antibody and PD-1 blocking antibody (downward triangle). All antibodies were mouse IgG1-D265A (Fc indolent) isotypes. (See also...) Figures 15A to 15D These data represent three independent experiments. Figures 15A to 15D The tumor volume is displayed over time. Each group has n = 10. “TF” indicates mice that rejected their tumors. Figure 15E and Figure 15F This shows the frequency of CD8+ T cells and CD4+ T cells within the tumor 7 days after the start of treatment. n=5 per group. Univariate ANOVA combined with Dunnett's multiple comparison showed a p-value of 0.0001. Figure 15G and Figure 15H show Figures 15A to 15D The results for a single mouse are shown in the image. Figure 15IVISTA knockout mice and wild-type littermates were implanted with MC38 tumors and treated with either a non-binding isotype-matched control antibody (upper two curves, 0 / 7 TF and 0 / 5 TF, marked with circles and downward triangles) or a mouse PD-1 blocking antibody (lower two curves, 0 / 5 TF and 5 / 8 TF, marked with squares and downward 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 next to each curve (e.g., 0 / 7 TF). These data represent two independent experiments. Error bars depict interquartile ranges. Figures 15J to 15M The results showed that MC38 was implanted into the tumor, and a non-binding isotype-matched control antibody was used. Figure 15J ), mouse PD-1 blocking antibody ( Figure 15K A combination of mouse PD-1 blocking antibody and non-pH selective human VISTA blocking antibody P1-061029 ( Figure 15L ), or a combination of mouse PD-1 blocking antibody and acidic pH selective human VISTA blocking antibody P1-068767 ( Figure 15M Tumor volume in treated human VISTA knock-in (KI) mice. All antibodies were mouse IgG1-D265A isotypes. Tumor volume over time is shown. n = 5–8 per group. These data represent an independent experiment. Figure 15N Serum antibody concentrations in human VISTA KI and wild-type littermate (WT) mice following intravenous injection of 5 mg / kg of P1-061029 (WT, down triangle; KI, square) or P1-068767 (WT, up triangle; KI, rhombus) are shown. The calculated mean serum retention time (MRT) for P1-061029 and P1-068767 in KI mice was estimated to be 4.1 and 71 hours, respectively. For each antibody, n = 4 KI mice and 1–2 WT mice. These data represent a single experiment. Figure 15O The table shows serum antibody concentrations in cynomolgus macaques following intravenous injection of 5 mg / kg VISTA.4 (circles) or P1-068767 (squares). The calculated mean serum retention time (MRT) for VISTA.4 and P1-061029 was estimated to be 7.6 h and 717 h, respectively. n = 1 macaque for each antibody. These data represent a single experiment. Error bars depict the standard error of the mean unless otherwise indicated.

[0025] Figures 16A to 16C It was shown that PD-1 ( ) was significantly reduced 7 days after the start of treatment. Figure 16A ), LAG-3 ( Figure 16B ) and TIM-3 ( Figure 16C A representative histogram of CD8+ T cell expression within the tumor. Error bars depict the standard error of the mean.

[0026] Figures 17A to 17C The results showed that VISTA binds to PSGL-1 at acidic pH, and this interaction was blocked by VISTA antibodies P1-061029, P1-068761, P1-068767 and VISTA.4. Figure 17A BLI binding sensing plots showing the binding of P-selectin-Fc and VISTA-Fc to trapped PSGL1 at pH 6.0 and pH 7.4. Figure 17B This is a histogram showing the inhibition of PSGL-1 binding to hVISTA by antibodies P1-061029, P1-068761, P1-068767, and VISTA.4. Figure 17C The data show antibody blocking of VISTA-Fc to CHO-PSGL-1 cells by VISTA.4 (upward triangle) and by the anti-PSGL-1 antibody KPL-1 (circle). These data represent two independent experiments. Error bars depict the standard error of the mean.

[0027] Figures 18A to 18E Display P1-068767 Fab and hVISTA or (in Figure 18E Images of the co-crystal structures of the non-blocking antibodies VISTA.5 and hVISTA. The VISTA IgV domain is characterized by an unusually histidine-rich extension of its central β-sheet. The VISTA IgV domain is co-crystallized with the P1-068767 antigen-binding fragment (Fab). The crystal structure of the VISTA+P1-068767 complex was determined at high resolution. Figure 18A Displaying the VISTA IgV structural domain: P1-068767Fab eutectic structure. Figure 18A Shows the overall structure of the VISTAIgV domain compounded with P1-068767 Fab (heavy chain, dark gray; light chain, light gray). Figure 18B The superposition of the VISTA and PD-L1 IgV domains is shown. The VISTA histidine residues are depicted as stick representations. Figure 18B The IgV domain of VISTA is shown to have an unusually histidine-rich β-sheet extension. Figure 18C The molecular surface (light gray electrostatic surface) of the P1-068767 epitope is shown as revealed by the crystal structure of VISTA+P1-068767. Figure 18C This shows the blocking antibody binding to the histidine-rich β-sheet extension of VISTA. Figure 18DA magnified view showing the interface between VISTA (gray striped cartoon, where epitope residues H121, H122, and H123 are depicted as sticks) and P1-068767 (depicted as an electrostatic surface, where its residues E100 and D102 are depicted as sticks). Figure 18D The acid pH selectivity of P1-068767 is demonstrated by linking VISTA histidine with an acidic residue. Figure 18E The non-blocking antibody VISTA.5 binds in a region of hVISTA that differs from the binding region of P1-068767.

[0028] Figure 19 The epitopes of VISTA.4 are shown as determined by MS-HDX (MS traces).

[0029] Figure 20 (SEQ ID NO: 738) shows based on Figure 19 The data shows the position of the VISTA.4 epitope in the amino acid sequence of hVISTA. Also... Figure 19 Residues 57-68, 86-97, and 148-165, highlighted in the text, are depicted in light gray and underlined. Figure 20 middle.

[0030] Figure 21A and Figure 21B The binding of VISTA multimers to activated human CD4+ T cells is shown at pH 6.0 in the presence of antibodies VISTA.4 (triangle), VISTA.5 (square), and non-VISTA-binding antibody (control, circle). Figure 21B Shows the blocking efficiency of each antibody relative to unblocked T cells. One-way ANOVA with Dunnett's multiple comparisons, ***, P < 0.001. These data represent more than four independent experiments. Error bars depict the standard error of the mean.

[0031] Figure 22 shows that the antibody blocking VISTA binding is functional under acidic pH. Blocking antibody VISTA.4 (square), non-blocking antibody VIST A.5 (triangle), and non-VISTA-binding (control, circle) antibody counteract the proliferation of human CD4+ T cells co-cultured with 293T cells engineered to express VISTA and TCR agonists (293T-OKT3-VISTA). Figure 22A ) and interferon-γ production ( Figure 22B The effect of ) was observed. One-way ANOVA combined with Dunnett's multiple comparisons, *, P < 0.05. These data represent more than four independent experiments.

[0032] Figure 23This shows the effect of VISTA.4 blockade on JurkatT cell activation after co-culturing with 293T-OKT3-VISTA cells at different pH levels (measured by NF-κB inhibition). These data represent a composite of three independent experiments.

[0033] Figure 24 This demonstrates the effect of pH on the inhibition of human CD4+ T cells by VISTA. Cells were stimulated with plates coated with OKT3 and VISTA-Fc at the indicated pH in the presence of VISTA.4 (upward triangle), VISTA.5 (downward triangle), or a non-VISTA-binding antibody (antibody control, square). Cells stimulated with plates coated with OKT3 and control IgG (VISTA control, black circle) or in the absence of OKT3 (no OKT3, gray diamond) are also shown. These data represent a single independent experiment.

[0034] Figures 25A to 25E The binding specificity of VISTA:PSGL-1 is determined by histidine and sulfotyrosine residues. For example... Figure 25A As shown, recombinant human PSGL-119 polymer-Fc proteins with or without sialylated Lewis X decorations (SLX+ and SLX-, respectively) were produced in cells. BLI binding values ​​for VISTA-Fc and P-selectin-Fc are shown as indicated at pH 6.0 (white) and 7.4 (black). Data represent single, independent experiments. Figure 25B As shown, the resulting human PSGL-119 polymer-Fc glycopeptide with sialylated Lewis X decoration was separated into fractions with greater than 90% tyrosine sulfate (SY-rich) and less than 1% tyrosine sulfate (SY-poor). BLI binding values ​​for VISTA-Fc and P-selectin-Fc at pH 6.0 (white) and 7.4 (black) are shown as indicated. These data represent a single independent experiment. Figures 25C to 25D The results provided produce human VISTA-Fc recombinant proteins with histidine residues at positions 153-155 intact (WT VISTA) or replaced by alanine (H2A mutant), aspartic acid (H2D mutant), or arginine (H2R mutant). Figure 25C The BLI binding values ​​show the binding of wild-type and mutant VISTA-Fc proteins to captured PSGL-1 at pH 6.0 and 7.4. These data represent a single experiment. Figure 25D The data show the binding of WT VISTA (circles), H2A mutant (squares), H2D mutant (downward triangles), and H2R mutant (gray upward triangles), as well as the control (diamonds), to CHO-PSGL-1 cells at pH 6.0. These data represent two independent experiments. Figure 25EThe computational model shows the PSGL-119 polymeric glycopeptide (top) complexed with the histidine-rich ligand interface of VISTA (gray band, bottom). VISTA residues H98, H100, H153, and H154 are labeled. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also labeled.

[0035] Figures 26A to 26F : Figure 26A The data show the BLI binding amounts of GP1BA-his (squares) and PSGL-119 polymer-Fc (circles) with the trapped VISTA-Fc at the indicated pH. These data represent one experiment. Figure 26B The histograms showing the binding of VISTA multimers to human platelets are displayed. Binding was performed at pH 7.4 and pH 6.0 in the presence of a non-VISTA binding control antibody, or at pH 6.0 in the presence of a VISTA.4 blocking antibody. Unstained platelets are also shown (gray-filled histograms). These data represent two independent experiments. Figure 26C The data show the BLI binding amounts of VSIG-3-Fc to the trapped VISTA-Fc at the indicated pH. These data represent two independent experiments. Figure 26D The BLI binding values ​​show the amount of VSIG-3-Fc bound to the captured VISTA-Fc at the indicated concentration at pH 6.0. Competition was provided by individual buffer (leftmost bar), non-binding isotype-matched control antibody, human PSGL-119 polymer-Fc, P1-061029, P1-061767, or VISTA.5 (rightmost bar). These data represent a single experiment. Figure 26E The binding of VSIG-3-Fc to activated human PBMC T cells is shown at pH 6.0 (circles) or pH 7.4 (squares). Binding of an isotype-matched control antibody at pH 6.0 (black diamonds) and pH 7.4 (grey triangles) is also shown. These data represent two independent experiments. Error bars depict the standard error of the mean. Figure 26F The BLI binding values ​​show the binding amounts of VISTA-Fc (left) and PSGL-119 polymer-Fc (right) to the trapped VISTA-Fc at the indicated pH. These data represent a single experiment.

[0036] Figures 27A to 27F : Figures 27A to 27D The images show sensory maps of antibodies VISTA.4 and P1-068767 in humans and cynomolgus monkeys at pH 7.4 (left) and pH 6.0 (right). These data represent two independent experiments. Figure 27A VISTA.4 human VISTA sensor image. Figure 27B: P1-068767's VISTA sensor image. Figure 27C VISTA.4 image of a crab-eating macaque with VISTA sensors. Figure 27D VISTA sensor image of crab-eating macaques (P1-068767). Figure 27E This shows the levels of labeled P1-061029 or P1-068767 antibodies in various organs, blood, or tumors of human VISTA knock-in mice implanted with MC38 tumors and treated with the corresponding antibodies. Figure 27F This shows tumor growth in mice treated only with P1-061029 (left, downward triangle) or only with P1-068767 (right, upward triangle). n = 16 per group. These data are composites of two independent experiments. Error bars depict the standard error of the mean.

[0037] Figure 28 The results show the effects of experiments in which human VISTA knock-in mice were implanted with MC38 tumor cells and treated with fluorescently labeled P1-061029 (left) or P1-068767 (right). The radiation efficiency (×10⁻¹⁰) in the indicated organs is shown at 51 hours post-injection. 9 These data represent a single experiment.

[0038] Figures 29A to 29C The results of the SPR assay show the binding of anti-hVISTA antibody to hVISTA. Figure 29A The results show the relative hVISTA binding (Rmax%) of the P1-068744 revertant mutant to the parental P1-061015 antibody at pH 6.0 (black bar) and pH 7.4 (gray bar) according to SPR. Figure 29B The results show the relative hVISTA binding (Rmax%) of the P1-068748 revertant mutant to the parental P1-061015 antibody at pH 6.0 (black bar) and pH 7.4 (gray bar) according to SPR. Figure 29C(Poss. 26-35, 50-66, 99-110 of SEQ ID NO: 95) shows an overview of the alignment results for the antibody heavy chain CDR. The bolded amino acid residues depicted in P1-068744 and P1-068748 are those amino acid residues that, when reverted to their corresponding residues in P1-061015, maintain pH selectivity and improve KD at pH 6.0 compared to either P1-068744 or P1-068748 antibody. The double-underlined amino acid residues are those amino acid residues that, when reverted to their corresponding residues in P1-061015, cause the loss of pH selectivity in P1-068744 or P1-068748, and are therefore important residues in these antibodies for pH-selective binding to hVISTA. The amino acid residues with serrated underlines are those that, when reverting to their corresponding residues in P1-061015, are detrimental to the kinetics and / or binding of P1-068744 or P1068748 with hVISTA, and are therefore important residues in these antibodies for binding to hVISTA. Aliases for the amino acid residues depicted in the P1-068744 and P1-068748 antibodies are shown.

[0039] Figures 30A to 30D The results are shown from a assay of the binding of anti-hVISTA antibody (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 binding percentages normalized to 100% relative to P1-061029. Figure 30A , Figure 30B , Figure 30C and Figure 30D Each bar chart displays the results for a specific group of antibodies, as shown below each bar. Detailed Implementation

[0040] definition

[0041] In this application, unless otherwise stated, the use of “or” means “and / or”. In the case of multiple dependent claims, the use of “or” is only to alternatively refer back to more than one preceding independent or dependent claim. The terms “comprising,” “including,” and “having” are used interchangeably herein. According to the invention, a “separated” molecule is a molecule that has been removed from its natural environment. Therefore, the term “separated” does not necessarily reflect the degree to which the molecule has been purified.

[0042] The term "peptide" refers to a polymer of amino acid residues and is not limited to a minimum length. A "protein" may contain one or more peptides. Such polymers of amino acid residues may contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers composed of amino acid residues. Both full-length proteins and fragments are covered by this definition. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" or "protein" respectively refers to a peptide or protein that includes modifications to its native sequence, such as deletions, additions, and substitutions (generally conserved in nature), provided that the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or due to errors in PCR amplification. A protein may contain two or more peptides.

[0043] “VISTA” is an abbreviation for T-cell activation inhibitory factor protein containing the V domain of immunoglobulin, a member of the B7 immune checkpoint regulator family. VISTA is also known as PD-1 homolog (PD1H), B7-H5, C10orf54, ESC differentiation-1 (Dies-1), platelet receptor Gi24 precursor, and death domain 1α (DD1α). The term “hVISTA” or “huVISTA” as used herein refers to the human VISTA protein. The amino acid sequence of hVISTA, including its signal peptide, is provided in SEQ ID NO: 1, and the sequence without the signal peptide is provided in SEQ ID NO: 2. (See the sequence listing below.) The extracellular domain of VISTA, or “ECD” or “VISTA-ECD”, refers to the portion of the VISTA protein located in the extracellular space, which, in the case of hVISTA, contains amino acids 1-162 of SEQ ID NO: 2. (See also...) Figure 1B The "IgV domain" portion of hVISTA contains residues 5-135 of SEQ ID NO: 2.

[0044] The term "lead peptide" or "lead sequence" refers to the sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates the secretion of the polypeptide from mammalian cells. The leader sequence can be cleaved after the polypeptide is exported from mammalian cells, resulting in the mature protein. Leader sequences can be natural or synthetic, and they can be heterologous or homologous to the protein to which they are attached.

[0045] The term "antibody" or "Ab" as used herein is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule comprising at least complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein said molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding antigens, 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 from various species such as mice, cynomolgus monkeys, etc.

[0046] The term "heavy chain" or "HC" refers to a polypeptide containing at least a heavy chain variable region and having or not having a leader sequence. In some embodiments, the heavy chain contains at least a portion of the heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide containing both a heavy chain variable region and a heavy chain constant region, having or not having a leader sequence, and having or not having a C-terminal lysine (K).

[0047] The term "heavy chain variable region" or "VH" refers to the region comprising the heavy chain complementarity-determining region (CDR) 1, framework region (FR) 2, CDR 2, FR 3, and CDR 3 of the heavy chain. In some embodiments, the heavy chain variable region also comprises at least a portion of FR 1 and / or at least a portion of FR 4. As specified below, in some embodiments, heavy chain CDR 1 comprises residues 26-35 of VH SEQ ID NO herein; heavy chain CDR 2 comprises residues 50-66 of VH SEQ ID NO herein; and heavy chain CDR 3 comprises residues 99-110 of VH SEQ ID NO herein. In other embodiments, if specified, heavy chain CDR 1 corresponds to Kabat residues 31-35; heavy chain CDR 2 corresponds to Kabat residues 50-65; and heavy chain CDR 3 corresponds to Kabat residues 95-102. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.). In some implementations, the heavy chain CDR is as specified herein, such as in the following sequence listing or Table 2.

[0048] The term "light chain" or "LC" refers to a polypeptide containing at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain contains at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide containing both a light chain variable region and a light chain constant region, with or without a leader sequence.

[0049] The term "light chain variable region" or "VL" refers to the region comprising light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also comprises FRI and / or FR4. As specified below, in some embodiments, light chain CDR1 comprises residues 24-35 of VL SEQ ID NO herein; light chain CDR2 comprises residues 51-57 of VL SEQ ID NO herein; and light chain CDR3 comprises residues 90-98 of VL SEQ ID NO herein. In other embodiments, if specified, then light chain CDR1 corresponds to Kabat residues 24 to 34; light chain CDR2 corresponds to Kabat residues 50 to 56; and light chain CDR3 corresponds to Kabat residues 89 to 97. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.). In some embodiments, light chain CDRs are as specified herein, such as in the sequence listing.

[0050] A "chimeric antibody" is an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. In some embodiments, a "chimeric antibody" is an antibody comprising at least one variable region from a first species (such as a mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as a human, cynomolgus monkey, etc.). In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all variable regions of the chimeric antibody are derived from the first species, and all constant regions of the chimeric antibody are derived from the second species.

[0051] A "humanized antibody" is an antibody in which at least one amino acid in the framework region of the non-human variable region has been replaced with a corresponding amino acid from the human variable region. In some embodiments, the humanized antibody contains at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, scFv, (Fab′)2, etc.

[0052] As used in this article, "human antibody" refers to antibodies produced in humans and in non-human animals containing human immunoglobulin genes, such as... Antibodies generated in the body, as well as selected antibodies using in vitro methods such as phage display, wherein the antibody lineage is based on human immunoglobulin sequences.

[0053] As used herein, "VISTA antibody" or "anti-VISTA antibody" 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 "huVISTA antibody" or "anti-huVISTA antibody," indicating that it specifically binds to human VISTA protein under at least some conditions, such as acidic pH. A VISTA antibody that specifically binds to the extracellular domain (ECD) of VISTA may, for example, be referred to as a "VISTA-ECD antibody."

[0054] In some implementations, the antibodies described herein may contain one or more “conserved substitutions” relative to a specific designated sequence. “Conserved amino acid substitution” as used herein refers to the substitution 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, the predicted non-essential amino acid in the antibody described herein is replaced by another amino acid residue from the same side chain family (e.g., basic, acidic, β-branched, aromatic, uncharged polar). Methods for identifying nucleotide and amino acid conserved substitutions that do not eliminate antigen binding have been 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).

[0055] In some embodiments, the antibody binds to VISTA with higher affinity at acidic pH compared to at neutral and / or physiological pH. In some embodiments, the antibody binds to VISTA with higher affinity at acidic pH and binds only negligibly or nonspecifically at neutral and / or physiological pH.

[0056] The "K" binding of antibodies to proteins such as VISTA-ECD protein D The "dissociation constant" or "dissociation constant" is a measure of the affinity or specificity of the antibody for the protein, such as the VISTA-ECD protein. Compared to a higher K...D Lower K D Indicates improved binding or affinity. K D The "dissociation rate" or kJ of antibodies and peptides 解离 Or kd and "association rate" or k 缔合 The ratio between K and ka constitutes the relationship. Dissociation rate and association rate are the rates at which two bound pairs in the system associate and dissociate. Therefore, when the association rate remains approximately constant, a slower dissociation rate leads to a higher overall affinity, and thus a lower K. D As used in this article, k has a specific value "or less". 解离 Indicator k 解离 The "dissociation rate" may be as specified or slower than the specified rate.

[0057] The term "specific binding" or "specifically binding" or similar terms refers to the binding of two polypeptides, such as an antibody and its polypeptide target, to K. D It is less than the case where two random polypeptides would exist under the same conditions. In other words, K D The smaller value is attributed to the non-specific aggregation of peptides in the system. D .

[0058] In some implementations, the antibody specifically binds to the VISTA-ECD protein at a specific pH or pH range. "Acidic" pH as used herein generally refers to a pH less than 7.0, "alkaline" pH generally refers to a pH greater than 7.0, and "neutral" pH generally refers to a pH of approximately 7.0. "Physiological pH" as used herein refers to pH under normal (i.e., non-cancerous) physiological conditions, such as from 7.35 to 7.45, or from 7.3 to 7.4, such as approximately 7.4. Phrases such as "binding under acidic conditions" or "binding under physiological conditions" used herein in cases involving the binding of two molecules, such as VISTA and its mate or VISTA and T cells, refer to binding at acidic pH and binding at physiological pH, respectively.

[0059] When referring to antibodies that "block the binding of a ligand (or receptor) or competitive antibody to a receptor (or ligand) alone or on a cell" or "inhibit the binding of a ligand (or receptor) or competitive 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, such as a 50% or greater overall reduction, or a 75%, 80%, 85%, 90%, 95%, or greater overall reduction. An "anti-VISTA blocking antibody" is, for example, an antibody that can block the binding of VISTA to PSGL-1 or another VISTA ligand or receptor or heparan sulfate proteoglycan under at least some conditions, such as acidic pH.

[0060] When referring to the accumulation of the antibody in a patient's tumor in relation to its accumulation in an organ or tissue, such as the blood, of the patient to which the antibody was administered, "better accumulation" or "preferred accumulation" means that the accumulation in the tumor is at least 50%, 75%, 100% (i.e., twice as much), five times or more, relative to the accumulation in an organ or tissue. For example, the antibody may accumulate at least twice as much in the subject's tumor relative to the accumulation in the blood of an acidic pH-selectively binding antibody.

[0061] As used in this article, "tumor model" refers to an in vivo preclinical assay that can be used to study the bioactivity of the VISTA-ECD antibody and includes xenograft or native mouse tumor assay systems. In some cases, tumor models can allow tracking of tumor size or growth after antibody treatment, and / or tracking of the presence of immune cells in the tumor, such as specific types of T cells or NK cells, to determine whether the antibody has triggered or enhanced an immune response.

[0062] As used herein, the term "immunostimulant" refers to a molecule that stimulates the immune system by acting as an agonist of an immunostimulatory molecule that includes a co-stimulatory molecule, or as an antagonist of an immunosuppressive molecule that includes a co-inhibitory molecule. Immunostimulatory or immunosuppressive molecules may be immune checkpoint regulators such as VISTA or another member of the B7 family, or another molecule as further described below. Immunostimulants may be biological agents such as antibodies or antibody fragments, other proteins or vaccines, or may be small molecule drugs. "Immunostimulatory molecules" include receptors or ligands that enhance, stimulate, induce, or otherwise "turn on" an immune response. As defined herein, immunostimulatory molecules include co-stimulatory molecules. "Immunosuppressive molecules" include receptors or ligands that reduce, inhibit, suppress, or otherwise "cut off" an immune response. As defined herein, immunosuppressive molecules include co-inhibitory molecules. Such immunostimulatory and immunosuppressive molecules may 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.

[0063] The "percentage of amino acid sequence identity (%)" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conservation substitutions as part of sequence identity. Alignments for the purpose of determining the percentage of amino acid sequence identity can be performed in various ways that are within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0064] The terms “trigger” or “enhancement” refer to the initiation or enhancement of any event (such as protein ligand binding), or the initiation or enhancement of any biological activity (such as an immune response) or phenotypic characteristic, or the initiation or enhancement of the occurrence, extent, or likelihood of said activity or characteristic. “Trigger” or “enhancement” is the initiation or increase of activity, function, and / or quantity relative to a reference. The trigger or enhancement need not be complete. For example, in some embodiments, “enhancement” means being able to result in an overall increase of 20% or greater. In another embodiment, “enhancement” means being able to result in an overall increase of 50% or greater. In yet another embodiment, “enhancement” means being able to result in an overall increase of 75%, 85%, 90%, 95%, or greater.

[0065] The term "inhibition" or "inhibit" more generally refers to reducing or stopping any event (such as protein ligand binding), or reducing or stopping any phenotypic trait, or reducing or stopping the occurrence, extent, or likelihood of said trait. "Reduction" or "inhibition" means a reduction, decrease, or cessation of activity, function, and / or quantity relative to a reference. Inhibition or reduction need not be complete. For example, in some embodiments, "reduction" or "inhibit" means an overall reduction of 20% or greater. In another embodiment, "reduction" or "inhibit" means an overall reduction of 50% or greater. In yet another embodiment, "reduction" or "inhibit" means an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0066] As used herein, “treatment” encompasses any application or administration of a therapeutic agent for a disease in a person, and includes suppressing the disease or its progression or one or more symptoms, suppressing or slowing the disease or its progression or one or more symptoms, halting its development, partially or completely relieving the disease or one or more symptoms, or preventing the recurrence of one or more symptoms of the disease.

[0067] The terms “subject” and “patient” are used interchangeably in this article to refer to people.

[0068] The term "effective amount" or "therapeutic effective amount" refers to the amount of a drug that is effective in treating a disease or condition in a subject, such as by partially or completely relieving one or more symptoms. In some implementations, the effective amount refers to the amount that, at the necessary dose and for the necessary duration, effectively achieves the desired therapeutic or preventative outcome.

[0069] The term "cancer" is used herein to refer to a group of cells exhibiting an abnormally high level of proliferation and growth. Cancer can be benign (also known as a benign tumor), 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 the proliferation or growth achieved by one or more cells constituting cancer, resulting in a corresponding increase in the size or extent of said cancer.

[0070] Examples of cancers to which the treatments described herein may be applicable include, but are not limited to, epithelial carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary carcinoma, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, squamous cell lung cancer, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancers (including squamous cell carcinoma of the head and neck).

[0071] Administering in combination with one or more other therapeutic agents includes simultaneous (parallel) administration and sequential (in order) administration in any order.

[0072] 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, intended for use with a therapeutic agent to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier should be non-toxic to the recipient at the dose and concentration used and compatible with other components of the formulation. A pharmaceutically acceptable carrier should be suitable for the formulation used. For example, if the therapeutic agent will be administered orally, the carrier could be a gel capsule. If the therapeutic agent will be administered subcutaneously, ideally, the carrier should be non-irritating to the skin and should not cause an injection site reaction.

[0073] "Chemotherapy agents" are compounds that can be used to treat cancer. Examples of chemotherapy agents that can be applied in the methods described herein include, but are not limited to, alkylating agents such as thiotepa and... Cyclophosphamides; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trimethylolamine; polyacetogenins (especially bullatacin and buprofen). Bullatacinone; camptothecin (including its synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, fifosfamide, and mechloretha mine), dichloromethyldiethylamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil nitrogen mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, and atrazomycin. amycin, azaserine, bleomycin, cactinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine; Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, and mycophenolic acid (Acid), nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, triamcinolone acetonide, etc. Trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folic acid.Acetaldehyde lactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, for example Pacific paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) (A cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel) (American Pharmaceutical Partners, Schaumberg, Illinois) and Doxetaxel ( -Poulenc Rorer, Antony, France); Chloranbucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; Novantrone; Teniposide; Edatraxate; Donomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including irinotecan combined with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid. (acid); capecitabine; cobretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin-based treatment regimens (FOLFOX); PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib). Inhibitors of cell proliferation such as VEGF-A and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0074] Other non-limiting exemplary chemotherapeutic agents that may be applied in the methods described herein include anti-hormonal agents for modulating or inhibiting the effects of hormones on cancer, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including...). Tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Toremifene; an aromatase inhibitor that inhibits aromatase, the enzyme that regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, amylglutathione, etc. Medroxyprogesterone acetate, eXemestane, formestanie, fadrozole Vorozole Letrozole and Anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes involved in signaling pathways in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ribozymes and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, for example vaccine, Vaccines and vaccine; rIL-2; Topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0075] "Anti-angiogenic agents" or "angiogenic inhibitors" refer to small molecular weight substances, polynucleotides (including, for example, repressive RNAs (RNAi or siRNA)), peptides, isolated proteins, recombinant proteins, antibodies, or conjugates or fusion proteins thereof that directly or indirectly inhibit angiogenesis, vascularization, or unwanted vascular permeability. It should be understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents that may be applied in the methods described herein may include antibodies or other antagonists against angiogenic agents, such as antibodies against VEGF-A (e.g., bevacizumab). ) or antibodies targeting VEGF-A receptors (such as KDR receptors or Flt-1 receptors), anti-PDGFR inhibitors such as (Imatinib Mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668) / SU11248 (sunitinib malate), AMG706, or those described, for example, in international patent application WO2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53: 217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179 (e.g., Table 3 listing anti-angiogenic therapies in the case of malignant melanoma); Ferrara and Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8: 200-206 (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

[0076] As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Therefore, a growth inhibitor that can be applied in the methods described herein can be a growth inhibitor that significantly reduces the percentage of cells in the S phase (such as cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, agents that block cell cycle progression (in situations other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincristine (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that block G1 also affect S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, dichloromethyldiethylamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., *The Molecular Basis of Cancer*, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs," written by Murakami et al. (WBSaunders, Philadelphia, 1995), for example, page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel, derived from the European yew (… Rhone-Poulenc Rorer is a paclitaxel (Pacific paclitaxel) A semi-synthetic analogue of Bristol-Myers Squibb. Pacific paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of mitosis in cells.

[0077] The term "anti-hypertrophic composition" refers to a composition containing at least one active therapeutic agent that can be used to treat cancer. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapy agents, apoptosis agents, anti-microtubule agents, and other agents used to treat 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). ), platelet-derived growth factor inhibitors (e.g.) (imatinib mesylate), COX-2 inhibitors (e.g., celecoxib), interferon, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab) ), PD-1 or PD-L1 inhibitors (e.g. 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-β, BlyS, APRIL, BCMA, CTLA4, TIM3 or one or more VEGF receptors, TRAIL / Apo2, and other bioactive agents and organic chemical agents, etc. Combinations of these are also included in this disclosure.

[0078] Antibodies that specifically bind to VISTA-ECD at acidic pH.

[0079] Because VISTA has many histidine residues in its extracellular domain (ECD), its folding and overall structure, as well as the surface available for binding to ligands such as antibodies, can differ at acidic pH compared to neutral pH, particularly around pH 6.5, where the pKa of histidine is located. Since the tumor microenvironment is typically acidic, antibodies may need to specifically bind to VISTA at acidic pH for binding to these microenvironments, where at least some surface histidine residues are more likely to be protonated.

[0080] The following sequence listing provides the amino acid sequences of human VISTA (hVISTA) with or without a signal peptide (SEQ ID NO: 1 and SEQ ID NO: 2 (mature hVISTA)). The signal peptide constitutes 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 constitutes amino acid residues 37-167 of SEQ ID NO: 1 and amino acid residues 5-135 of SEQ ID NO: 2. The stem 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; 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 residue 187 of SEQ ID NO: 1 and amino acid residue 155 of SEQ ID NO: 2 (bold and underlined) can be D or E, representing polymorphism in hVISTA. The residues are shown in bold and underlined. Therefore, SEQ ID NO: 1 and SEQ ID NO: 2 cover the two human polymorphisms at said residues. Histidine residues in the ECD of VISTA are gray-shaded.

[0081] An anti-VISTA antibody (Ab) can specifically bind to VISTA-ECD or a fragment thereof at an acidic pH, said fragment being, for example, containing the IgV domain of VISTA or a region from hVISTA containing, for example, amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 7.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 6.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 6.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 6.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 6.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 5.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.0.

[0082] Certain abs specifically bind to the VISTA-ECD protein within a pH range of 5.0-7.0. Certain abs specifically bind to the VISTA-ECD protein within a pH range of 5.0-6.5. Certain abs specifically bind to the VISTA-ECD protein within a pH range of 5.0-6.0. Certain abs specifically bind to the VISTA-ECD protein within a pH range of 5.5-7.0. Certain abs specifically bind to the VISTA-ECD protein within a pH range of 5.5-6.5. Certain abs specifically bind to the VISTA-ECD protein within a pH range of 6.0-6.5.

[0083] This article also provides information on using 10 at pH 6.5 or lower. -6 M or smaller K D An Ab binds to a VISTA-ECD protein, such as hVISTA-ECD or a fragment thereof, said fragment comprising the IgV domain of VISTA or a region from hVISTA comprising, for example, amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2. In some embodiments, the Ab is in 10 -7 M or smaller K D Combined. In some implementations, Ab is in 10 -8 M or smaller K D Combined. In some implementations, Ab is in 10 -9 M of K D Combined. In some implementations, Ab is in 10 -10 M or smaller K D Binding. For example, Ab can bind at pH 6.5 or lower at a concentration of 10. -8 M or smaller K D It binds to the VISTA-ECD protein.

[0084] This article also provides information on using 10 in a pH range of 6.0–6.5. -6 M or smaller K D Ab binds to the VISTA-ECD protein. In some embodiments, the Ab is in the form of 10... -7 M or smaller K D Combined. In some implementations, Ab is in 10 -8 M or smaller K D Combined. In some implementations, Ab is in 10 -9 M of K DCombined. In some implementations, Ab is in 10 -10 M or smaller K D Combining. For example, Ab can be used at pH 6.5 or lower, such as in the pH range of 6.0-6.5, at a concentration of 10. -7 M or smaller K D It binds to the VISTA-ECD protein. Furthermore, Ab can be applied at pH 6.5 or lower, for example, in a pH range of 6.0–6.5, at a concentration of 10. -8 M or smaller K D Binds to the VISTA-ECD protein. Ab can be used at pH 6.5 or lower, for example, in a pH range of 6.0–6.5, at a concentration of 10. -9 M or smaller K D Combined with hVISTA-ECD.

[0085] This article also provides, for example, at 25°C or 37°C, at a pH of 6.5 or lower, at 10 -5 s -1 or smaller k 解离 An Ab that specifically binds to a VISTA-ECD protein, such as hVISTA-ECD or a fragment thereof, said fragment comprising the IgV domain of VISTA or a region from hVISTA comprising, for example, amino acids 20-95, 20-70, 35-70, 35-95, 35-127, or 37-125 of SEQ ID NO: 2. In some embodiments, the Ab has a 10% binding affinity at 25°C or 37°C. -4 s -1 or smaller k 解离 In some implementations, Ab has a strength of 210 at 25°C or 37°C. -4 s -1 or smaller k 解离 In some implementations, Ab exhibits a strength of 5-10 at 25°C or 37°C. -4 s -1 or smaller k 解离 In some implementations, Ab exhibits a strength of 7-10 at 25°C or 37°C. -4 s -1 or smaller k 解离 In some implementations, Ab has a strength of 10 at 25°C or 37°C. -3 s -1 or smaller k 解离 In some implementations, Ab has a strength of 210 at 25°C or 37°C. -3 s -1 or smaller k 解离In some implementations, Ab exhibits a strength of 5-10 at 25°C or 37°C. -3 s -1 or smaller k 解离 In some implementations, Ab exhibits a strength of 7-10 at 25°C or 37°C. -3 s -1 or smaller k 解离 In some implementations, Ab has a strength of 10 at 25°C or 37°C. -2 s -1 k 解离 In some implementations, Ab has a strength of 10 at 25°C or 37°C. -1 s -1 or smaller k 解离 For example, Ab can be administered at 25°C or 37°C, at a pH of 6.5 or lower, at a concentration of 10. -3 s -1 or smaller k 解离 It specifically binds to the VISTA-ECD protein. Ab can be applied at 25°C or 37°C at a pH of 6.5 or lower, at a concentration of 10... -3 s -1 or smaller k 解离 It specifically binds to hVISTA-ECD. Furthermore, the Ab can be administered at 25°C or 37°C at a pH of 6.5 or lower, at a concentration of 10. -2 s -1 or smaller k 解离 It specifically binds to the VISTA-ECD protein.

[0086] This article provides, for example, at pH 6.5 or lower, at (i)10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller or 10 -10 M or smaller K D and (ii) such as 10 measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 4 (or 2, 5 or 710) -4 )s -1 or smaller, 10 -3 (or 2, 5 or 710) -4 )s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离An Ab binds to a VISTA-ECD protein, such as hVISTA-ECD or a fragment thereof, at a rate comprising the IgV domain of VISTA or a region from hVISTA containing, for example, amino acids 20-95, 20-70, 35-70, 35-95, 35-95, 35-127, or 37-125 of SEQ ID NO: 2. For example, the Ab can bind at a rate of 10 at pH 6.5 or lower. -7 M or smaller K D And, for example, 10 measured at 25°C or 37°C -3 s -1 or smaller k 解离 The rate at which it binds to the VISTA-ECD protein is [not specified]. Ab can bind at pH 6.5 or lower at a rate of 10 [units not specified]. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C -3 s -1 or smaller k 解离 The rate at which it binds to the VISTA-ECD protein is [not specified]. Ab can bind at pH 6.5 or lower at a rate of 10 [units not specified]. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C -2 s -1 or smaller k 解离 The rate at which it binds to the VISTA-ECD protein. For example, Ab can bind at a rate of 10 at pH 6.5 or lower. -7 M or smaller K D And, for example, 10 measured at 25°C or 37°C -3 s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -9 M or smaller K D And, for example, 10 measured at 25°C or 37°C -3 s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -9 M or smaller K D And, for example, 10 measured at 25°C or 37°C -2 s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C4 (or 2, 5 or 710) -4 )s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C -5 (or 2, 5 or 710) -5 )s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -9 M or smaller K D And, for example, 10 measured at 25°C or 37°C -4 (or 2, 5, or 7, 10) -4 )s -1 or smaller k 解离 The rate of binding to hVISTA-ECD is [missing information]. Ab can bind at pH 6.5 or lower at a rate of 10 [missing information]. -9 M or smaller K D And, for example, 10 measured at 25°C or 37°C -5 (or 2, 5 or 710) -5 )s -1 or smaller k 解离 Rate is incorporated into hVISTA-ECD.

[0087] This article provides, for example, at 25°C or 37°C, at a pH of 6.5 or lower, at 10 4 M -1 s -1 or higher k 缔合 An Ab that specifically binds to the VISTA-ECD protein. In some such embodiments, the Ab can be 10 5 M -1 s -1 or higher k 缔合 Combined. In some such implementations, Ab can be 10 6 M -1 s -1 or higher k 缔合 Combined. In some such implementations, Ab can be 10 7 M -1 s -1 or higher k 缔合 Combined. For example, Ab can be measured at pH 6.5 or lower, such as at 25°C or 37°C, with a concentration of 10. 6 M -1 s-1 or higher k 缔合 Binds to the VISTA-ECD protein. For example, Ab can be measured at pH 6.5 or lower, such as at 25°C or 37°C, with a concentration of 10. 6 M -1 s -1 or higher k 缔合 Combined with hVISTA's ECD.

[0088] This article provides, for example, at pH 6.5 or lower, at (i)10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller or 10 -10 M or smaller K D and (ii) such as 10 measured, for example, at 25°C or 37°C. 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Abs bind to the VISTA-ECD protein. For example, the Abs can bind at pH 6.5 or lower at a concentration of 10. -7 M or smaller K D And, for example, 10 measured at 25°C or 37°C 6 M -1 s -1 or higher k 缔合 The rate at which it binds to the VISTA-ECD protein. For example, Ab can bind at a rate of 10 at pH 6.5 or lower. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C 6 M -1 s -1 or higher k 缔合 The rate at which it binds to the VISTA-ECD protein. For example, Ab can bind at a rate of 10 at pH 6.5 or lower. -7 M or smaller K D And, for example, 10 measured at 25°C or 37°C 6 M -1 s -1 or higher k 缔合The rate at which it binds to hVISTA-ECD. For example, the Ab can bind at a rate of 10 at pH 6.5 or lower. -8 M or smaller K D And, for example, 10 measured at 25°C or 37°C 6 M -1 s -1 or higher k 缔合 Rate is incorporated into hVISTA-ECD.

[0089] In some implementations, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -7 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 2 10 -5 s -1 Or smaller, 5 10 -5 s -1 Or smaller, 7 10 -5 s -1 or smaller, 10 -4 s -1 or smaller, 2 10 -4 s -1 Or smaller, 5 10 -4 s -1 Or smaller, 7 10 -4 s -1 or smaller, 10 -3 s -1 Or smaller, 210 -3 s -1 Or smaller, 5 10 -3 s -1 Or smaller, 7 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 Binds to the VISTA-ECD protein. In some embodiments, the Ab can be used at pH 6.5 or lower at a concentration of 10. -9 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s-1 or smaller or 10 -1 s -1 or smaller k 解离 Binds to the VISTA-ECD protein. In some such embodiments, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -10 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 It binds to the VISTA-ECD protein.

[0090] In some implementations, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -7 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Binds to the VISTA-ECD protein. In some embodiments, the Ab can be used at pH 6.5 or lower at a concentration of 10. -8 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合Binds to the VISTA-ECD protein. In some embodiments, the Ab can be used at pH 6.5 or lower at a concentration of 10. -9 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Binds to the VISTA-ECD protein. In some such embodiments, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -10 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 It binds to the VISTA-ECD protein.

[0091] In some implementations, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -7 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 And, for example, 10 measured at 25°C or 37°C 4 M -1 s -1 or higher, 10 5 M -1s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Binds to the VISTA-ECD protein. In some embodiments, the Ab can be used at pH 6.5 or lower at a concentration of 10. -8 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 And, for example, 10 measured at 25°C or 37°C 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Binds to the VISTA-ECD protein. In some embodiments, the Ab can be used at pH 6.5 or lower at a concentration of 10. - 9 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 And, for example, 10 measured at 25°C or 37°C 4 M -1 s -1 or higher, 10 5M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 Binds to the VISTA-ECD protein. In some such embodiments, the Ab can be used at a pH of 6.5 or lower at a concentration of 10. -10 M or smaller K D and 10, as measured, for example, at 25°C or 37°C. -5 s -1 or smaller, 10 -4 s -1 or smaller, 10 -3 s -1 or smaller, 10 -2 s -1 or smaller or 10 -1 s -1 or smaller k 解离 And, for example, 10 measured at 25°C or 37°C 4 M -1 s -1 or higher, 10 5 M -1 s -1 or higher, 10 6 M -1 s -1 or higher, 10 7 M -1 s -1 or higher k 缔合 It binds to the VISTA-ECD protein.

[0092] As indicated above, in some of the above embodiments, the VISTA-ECD protein is hVISTA-ECD, or a portion of hVISTA-ECD, such as an IgV domain. In some of the above embodiments, the Ab can specifically bind to an epitope containing amino acids 20-95 of SEQ ID NO: 2. In some of the above embodiments, the Ab can specifically bind to an epitope containing amino acids 20-70 of SEQ ID NO: 2. In some of the above embodiments, the Ab can specifically bind to an epitope containing amino acids 35-95 of SEQ ID NO: 2. In some of the above embodiments, the Ab can specifically bind to an epitope containing amino acids 35-70 of SEQ ID NO: 2. In some of the above embodiments, the epitope is a three-dimensional epitope that not only contains one of the above portions of SEQ ID NO: 2 from residues 20-95, 20-70, 35-95, or 35-70, but also contains another portion of SEQ ID NO: 2, such as residues 95-105 of SEQ ID NO: 2. In some embodiments, the Ab binds to an epitope of hVISTA as described in WO2015 / 097536. For example, the Ab may compete or cross-compete with the Ab disclosed in WO2015 / 097536 for binding to hVISTA. In some embodiments, the Ab binds to a conformational epitope of human VISTA. In some embodiments, the Ab binds to a conformational epitope of human VISTA comprising, or present within, residues 103-111 and 136-146 of SEQ ID NO: 2. In some embodiments, the Ab binds to a conformational epitope of human VISTA comprising, or present within, residues 24-36, 54-65, and 100-102 of SEQ ID NO: 2. In some embodiments, the Ab binds to a conformational epitope comprising an amino acid residue in the FG ring of human VISTA. In some embodiments, Ab binds to a polypeptide containing amino acid residues 35 to 127 and / or 37-125 of SEQ ID NO: 2.In some embodiments, the antibody binds to a VISTA ECD polypeptide or a portion thereof comprising amino acid residues 350-127 of SEQ ID NO: 2, but the antibody does not bind or binds with reduced affinity to the VISTA ECD polypeptide or a portion thereof comprising amino acid substitutions, wherein the substitution (1) is a substitution of 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) is a substitution of 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 significantly the same binding characteristics) as the antibodies described herein, such as those set forth in the examples and / or claims.

[0093] Some of the above antibodies can exhibit differential binding affinity to the VISTA-ECD protein depending on pH. Some antibodies that specifically bind to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or lower, also specifically bind to the VISTA-ECD protein with similar affinity at neutral and / or alkaline pH (i.e., they are "full binders"). For example, some of these antibodies can bind to the VISTA-ECD protein with an affinity of 10 at both pH 6.5 and pH 7.0 (at a constant temperature, such as 25°C or 37°C). -7 M or smaller K D Binding to the VISTA-ECD protein, resulting in K at pH 6.5. D K at pH 7.0 D Within 1.5 times. Some of these Abs can be expressed at pH 6.5 and at pH 7.0 (at a constant temperature, such as 25°C or 37°C) at 10. -8 M or smaller K D Binding to the VISTA-ECD protein, resulting in K at pH 6.5. D K at pH 7.0 D Within 1.5 times. Some of these Abs can be expressed at pH 6.5 and at pH 7.0 (at a constant temperature, such as 25°C or 37°C) at 10. -8 M or smaller K D Binding to hVISTA-ECD, resulting in K at pH 6.5 D K at pH 7.0 D Within 1.5 times.

[0094] Certain Abs that specifically bind to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or lower, may bind to the VISTA-ECD protein with lower affinity under neutral, physiological, and / or alkaline conditions (“pH-sensitive binders” or “pH-sensitive Abs”). Some Abs that specifically bind to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or lower, may have non-significant, for example, almost undetectable binding to the VISTA-ECD protein under neutral, physiological, and / or alkaline conditions. For example, in some embodiments, the Ab may bind to the VISTA-ECD protein with a 10% affinity at pH 6.5. -8 M or smaller K D And at pH 7.0 and / or pH 7.4 with a concentration greater than 10 -8 M of K D Binds to the VISTA-ECD protein. In some such embodiments, Ab can be used at pH 6.5 at a concentration of 10... -8 M or smaller K D , and at pH 7.0 and / or pH 7.4 with a greater K than at pH 6.5. D 1.5 times K D Binds to the VISTA-ECD protein. In some embodiments, a concentration of K is provided at up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 300, 1 / 500, 1 / 1000, or 1 / 5000 compared to that at pH 7.0 and at pH 6.5. D pH-sensitive Abs that specifically bind to the VISTA-ECD protein (at a constant temperature, such as 25°C or 37°C). For example, in some cases, Abs bind at pH 6.0 with up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 300, 1 / 500, 1 / 1000, or 1 / 5000 of K relative to pH 7.0 and / or pH 7.4 or higher. D Binds to the VISTA-ECD protein (at a constant temperature, for example, 25°C or 37°C).

[0095] In some implementations, relative to k under neutral, physiological, or alkaline conditions 解离 Ab under acidic conditions with a lower k 解离 It specifically binds to the VISTA-ECD protein. In some embodiments, it provides a contrast agent compared to that at pH 7.0 and / or pH 7.4. 解离 At pH 6.5, at up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000 of k解离 Abs bound to the VISTA-ECD protein under acidic conditions, such as as measured at 25°C or 37°C. In other words, the dissociation rate is slower at acidic pH compared to neutral pH. For example, in some embodiments, such as as measured at 25°C or 37°C, the Abs at pH 6.0 show a kJ / g at up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000 relative to pH 7.0 and / or pH 7.4. 解离 Rate-specific binding to the VISTA-ECD protein. In some embodiments, a method is provided that provides a ratio compared to K at pH 7.4. 解离 At pH 6.5, at up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000 of k 解离 Abs bound to the VISTA-ECD protein, as measured, for example, at 25°C or 37°C. In some embodiments, as measured, for example, at 25°C or 37°C, relative to pH 7.4, the Abs at pH 6.0 have a kJ of up to 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000. 解离 Rate-specific binding to the VISTA-ECD protein. In some embodiments, a method is provided that provides a rate-specific binding compared to kJ at pH 7.0-7.4. 解离 At pH 6.0-6.5, the kJ values ​​are at most 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000. 解离 Abs bound to the VISTA-ECD protein, as measured, for example, at 25°C or 37°C.

[0096] In some implementations, Ab exhibits a higher kJ / kJ value under acidic conditions compared to neutral, physiological, or alkaline conditions. 缔合 It specifically binds to the VISTA-ECD protein. In some embodiments, it provides a contrast agent compared to that at pH 7.0 and / or pH 7.4. 缔合 At pH 6.5, at at least 2, 5, 10, 20, 50, 100, or 1000 times the concentration of K 缔合 Abs bound to the VISTA-ECD protein under acidic conditions, such as as measured at 25°C or 37°C. For example, in some embodiments, as measured at 25°C or 37°C, the Abs at pH 6.0 show at least 2, 5, 10, 20, 50, 100, or 1000 times the kJ of the protein compared to pH 7.0 and / or pH 7.4. 缔合It specifically binds to the VISTA-ECD protein.

[0097] In some embodiments, at least one histidine residue of Ab in SEQ ID NO: 1, such as His 98, is specifically bound to the VISTA-ECD protein at a protonated pH. In some embodiments, most histidine residues of Ab in ECD are specifically bound to the VISTA-ECD protein at a protonated pH, which is expected to be pH 6.5 or lower, for example, between pH 6.0 and pH 6.5.

[0098] This article also covers Abs that bind to the VISTA-ECD protein with higher affinity and specificity at neutral, physiological, or alkaline pH levels compared to acidic pH, provided that the binding affinity remains high at acidic pH. For example, Abs can bind with an affinity of 10 at both pH 6.5 and pH 7.0. -8 M or smaller K D It binds to the VISTA-ECD protein, but compared to at pH 6.5, Ab at pH 7.0 has at most 1 / 1.5, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 300, 1 / 500, and 1 / 1000 of K. D Combine.

[0099] This article also covers Ab, which shares one or more of the properties mentioned above in this chapter. These properties, such as a specific K... D k 解离 k 缔合 Specific epitopes should not be treated in isolation. Therefore, Ab can bind to an epitope containing one of the regions described above (SEQ ID NO: 2), and can also possess the fully binding, pH-sensitive, or pH-selective binding properties described above, such as through its K at different pH values. D k 解离 or k 缔合 One or more of its characteristics are displayed.

[0100] In any of the above embodiments, Ab may be, for example, a full-length antibody (i.e., containing 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, a scFv fragment, an Fv fragment, or Ab may be a chimeric antibody, a humanized antibody, or a human antibody, or Ab may be a bispecific or multispecific antibody.

[0101] Several different methods can be used to determine how well an Ab binds to the VISTA-ECD protein at a given pH. For example, by surface plasmon resonance (SPR), such as through... Assay. An exemplary SPR assay involves capturing one or more antibodies onto a CM4 sensor chip using an immobilization trapping reagent (e.g., using...). Anti-human Fc trapping kit, GE Healthcare catalog number BR-1008-39, or The anti-mouse trapping kit (GE Healthcare catalog number BR-1008-39) is used to determine the binding kinetics and affinity of VISTA antigen as an analyte by serially flowing it at a specific concentration in a run buffer with the desired pH. In one embodiment, VISTA is injected at two to five concentrations (e.g., 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM) in the range of 0.1 nM to 500 nM at a flow rate of 30 μL / min, with a maximum association time of four minutes and a maximum dissociation time of ten minutes. Between binding cycles, the trapping surface is regenerated according to the manufacturer's instructions for the respective trapping kit. All data are double-referenced using a reference flow cell and a blank injection. The T200 evaluation software fits data with simple 1:1 kinetics to a Langmuir combined model with mass transfer. The SPR method described in the examples can also be used.

[0102] The affinity of an Ab for the VISTA ECD peptide can be determined using cells expressing the VISTA ECD peptide, PSGL-1, or heparan sulfate on their surfaces. The method includes flow cytometry, wherein the binding of the Ab to the cell-bound VISTA-ECD is measured at a given pH, such as pH 6.5 or lower. An exemplary flow cytometry assay includes resuspending 293T cells or other cells ectopically expressing hVISTA ECD in a buffer consisting of HBSS + 1% BSA adjusted to a desired pH, such as pH 6.0 adjusted with MES or pH 7.4 adjusted with HEPES. An Ab against hVISTA (e.g., human IgG) is serially diluted from about 20 μg / mL and incubated with the resuspended cells at 4°C for 30 min. The cells are then washed twice with the same buffer to maintain the desired pH, such as 6.0 or 7.4, and incubated with a secondary antibody that recognizes a primary antibody (e.g., human IgG) and is stable at the reduced pH. Cells were then washed as described above and immediately acquired on a BD Fortessa or other flow cytometer without fixation. The affinity of Ab for the VISTA_ECD peptide was determined as described in the examples.

[0103] In some embodiments, the Ab bound to hVISTA ECD blocks the binding of hVISTA to its binding partner, such as the cellular binding pair (e.g., the VISTA receptor). Inhibition or blockage may be 100% or at least 99%, 95%, 90%, 85%, 80%, 75%, or 50%. In some embodiments, the Ab binds to the VISTA-ECD protein at an acidic pH, such as pH 6.5 or lower, and inhibits the binding of VISTA to its binding partner by at least 50%, such as at least 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 7.0 and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than 6.8 and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 6.5, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 6.3, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 6.0, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.8, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.5, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH values ​​below 5.3, and inhibits the binding of VISTA to its binding partner by at least 50%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at pH less than 5.0 and inhibits the binding of VISTA to its binding partner by at least 50%.

[0104] Certain abs specifically bind to the VISTA-ECD protein at a pH range of 5.0–7.0, inhibiting the binding of VISTA to its binding partner by at least 50%. Certain abs specifically bind to the VISTA-ECD protein at a pH range of 5.0–6.5, inhibiting the binding of VISTA to its binding partner by at least 50%. Certain abs specifically bind to the VISTA-ECD protein at a pH range of 5.0–6.0, inhibiting the binding of VISTA to its binding partner by at least 50%. Certain abs specifically bind to the VISTA-ECD protein at a pH range of 5.5–6.5, inhibiting the binding of VISTA to its binding partner by at least 50%. Certain Abs specifically bind to the VISTA-ECD protein at a pH range of 6.0–6.5, and inhibit the binding of VISTA to its binding partner by at least 50%. The inhibition of binding can be determined as described in the examples.

[0105] The VISTA binding partner can be PSGL-1, such as human PSGL-1. Sequences of human PSGL-1 isoforms are provided herein as SEQ ID NO: 3-10. VISTA binds to PSGL-1 with or without sialylated Lewis X. The binding partner can also be, for example, heparan sulfate proteoglycans present on certain cells.

[0106] Inhibition of binding to VISTA binding partners can be determined by measuring the inhibition of binding of VISTA (or VISTA ECD or VISTA IgV domain or VISTA-positive cells) to cells that bind to VISTA, such as T cells (e.g., activated or inactivated CD4+ T cells, CD8+ T cells), NK cells, or other cells that bind to VISTA, in the presence and absence of the antibody. An exemplary experiment that can be used to determine whether an antibody inhibits the binding of VISTA to its binding partners or T cells expressing the binding partners is a flow cytometry assay, for example, comprising the following assay: resuspending human peripheral blood mononuclear cells from donor blood, erythrocyte sedimentation rate (ESR), or apheresis products in a buffer of HBSS + 1% BSA adjusted to a desired pH, such as pH 6.0 adjusted with MES or pH 7.4 adjusted with HEPES. Cells were then incubated at 4°C with 20 μg / mL of a recombinant chimeric protein (VISTA-Fc) consisting of hVISTA ECD fused to human IgG1 Fc, and with different concentrations of candidate VISTA blocking antibodies or control antibodies for 30 minutes. Cells were then washed twice in the same buffer to maintain the desired pH, for example, 6.0 or 7.4, and further incubated at 4°C with a secondary antibody that recognizes VISTA-Fc but not the candidate blocking antibody or control antibody, and is stable at a lower pH for 30 minutes. Cells were then washed as described above and immediately harvested on a BD Fortessa or other flow cytometer without fixation. Inhibition of binding can be measured, for example, as described in the examples.

[0107] In specific embodiments, the Abs described herein can trigger or enhance immune responses, such as antigen-specific immune responses. In some embodiments, the Abs stimulate T cell activity, particularly at acidic pH conditions, such as those found 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 using, for example, the Jurkat assay described in the examples. Stimulation of T cell activity can also be measured by measuring IFN-γ secretion from T cells, wherein enhanced IFN-γ secretion indicates T cell stimulation. The secretion of other cytokines from activated T cells can also be measured. In some embodiments, signal transduction of activated T cells, such as NF-κB levels, is measured. In specific embodiments, the Abs described herein inhibit cell adhesion, which can be measured as described in the examples.

[0108] It can also demonstrate anti-VISTA Ab activity in monocyte assays, ADCC assays and ADCP assays, especially at acidic pH conditions such as those found in the tumor microenvironment.

[0109] In some implementations, anti-VISTA Ab inhibits tumor growth in tumor models, such as human VISTA knock-in tumor models.

[0110] As illustrated in the examples herein, recycling of anti-VISTA Abs in vivo, such as for enhancing the pharmacokinetic (PK) properties of the antibody, i.e., its half-life, requires the anti-VISTA antibody to bind to VISTA under acidic conditions. Therefore, as further described herein, anti-VISTA Abs that bind to VISTA at low pH, such as 6.5 or lower, are also expected to have a longer acceptable half-life compared to VISTA antibodies that do not bind to VISTA at acidic pH.

[0111] In some embodiments, the VISTA antibody preferably binds to one or more tumors in vivo, compared to myeloid cells. The specific anti-VISTA Ab described herein has been shown to preferentially localize to tumors compared to blood in VISTA knock-in mice. For example, in some embodiments, the antibody described herein accumulates in tumors at at least twice the level of blood, 24 to 51 hours after administration of the Ab to the subject. In some embodiments, the anti-VISTA Ab does not accumulate significantly more in the liver or lungs compared to the subject's blood. In some embodiments, the anti-VISTA Ab in tumors may be maintained over time, for example, up to at least 51 hours after administration of the VISTA Ab to the subject. In some embodiments, the localization of the anti-VISTA Ab in the subject is determined by administering the Ab labeled with a PET tracer.

[0112] Example hVISTA-ECD combined with Ab

[0113] This article provides Abs that preferentially bind to hVISTA (ECD) at acidic pH (e.g., under acidic conditions) relative to physiological or neutral pH.

[0114] This disclosure describes the following anti-hVISTA Abs: 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_E59 Y_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_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 a heavy chain CDR of P1-068744 or P1-068748Ab modified to restore at least one H, D, or E residue to the same position found in the parent antibody P1-061015. In some embodiments, the antibody may contain a light chain CDR of antibody P1-061015.

[0115] More generally, this disclosure includes anti-hVISTA Abs containing heavy chain variable regions (VHs) comprising VH CDR1 containing GFTFSX1YAMH, where X1 is E or S (SEQ ID NO: 690); VH CDR2 containing X2IWYDGSNKYX3ADSVKG, where X2 is H or I, and X3 is E or Y (SEQ ID NO: 691); and / or VHCDR3 containing DSGFYX4SYYFDX5, where 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, for example, see...) Figure 29C In such cases, the antibody may contain VH CDR1, CDR2, and / or CDR3 of P1-068744, but with the following specified responses to the position of P1-61015 (see Tables 11 and 27): P1-068744_E31S (i.e., containing CDRs of P1-068744, with the exception that X1 is S), P1-68744_H50I (i.e., X2 is I), P1-68744_E59Y (i.e., X3 is Y), P1-068 744_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 naming of these antibodies used above is based on the Kabat numbering scheme, which numbers residues partly by their structural position rather than by their sequence position. Because antibodies can have CDRs of varying lengths, the numbering scheme may not necessarily correspond to the positional numbering of residues in the antibody sequence. The above substitutions at X1, X2, X3, X4, and X5 are for positions 31, 50, 60, 104, and 110 of the heavy chain sequence located in SEQ ID Nos: 95 and 103 herein, as can also be readily seen from reviewing Tables 11 and 27. For further information on antibody residue numbering formats, see, for example, M. Dondelinger et al., Front. Immunol. 9:2278 (2018).

[0116] In some embodiments, the antibody may contain a heavy chain CDR of P1-068744 or one of the above mutants where X2 is H. In some embodiments, the antibody may contain a heavy chain CDR of P1-068744 or one of the above mutants where X2 is H and X4 is E (i.e., does not contain the H50I or E100S mutation). Examples include antibodies containing 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 also have (a) a composition of <5 × 10 -8M, <1×10 -8 M, <5×10 -9 M or <3×10 -9 M represents the affinity for hVISTA as indicated by Kd (e.g., based on surface plasmon resonance (SPR)); (b) 1 × 10 at pH 7.4 -8 and 1×10 -6 The Kd (e.g., measured by SPR); and / or (c) the antibody also has a Kd < 5 × 10⁻⁶ at pH 6.0. -3 (1 / s), <4×10 -3 (1 / s) or <3×10 -3 (1 / s) k dissociation (e.g., as measured by SPR). In other embodiments, the antibody retains H at position X2 and E at position X3. In some embodiments, the antibody retains E at both positions X3 and X4. And, in some embodiments, the antibody retains H at X2, E at X3, and E at X4, compared to P1-068744. Examples include antibodies comprising the heavy chain CDR 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 CDR of one of these antibodies and the light chain CDR of the P061015 antibody. In some embodiments, the anti-hVISTA antibody is one of those described herein, for example, that does not have other amino acid substitutions in the CDR or variable region.

[0117] Furthermore, this disclosure includes anti-hVISTA Abs containing heavy chain variable regions (VHs) comprising VH CDR1 containing GFTFSX1X2AMH, where X1 is H or S and X2 is H or Y (SEQ ID NO: 693); VH CDR2 containing IIwYDGSNX3X4YADSVKG, where X3 is D or K and X4 is D or Y (SEQ ID NO: 694); and / or VH CDR3 containing DSGFYX5SYYFDY, where 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 also...) Figure 29CIn such cases, the antibody may contain VH CDR1, CDR2, and / or CDR3 of P1-068748, but with the following designated responses to the position of P1-61015 (see Tables 12 and 28): P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K VH CDR1, CDR2, and / or CDR3 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. Note that the naming in this document 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 sequence of SEQ ID No: 95 and 99, as can also be seen by reviewing Tables 12 and 28 and SEQ ID No: 95 and 99.

[0118] In some such embodiments, the heavy chain CDR may be retained at position X5 (i.e., at amino acid sequence position 104; i.e., they do not contain the D100S substitution as described in the nomenclature above). In some cases, the antibody retains the D at position X5 and also exhibits pH-selective binding to hVISTA. In some cases, the antibody retains the D at position X4. In some cases, the antibody retains the D at both positions X4 and X5 (i.e., it does not include the D58Y or D100S substitution relative to P1-068744). In some cases, the antibody comprises a heavy chain CDR 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 a heavy chain CDR of one of these antibodies and a light chain CDR of the P061015 antibody. In some embodiments, the antibody also has (a) a composition of <1×10⁻⁶… 8 M, <7×10 -9 M or <5×10 -9M's Kd represents the affinity for hVISTA (e.g., based on surface plasmon resonance (SPR)); (b) 5 × 10 at pH 7.4 -8 and 1×10 -6 The Kd (e.g., measured by SPR); and / or (c) the antibody also has a Kd < 5 × 10⁻⁶ at pH 6.0. -3 (1 / s), <4×10 -3 (1 / s) or <3×10 -3 (1 / s) k dissociation (e.g., as measured by SPR). In some embodiments, the anti-hVISTA antibody is, for example, one described herein above, without other amino acid substitutions in the CDR or variable region.

[0119] In some embodiments, the anti-hVISTA antibody comprises VH and VL CDRs of P1-068744 or P1-068748, or comprises VH and VL of P1-068744 or P1-068748 containing amino acid reversions to those amino acids in P1-061015, wherein such amino acid reversions do not affect the desired characteristics of the antibody. For example, the anti-hVISTA antibody may comprise VH and VL or VH and VL CDRs of P1-068744 or P1-068748 with reversion mutations that do not affect pH selectivity and / or binding kinetics, as further described herein.

[0120] In some embodiments, the anti-hVISTA Ab comprises a heavy chain variable region (“VH”) containing VHCDR1, CDR2, and / or CDR3 of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH containing VH CDR1, CDR2, and CDR3 of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH containing the following: VH of CDR1, CDR2 and / or CDR3: P1-061029 or P1-061015 or their offspring, 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, P1-069063, 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_H 100G, 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_E100 fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-0687 67_E100fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061 029_F100fE, P1-061029_V102D, P1-061029_Y32E, P1-061029_Y32E_F10 0fE、P1-068744_E31S、P1-68744_H50I、P1-68744_E59Y、P1-068744_E100 S, 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_E59 Y_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_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 types contain amino acid positions 26-35 (VH CDR1) of the VH sequence of each of the above antibody types provided in the following sequence listing.50-66 (VHCDR2) and 99-110 (VH CDR3). For each of the VH sequences of the above antibody types provided in the sequence listing below, the CDR is underlined and indicated in bold.

[0121] In some embodiments, the anti-hVISTA Ab comprises a VL containing any of the anti-hVISTA Abs provided herein, such as VLCDR1, CDR2, and CDR3. In some embodiments, the anti-hVISTA... Ab contains VLs with one of the following VLCDR1, CDR2, and CDR3: 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, P1-0690 75. 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, P1-06 8761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-06876 1_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_E32 Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E 32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1-068767_E5 5A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068767_E30D_D52 N. 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_Y 32E_F100fE, P1-068744_E31S, P1-68744_H50I, P1-68744_E59Y, P1-068744_E100S, P1-068744_E102Y, P1-06874 4_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_E59 Y_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_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. Each of these types, VL CDR1, CDR2, and CDR3, comprises amino acid positions 24-35 (VL CDR1), 51-57 (VL CDR2), and 90-98 (VL CDR3) of the VL sequence for each of the above antibody types, as provided in the following sequence listing. In each of these sequences, the CDR is underlined and indicated in bold.

[0122] In some embodiments, the anti-hVISTA Ab comprises a VH containing VHCDR1, CDR2, and / or CDR3 of any anti-hVISTA Ab provided herein and a VL containing CDR1, CDR2, and / or CDR3 of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH containing VH, CDR1, CDR2, and CDR3 of any anti-hVISTA Ab provided herein and a VL containing CDR1, CDR2, and CDR3 of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH containing the following... VH of CDR1, CDR2 and / or CDR3: P1-061029 or P1-061015 or their offspring, 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, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-0 68738, P1-068740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-068754, P1-068761 _E55A, P1-068761H100G, 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_E10 0fF、P1-068761_H100G_E100fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-06876 1_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, P1-06 8767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-068767_E10 0fF_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; and VL containing the following items VL for CDR1, CDR2, and CDR3: P1-061029 or P1-061015 or their offspring, 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, P 1-069063, 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, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1 -068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068 761_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_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_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.

[0123] In some implementations, anti-hVISTA Ab may include:

[0124] (a) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-061029 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-061029;

[0125] (b) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-061015 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-061015;

[0126] (c) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068757 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068757;

[0127] (d) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068759 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068759;

[0128] (e) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068761;

[0129] (f) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068763 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068763;

[0130] (g) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-068765 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-068765;

[0131] (h) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068767 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068767;

[0132] (i) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068769 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068769;

[0133] (j) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068771 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068771;

[0134] (k) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068773 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068773;

[0135] (l) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-068775 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-068775;

[0136] (m) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069059 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069059;

[0137] (n) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069061 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069061;

[0138] (o) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069063 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069063;

[0139] (p) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069065 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069065;

[0140] (q) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069067 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069067;

[0141] (r) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069069 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069069;

[0142] (s) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-069071 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-069071;

[0143] (t) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069073 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069073;

[0144] (u) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-069075 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-069075;

[0145] (v) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-069077 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-069077;

[0146] (w) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068736 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068736;

[0147] (x) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068738 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068738;

[0148] (y) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068740 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068740;

[0149] (z) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068742 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068742;

[0150] (aa) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068744 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068744;

[0151] (bb) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-068746 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-068746;

[0152] (cc) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH containing P1-068748 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL containing P1-068748;

[0153] (dd) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-068750 and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-068750;

[0154] (ee) VH containing the amino acid sequence of VH CDR1, CDR2 and CDR3 of P1-068752 and VL containing the amino acid sequence of VL CDR1, CDR2 and CDR3 of P1-068752;

[0155] (ff) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068754 and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068754.

[0156] (gg) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH containing P1-068761_E55A and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068761_E55A;

[0157] (hh) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_H100G and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_H100G;

[0158] (ii) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761_E56N and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068761_E56N;

[0159] (jj) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068761_E55A_E56N and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068761_E55A_E56N;

[0160] (kk) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of VH with P1-068761_E30D and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of VL with P1-068761_E30D:

[0161] (ll) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761_E30D_E55A and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068761_E30D_E55A;

[0162] (mm) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068761_E56N_H100G and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068761_E56N_H100G;

[0163] (nn) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761_E30D_H100G and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068761_E30D_H100G;

[0164] (oo) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761_E30D_E56N and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068761_E30D_E56N;

[0165] (pp) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068761_E100fF and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068761_E100fF;

[0166] (qq) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E55A_E100fF;

[0167] (rr) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_H100G_E100fF;

[0168] (ss) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E30D_E100fF;

[0169] (tt) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E56N_E100fF;

[0170] (uu) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y;

[0171] (vv) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y_E55A and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y_E55A;

[0172] (ww) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y_E56N;

[0173] (xx) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068761_E30D_E32Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068761_E30D_E32Y;

[0174] (yy) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068761_E32Y_H100G and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068761_E32Y_H100G;

[0175] (zz) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068761_E32Y_E100fF;

[0176] (aaa) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_D52N_D102V;

[0177] (bbb) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068767_D52N and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068767_D52N;

[0178] (ccc) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_D52N_E55A;

[0179] (ddd) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E55A_D102V;

[0180] (eee) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068767_D102V;

[0181] (fff) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068767_E55A and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068767_E55A;

[0182] (ggg) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068767_E30D_D52N and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068767_E30D_D52N;

[0183] (hhh) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E30D_D102V;

[0184] (iii) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068767_E30D and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL containing P1-068767_E30D;

[0185] (jjj) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068767_E30D_E55A and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068767_E30D_E55A;

[0186] (kkk) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E100fF_D102V and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E100fF_D102V;

[0187] (lll) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E55A_E100fF;

[0188] (mmm) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_D52N_E100fF;

[0189] (nnn) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068767_E100fF and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068767_E100fF;

[0190] (ooo) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068767_E30D_E100fF;

[0191] (ppp) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-061029_F100fE_V102D;

[0192] (qqq) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-061029_F100fE and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-061029_F100fE;

[0193] (rrr) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of P1-061029_V102D:

[0194] (sss)VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH containing P1-061029_Y32E and VL containing CDR1, CDR2 and CDR3 of VL containing P1-061029_Y32E;

[0195] (ttt)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-061029_Y32E_F100fE;

[0196] (uuu) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068744_E31S and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068744_E31S;

[0197] (vvv) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I;

[0198] (www) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E59Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E59Y;

[0199] (xxx) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068744_E100S and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068744_E100S;

[0200] (yyy) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E102Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E102Y;

[0201] (zzz) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068744_E31S_H50I and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068744_E31S_H50I;

[0202] (aaaa) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I_E59Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I_E59Y;

[0203] (bbbb)VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068744_E59Y_E100S and VL containing CDR1, CDR2 and CDR3 of P1-068744_E59Y_E100S;

[0204] (cccc) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E100S_E102Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E100S_E102Y;

[0205] (dddd)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E31S_E102Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E31S_E102Y;

[0206] (eeee) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH with P1-068744_E31S_E59Y and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of VL with P1-068744_E31S_E59Y;

[0207] (ffff) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E31S_E100S and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_E31S_E100S;

[0208] (gggg) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I_E100S and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I_E100S;

[0209] (hhhh) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068744_H50I_E102Y;

[0210] (iiii) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068744_E59Y_E102Y and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068744_E59Y_E102Y;

[0211] (jjjj) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H31S and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H31S;

[0212] (kkkk)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H32Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H32Y;

[0213] (llll) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068748_D57K and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068748_D57K;

[0214] (mmmm) VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068748_D58Y and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068748_D58Y;

[0215] (nnnn) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D100S and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D100S;

[0216] (oooo) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH with P1-068748_H31S_H32Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of VL with P1-068748_H31S_H32Y;

[0217] (pppp) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH containing P1-068748_H32Y_D57K and VL containing CDR1, CDR2 and CDR3 of VL containing P1-068748_H32Y_D57K;

[0218] (qqqq) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D57K_D58Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D57K_D58Y;

[0219] (rrrr) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D58Y_D100S;

[0220] (ssss)VH containing the amino acid sequence of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequence of CDR1, CDR2 and CDR3 of P1-068748_H31S_D57K;

[0221] (tttt)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H31S_D58Y;

[0222] (uuuu)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H31S_D100S andVL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H31S_D100S;

[0223] (vvvv)VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H32Y_D58Y and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_H32Y_D58Y;

[0224] (www)VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of VH and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of P1-068748_H32Y_D100S; or

[0225] (xxxx) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D57K_D100S and VL containing the amino acid sequences of CDR1, CDR2 and CDR3 of P1-068748_D57K_D100S.

[0226] Additionally, the following sequence listing provides the heavy and light chain variable region sequences, as well as the full-length heavy and light chain sequences, of the antibodies listed above that have the IgG1.3 heavy chain constant region (unless a different HC constant region is indicated in the table). The positions of VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, are indicated by amino acid residues and by bolding and underlining the CDRs in each VH and VL sequence. For example, VH CDR1 of P1-061029 contains amino acids 26-35 of SEQ ID NO: 67, while VH CDR2 contains amino acids 50-66 of SEQ ID NO: 67, and VH CDR3 contains amino acids 99-110 of SEQ ID NO: 67, and so on, as indicated by the bolded and underlined amino acids of SEQ ID NO: 67 shown in the sequence listing.

[0227] In some embodiments, the anti-hVISTA Ab comprises a VH containing the amino acid sequence of the VH of any anti-hVISTA Ab provided herein. Individual VH sequences for specific antibody species provided herein are listed in the sequence listing. In some embodiments, the anti-hVISTA... Ab contains a VH with an amino acid sequence containing the following: P1-061029 or P1-061015 or their 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, P1-069063, P1-069065, P1-069067, P1-069069, P1-06 9071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-06874 8. 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_E 100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-06 8761_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, P 1-068767_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_E102Y P1-068744_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_H3 1S_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.

[0228] In some implementation schemes, anti-hVISTA Ab contains 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, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, and 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_E 56N, 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, P 1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1- 068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-0 68761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1 -068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-0687 67_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D1 02V, 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_V 102D, 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_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-06 8748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K The VH of any of the following sequences: 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 1, 2, 3, 4, or 5 amino acid substitutions in the frame region of the VH sequence, such as 1, 2, 3, 4, or 5 conserved substitutions. Optionally, P1-061029 or its progeny may contain substitutions of K16R and / or T84A in the VH frame region (P1-061015 already has R and A at these positions).

[0229] In some embodiments, the anti-hVISTA Ab comprises VH CDR1, CDR2, and CDR3 containing the amino acid sequence of the VH CDR of any anti-hVISTA Ab provided herein, and comprises a VH 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 anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab... Ab contains a VH containing 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% of the amino acid sequence identical to the VH 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, P1-06 1015, 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, P 1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P 1-068761_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_D 102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D102V, P1-0687 67_E30D, P1-068767_E30D_E55A, P1-068767_E100fF_D102V, P1-068767_E55A_E 100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P 1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y3 2E, 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_E102Y P1-068744_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_H3 1S_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 some embodiments, due to the substitution of 1, 2, 3, 4, or 5 amino acids in the frame region of the VH sequence, such as 1, 2, 3, ...Four or five conserved substitutions, or one or both of the K16R and / or T84A substitutions, such as in P1-061029 or its progeny, so the VH of the antibody differs from the VH sequence shown in the sequence listing.

[0230] In some embodiments, the anti-hVISTA Ab comprises a VH consisting of the amino acid sequence of the VH of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA... Ab contains a VH consisting of the amino acid sequence of the following: P1-061029 or P1-061015 or their 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, P1-069063, P1-069065, P1-069067, P1-069069, P1-06907. 1. 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, P 1-068761_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_E 56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D5 2N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55 A. 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_E1 00fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E, P 1-061029_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_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_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, optionally, in the case of P1-061029 or its offspring, having one or both of the substitutions K16R and / or T84A.

[0231] In some embodiments, the anti-hVISTA Ab comprises a VL containing the amino acid sequence of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VL containing the amino acid sequence of any of the following: P1-061029 or P1-061015 or their 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. , P1-069063, 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, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A _E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1-06876 1_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100f F. P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P 1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E 32Y, 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_E1 00fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E, P 1-061029_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_E102Y P1-068744_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_H3 1S_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 some embodiments, the anti-hVISTA Ab comprises VL CDR1, CDR2, and CDR3 containing the amino acid sequence of the VL CDR of any anti-hVISTA Ab provided herein, and comprises a VL 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 anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises an amino acid sequence containing at least 90%, at least 91%, at least 92%, or at least 92% identical to the VL of any anti-hVISTA Ab provided herein.VLs with at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical amino acid sequences: 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, P1-0 69075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-06 8742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068752P1-06875 4. 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_E100f F、P1-068761_E56N_E100fF、P1-068761_E32Y、P1-068761_E32Y_E55A、P1- 068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-0 68761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-0687 67_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E5 5A, 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_E3 1S, 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_E59 Y_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_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 some embodiments, the VL of the antibody differs from the VL sequence shown in the sequence listing due to 1, 2, 3, 4, or 5 amino acid substitutions in the frame region of the VL sequence, such as 1, 2, 3, 4, or 5 conserved substitutions. For example, P1-061015 or its progeny may optionally have a T85V substitution in the VL frame region.

[0232] In some embodiments, the anti-hVISTA Ab comprises a VL consisting of the amino acid sequence of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA... Ab contains a VL consisting of the amino acid sequence of the following: P1-061029 or P1-061015 or their 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, P1-069063, P1-069065, P1-069067, P1-069069, P1-06907. 1. 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, P 1-068761_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_E 56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D5 2N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55 A. 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_E1 00fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E, P 1-061029_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_E102Y P1-068744_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_H3 1S_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.

[0233] In some embodiments, the anti-hVISTA Ab comprises a VH containing the amino acid sequence of the VH of any anti-hVISTA Ab provided herein, and a VL containing the amino acid sequence of the VL of any anti-hVISTA Ab provided herein. In some of these embodiments, the anti-hVISTA Ab... Ab contains a VH with an amino acid sequence containing the following: P1-061029 or P1-061015 or their 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, P1-069063, 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, P1-068 761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761E100fF, 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_D1 02V, 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, P1-0687 67_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102 D. 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_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E102Y, P1-068744_E59Y_E1 02Y, 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, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S_D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S or P1-068748_D57K_D100S; and P1-061029 (SEQ VL is a VL containing the amino acid sequence of P1-061015 (SEQ ID NO: 68) or P1-061015 (SEQ ID NO: 96) or P1-061015 containing a T85V substitution (see SEQ ID NO: 569; P1-061015T85V).

[0234] However, in some implementations, the VH of the antibody is 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, P1-0 68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E 55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E 56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E10 0fF、P1-068761_E30D_E100fF、P1-068761_E56N_E100fF、P1-068761_E32Y , P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P 1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V , P1-068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-06 8767_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D 102V, 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_V 102D, 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_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E10 0S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D100 S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57K_D58 The VH of Y, 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 1, 2, 3, 4, or 5 amino acid substitutions in the frame region of the VH sequence, such as 1, 2, 3, 4, or 5 conserved substitutions, and the VL is the VL of P1-061029, P1-061015, or P1-061015T85V.

[0235] In some embodiments, the anti-hVISTA Ab comprises VH and VL amino acid sequences containing the following: P1-061029 or P1-061015 or their 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, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-06 9073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-06 8740, P1-068742, P1-068744, P1-068766, P1-068748, P1-068750, P1-068 752P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1 -068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-06876 1_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068 761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-06 8761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068 761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-0687 61_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1- 068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-06876 7_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D10 2V, 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, P1-06 1029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E, P1-061029_Y32E_F100fE, P 1-068744_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_H3 1S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_ D100S, P1-068748_H31S_H32Y, P1-068748_H32Y_D57K, P1-068748_D57 K_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, optionally, wherein the VH of P1-061029 or its offspring contains one or both of K16R and / or T84A substitutions, and further optionally, wherein the VL of P1-061015 or its offspring contains T85V substitutions.

[0236] In some embodiments, the anti-hVISTA Ab comprises VH CDR1, CDR2, and CDR3 containing the amino acid sequence of the VH CDR of any anti-hVISTA Ab provided herein, and VL CDR1, CDR2, and CDR3 containing the amino acid sequence of the VL CDR of any anti-hVISTA Ab provided herein, and further comprises 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 to the corresponding VH and VL of any anti-hVISTA Ab provided herein. In some embodiments, the VH and VL of the antibody differ from the VH and VL sequences shown in the sequence listing due to 1, 2, 3, 4, or 5 amino acid substitutions in the frame region of the sequence, such as 1, 2, 3, 4, or 5 conserved substitutions, or one or both of the K16R and / or T84A substitutions in the VH sequence, such as the T85V substitution, such as the T85V substitution, in the case of P1-061015 or its progeny.

[0237] In some embodiments, the anti-hVISTA Ab comprises VH and VL consisting of the amino acid sequences of VH and VL of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA... Ab comprises the VH and VL amino acid sequences of each of the following: P1-061029 or P1-061015 or their 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, P1-069063, P1-069065, P1-069067, P1-06906... 9. 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_E3 0D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E5 5A_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_D52 N. 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, P 1-068767_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_E102Y P1-068744_E31S_E59Y, P1-068744_E31S_E100S, P1-068744_H50I_E1 00S, P1-068744_H50I_E102Y, P1-068744_E59Y_E102Y, P1-068748_H31 S, P1-068748_H32Y, P1-068748_D57K, P1-068748_D58Y, P1-068748_D 100S, 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, optionally, wherein the VH of P1-061029 or its descendants is modified by having one or both of K16R and / or T84A substitutions, and the VL of P1-061015 or its descendants is modified by having T85V substitutions.

[0238] Anti-hVISTA Ab may include:

[0239] (a) VH containing the amino acid sequence of VH and VL containing the amino acid sequence of VL of P1-061029.

[0240] (b) VH containing the amino acid sequence of VH with P1-061015 and VL containing the amino acid sequence of VL with P1-061015.

[0241] (c) VH containing the amino acid sequence of VH with P1-068757 and VL containing the amino acid sequence of VL with P1-068757.

[0242] (d) VH containing the amino acid sequence of VH with P1-068759 and VL containing the amino acid sequence of VL with P1-068759.

[0243] (e) VH containing the amino acid sequence of VH with P1-068761 and VL containing the amino acid sequence of VL with P1-068761.

[0244] (f) VH containing the amino acid sequence of VH with P1-068763 and VL containing the amino acid sequence of VL with P1-068763.

[0245] (g) VH containing the amino acid sequence of VH of P1-068765 and VL containing the amino acid sequence of VL of P1-068765.

[0246] (h) VH containing the amino acid sequence of VH with P1-068767 and VL containing the amino acid sequence of VL with P1-068767.

[0247] (i) VH containing the amino acid sequence of VH containing P1-068769 and VL containing the amino acid sequence of VL containing P1-068769.

[0248] (j) VH containing the amino acid sequence of VH with P1-068771 and VL containing the amino acid sequence of VL with P1-068771.

[0249] (k) VH containing the amino acid sequence of VH with P1-068773 and VL containing the amino acid sequence of VL with -068773;

[0250] (l) VH containing the amino acid sequence of VH with P1-068775 and VL containing the amino acid sequence of VL with P1-068775.

[0251] (m) VH containing the amino acid sequence of VH with P1-069059 and VL containing the amino acid sequence of VL with P1-069059.

[0252] (n) VH containing the amino acid sequence of VH of P1-069061 and VL containing the amino acid sequence of VL of P1-069061.

[0253] (o) VH containing the amino acid sequence of VH with P1-069063 and VL containing the amino acid sequence of VL with P1-069063.

[0254] (p) VH containing the amino acid sequence of VH with P1-069065 and VL containing the amino acid sequence of VL with P1-069065.

[0255] (q) VH containing the amino acid sequence of VH with P1-069067 and VL containing the amino acid sequence of VL with P1-069067.

[0256] (r) VH containing the amino acid sequence of VH with P1-069069 and VL containing the amino acid sequence of VL with P1-069069.

[0257] (s) VH containing the amino acid sequence of VH with P1-069071 and VL containing the amino acid sequence of VL with P1-069071.

[0258] (t) VH containing the amino acid sequence of VH with P1-069073 and VL containing the amino acid sequence of VL with P1-069073.

[0259] (u) VH containing the amino acid sequence of VH with P1-069075 and VL containing the amino acid sequence of VL with P1-069075.

[0260] (v) VH containing the amino acid sequence of VH with P1-069077 and VL containing the amino acid sequence of VL with P1-069077.

[0261] (w) VH containing the amino acid sequence of VH with P1-068736 and VL containing the amino acid sequence of VL with P1-068736.

[0262] (x) VH containing the amino acid sequence of VH with P1-068738 and VL containing the amino acid sequence of VL with P1-068738.

[0263] (y) VH containing the amino acid sequence of VH with P1-068740 and VL containing the amino acid sequence of VL with P1-068740.

[0264] (z) VH containing the amino acid sequence of VH with P1-068742 and VL containing the amino acid sequence of VL with P1-068742;

[0265] (aa) VH containing the amino acid sequence of VH with P1-068744 and VL containing the amino acid sequence of VL with P1-068744.

[0266] (bb) VH containing the amino acid sequence of VH with P1-068746 and VL containing the amino acid sequence of VL with P1-068746.

[0267] (cc) VH containing the amino acid sequence of VH with P1-068748 and VL containing the amino acid sequence of VL with P1-068748.

[0268] (dd) VH containing the amino acid sequence of VH with P1-068750 and VL containing the amino acid sequence of VL with P1-068750.

[0269] (ee) VH containing the amino acid sequence of VH with P1-068752 and VL containing the amino acid sequence of VL with P1-068752.

[0270] (ff) VH containing the amino acid sequence of VH with P1-068754 and VL containing the amino acid sequence of VL with P1-068754.

[0271] (gg) VH containing the amino acid sequence of VH with P1-068761_E55A and VL containing the amino acid sequence of VL with P1-068761_E55A;

[0272] (hh) VH containing the amino acid sequence of VH with P1-068761_H100G and VL containing the amino acid sequence of VL with P1-068761_H100G;

[0273] (ii) VH containing the amino acid sequence of VH with P1-068761_E56N and VL containing the amino acid sequence of VL with P1-068761_E56N;

[0274] (jj) VH containing the amino acid sequence of VH with P1-068761_E55A_E56N and VL containing the amino acid sequence of VL with P1-068761_E55A_E56N;

[0275] (kk) VH containing the amino acid sequence of VH with P1-068761_E30D and VL containing the amino acid sequence of VL with P1-068761_E30D;

[0276] (ll) VH containing the amino acid sequence of VH with P1-068761_E30D_E55A and VL containing the amino acid sequence of VL with P1-068761_E30D_E55A;

[0277] (mm) VH containing the amino acid sequence of VH with P1-068761_E56N_H100G and VL containing the amino acid sequence of VL with P1-068761_E56N_H100G;

[0278] (nn) VH containing the amino acid sequence of VH with P1-068761_E30D_H100G and VL containing the amino acid sequence of VL with P1-068761_E30D_H100G;

[0279] (oo) VH containing the amino acid sequence of VH with P1-068761_E30D_E56N and VL containing the amino acid sequence of VL with P1-068761_E30D_E56N;

[0280] (pp) VH containing the amino acid sequence of VH with P1-068761E100fF and VL containing the amino acid sequence of VL with P1-068761E100fF.

[0281] (qq) VH containing the amino acid sequence of VH with P1-068761_E55A_E100fF and VL containing the amino acid sequence of VL with P1-068761_E55A_E100fF;

[0282] (rr) VH containing the amino acid sequence of VH with P1-068761_H100G_E100fF and VL containing the amino acid sequence of VL with P1-068761_H100G_E100fF;

[0283] (ss) VH containing the amino acid sequence of VH with P1-068761_E30D_E100fF and VL containing the amino acid sequence of VL with P1-068761_E30D_E100fF;

[0284] (tt)VH containing the amino acid sequence of VH with P1-068761_E56N_E100fF and VL containing the amino acid sequence of VL with P1-068761_E56N_E100fF;

[0285] (uu) VH containing the amino acid sequence of VH with P1-068761_E32Y and VL containing the amino acid sequence of VL with P1-068761_E32Y;

[0286] (vv) VH containing the amino acid sequence of VH with P1-068761_E32Y_E55A and VL containing the amino acid sequence of VL with P1-068761_E32Y_E55A;

[0287] (ww) VH containing the amino acid sequence of VH with P1-068761_E32Y_E56N and VL containing the amino acid sequence of VL with P1-068761_E32Y_E56N;

[0288] (xx) VH containing the amino acid sequence of VH with P1-068761_E30D_E32Y and VL containing the amino acid sequence of VL with P1-068761_E30D_E32Y;

[0289] (yy) VH containing the amino acid sequence of VH with P1-068761_E32Y_H100G and VL containing the amino acid sequence of VL with P1-068761_E32Y_H100G;

[0290] (zz) VH containing the amino acid sequence of VH with P1-068761_E32Y_E100fF and VL containing the amino acid sequence of VL with P1-068761_E32Y_E100fF;

[0291] (aaa) VH containing the amino acid sequence of VH with P1-068767_D52N_D102V and VL containing the amino acid sequence of VL with P1-068767_D52N_D102V;

[0292] (bbb) VH containing the amino acid sequence of VH with P1-068767_D52N and VL containing the amino acid sequence of VL with P1-068767_D52N;

[0293] (ccc) VH containing the amino acid sequence of VH containing P1-068767_D52N_E55A and VL containing the amino acid sequence of VL containing P1-068767_D52N_E55A;

[0294] (ddd) VH containing the amino acid sequence of VH with P1-068767_E55A_D102V and VL containing the amino acid sequence of VL with P1-068767_E55A_D102V;

[0295] (eee) VH containing the amino acid sequence of VH with P1-068767_D102V and VL containing the amino acid sequence of VL with P1-068767_D102V;

[0296] (fff) VH containing the amino acid sequence of VH with P1-068767_E55A and VL containing the amino acid sequence of VL with P1-068767_E55A;

[0297] (ggg) VH containing the amino acid sequence of VH with P1-068767_E30D_D52N and VL containing the amino acid sequence of VL with P1-068767_E30D_D52N;

[0298] (hhh) VH containing the amino acid sequence of VH with P1-068767_E30D_D102V and VL containing the amino acid sequence of VL with P1-068767_E30D_D102V;

[0299] (iii) VH containing the amino acid sequence of VH with P1-068767_E30D and VL containing the amino acid sequence of VL with P1-068767_E30D;

[0300] (jjj)VH containing the amino acid sequence of VH with P1-068767_E30D_E55A and VL containing the amino acid sequence of VL with P1-068767_E30D_E55A;

[0301] (kkk) VH containing the amino acid sequence of VH with P1-068767_E100fF_D102V and VL containing the amino acid sequence of VL with P1-068767_E100fF_D102V;

[0302] (lll) VH containing the amino acid sequence of VH with P1-068767_E55A_E100fF and VL containing the amino acid sequence of VL with P1-068767_E55A_E100fF;

[0303] (mmm) VH containing the amino acid sequence of VH with P1-068767_D52N_E100fF and VL containing the amino acid sequence of VL with P1-068767_D52N_E100fF;

[0304] (nnn) VH containing the amino acid sequence of VH with P1-068767_E100fF and VL containing the amino acid sequence of VL with P1-068767_E100fF;

[0305] (ooo)VH containing the amino acid sequence of VH with P1-068767_E30D_E100fF and VL containing the amino acid sequence of VL with P1-068767_E30D_E100fF;

[0306] (ppp) VH containing the amino acid sequence of VH with P1-061029_F100fE_V102D and VL containing the amino acid sequence of VL with P1-061029_F100fE_V102D;

[0307] (qqq) VH containing the amino acid sequence of VH with P1-061029_F100fE and VL containing the amino acid sequence of VL with P1-061029_F100fE;

[0308] (rrr) VH containing the amino acid sequence of VH with P1-061029_V102D and VL containing the amino acid sequence of VL with P1-061029_V102D;

[0309] (sss)VH containing the amino acid sequence of VH with P1-061029_Y32E and VL containing the amino acid sequence of VL with P1-061029_Y32E;

[0310] (ttt)VH containing the amino acid sequence of VH with P1-061029_Y32E_F100fE and VL containing the amino acid sequence of VL with P1-061029_Y32E_F100fE;

[0311] (uuu) VH containing the amino acid sequence of VH with P1-068744_E31S and VL containing P1-068744_E31S;

[0312] (vvv) VH containing the amino acid sequence of VH containing P1-068744_H50I and VL containing P1-068744_H50I;

[0313] (www) VH containing the amino acid sequence of P1-068744_E59Y and VL containing P1-068744_E59Y;

[0314] (xxx) VH containing the amino acid sequence of VH containing P1-068744_E100S and VL containing P1-068744_E100S;

[0315] (yyy) VH containing the amino acid sequence of VH containing P1-068744_E102Y and VL containing P1-068744_E102Y;

[0316] (zzz) VH containing the amino acid sequence of VH containing P1-068744_E31S_H50I and VL containing P1-068744_E31S_H50I;

[0317] (aaaa)VH containing the amino acid sequence of VH with P1-068744_H50I_E59Y and VL containing P1-068744_H50I_E59Y;

[0318] (bbbb)VH containing the amino acid sequence of VH with P1-068744_E59Y_E100S and VL containing P1-068744_E5;9Y_E100S;

[0319] (cccc) VH containing the amino acid sequence of VH containing P1-068744_E100S_E102Y and VL containing P1-068744_E100S_E102Y;

[0320] (dddd)VH containing the amino acid sequence of VH containing P1-068744_E31S_E102Y and VL containing P1-068744_E31S_E102Y;

[0321] (eeee) VH containing the amino acid sequence of VH containing P1-068744_E31S_E59Y and VL containing P1-068744_E31S_E59Y;

[0322] (ffff) VH containing the amino acid sequence of VH containing P1-068744_E31S_E100S and VL containing P1-068744_E31S_E100S;

[0323] (gggg) VH containing the amino acid sequence of P1-068744_H50I_E100S and VL containing P1-068744_H50I_E100S;

[0324] (hhhh) VH containing the amino acid sequence of P1-068744_H50I_E102Y and VL containing P1-068744_H50I_E102Y;

[0325] (iiii) VH containing the amino acid sequence of VH containing P1-068744_E59Y_E102Y and VL containing P1-068744_E59Y_E102Y;

[0326] (jjjj)VH containing the amino acid sequence of VH containing P1-068748_H31S and VL containing P1-068748_H31S;

[0327] (kkkk)VH containing the amino acid sequence of P1-068748_H32Y and VL containing P1-068748_H32Y;

[0328] (llll) VH containing the amino acid sequence of VH containing P1-068748_D57K and VL containing P1-068748_D57K;

[0329] (mmmm) VH containing the amino acid sequence of P1-068748_D58Y and VL containing P1-068748_D58Y;

[0330] (nnnn) VH containing the amino acid sequence of P1-068748_D100S and VL containing P1-068748_D100S;

[0331] (oooo) VH containing the amino acid sequence of VH containing P1-068748_H31S_H32Y and VL containing P1-068748_H31S_H32Y;

[0332] (pppp) VH containing the amino acid sequence of VH containing P1-068748_H32Y_D57K and VL containing P1-068748_H32Y_D57K;

[0333] (qqqq) VH containing the amino acid sequence of VH and VL containing the amino acid sequence of P1-068748_D57K_D58Y;

[0334] (rrrr) VH containing the amino acid sequence of VH containing P1-068748_D58Y_D100S and VL containing P1-068748_D58Y_D100S;

[0335] (ssss)VH containing the amino acid sequence of VH containing P1-068748_H31S_D57K and VL containing P1-068748_H31S_D57K;

[0336] (tttt)VH containing the amino acid sequence of VH containing P1-068748_H31S_D58Y and VL containing P1-068748_H31S_D58Y;

[0337] (uuuu)VH containing the amino acid sequence of VH with P1-068748_H31S_D100S and VL containing P1-068748_H31S_D100S;

[0338] (vvvv)VH containing the amino acid sequence of VH with P1-068748_H32Y_D58Y and VL containing P1-068748_H32Y_D58Y;

[0339] (www)VH containing the amino acid sequence of VH containing P1-068748_H32Y_D100S and VL containing P1-068748_H32Y_D100S; or

[0340] (xxxx) VH containing the amino acid sequence of VH of P1-068748_D57K_D100S and VL containing VL of P1-068748_D57K_D100S, optionally, wherein the VH of (a) or (c)-(ttt) contains one or both of K16R and T84A substitutions, and optionally, wherein the VL of (b) or (uuu)-(xxxx) contains T85V substitutions.

[0341] Anti-hVISTA Ab may include:

[0342] (a) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-061029 and VL containing the amino acid sequence of VL containing P1-061029.

[0343] (b) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068757 and VL containing the amino acid sequence of VL containing P1-068757.

[0344] (c) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068759 and VL containing the amino acid sequence of VL containing P1-068759.

[0345] (d) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761 and VL containing the amino acid sequence of VL containing P1-068761.

[0346] (e) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068763 and VL containing the amino acid sequence of VL containing P1-068763.

[0347] (f) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068765 and VL containing the amino acid sequence of VL containing P1-068765.

[0348] (g) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767 and VL containing the amino acid sequence of VL containing P1-068767.

[0349] (h) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068769 and VL containing the amino acid sequence of VL containing P1-068769.

[0350] (i) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068771 and VL containing the amino acid sequence of VL containing P1-068771.

[0351] (j) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068773 and VL containing the amino acid sequence of VL containing -068773.

[0352] (k) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068775 and VL containing the amino acid sequence of VL containing P1-068775.

[0353] (l) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069059 and VL containing the amino acid sequence of VL containing P1-069059.

[0354] (m) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069061 and VL containing the amino acid sequence of VL containing P1-069061.

[0355] (n) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069063 and VL containing the amino acid sequence of VL containing P1-069063.

[0356] (o) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069065 and VL containing the amino acid sequence of VL containing P1-069065.

[0357] (p) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069067 and VL containing the amino acid sequence of VL containing P1-069067.

[0358] (q) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069069 and VL containing the amino acid sequence of VL containing P1-069069.

[0359] (r) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069071 and VL containing the amino acid sequence of VL containing P1-069071.

[0360] (s) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069073, and VL containing the amino acid sequence of VL containing P1-069073.

[0361] (t) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069075 and VL containing the amino acid sequence of VL containing P1-069075.

[0362] (u) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-069077 and VL containing the amino acid sequence of VL containing P1-069077.

[0363] (v) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E55A and VL containing the amino acid sequence of VL containing P1-068761_E55A.

[0364] (w) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_H100G and VL containing the amino acid sequence of VL containing P1-068761_H100G.

[0365] (x) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E56N and VL containing the amino acid sequence of VL containing P1-068761_E56N;

[0366] (y) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E55A_E56N and VL containing the amino acid sequence of VL.

[0367] (z) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E30D and VL containing the amino acid sequence of VL containing P1-068761_E30D.

[0368] (aa) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E30D_E55A and VL containing the amino acid sequence of VL containing P1-068761_E30D_E55A.

[0369] (bb) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E56N_H100G and VL containing the amino acid sequence of VL containing P1-068761_E56N_H100G.

[0370] (cc) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E30D_H100G and VL containing the amino acid sequence of VL containing P1-068761_E30D_H100G.

[0371] (dd) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E30D_E56N and VL containing the amino acid sequence of VL containing P1-068761_E30D_E56N;

[0372] (ee) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E100fF and VL containing the amino acid sequence of VL containing P1-068761E100fF.

[0373] (ff) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E55A_E100fF and VL containing the amino acid sequence of VL.

[0374] (gg) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_H100G_E100fF and VL containing the amino acid sequence of VL containing P1-068761_H100G_E100fF;

[0375] (hh) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E30D_E100fF and VL containing the amino acid sequence of VL.

[0376] (ii) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068761_E56N_E100fF and VL containing the amino acid sequence of VL.

[0377] (jj) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E32Y and VL containing the amino acid sequence of VL containing P1-068761_E32Y.

[0378] (kk) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E32Y_E55A and VL containing the amino acid sequence of VL containing P1-068761_E32Y_E55A;

[0379] (1l) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E32Y_E56N and VL containing the amino acid sequence of VL containing P1-068761_E32Y_E56N;

[0380] (mm) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E30D_E32Y and VL containing the amino acid sequence of VL.

[0381] (nn) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E32Y_H100G and VL containing the amino acid sequence of VL containing P1-068761_E32Y_H100G.

[0382] (oo) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068761_E32Y_E100fF and VL containing the amino acid sequence of VL.

[0383] (pp) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_D52N_D102V and VL containing the amino acid sequence of VL containing P1-068767_D52N_D102V;

[0384] (qq) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_D52N and VL containing the amino acid sequence of VL.

[0385] (rr) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_D52N_E55A and VL containing the amino acid sequence of VL containing P1-068767_D52N_E55A;

[0386] (ss) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E55A_D102V and VL containing the amino acid sequence of VL containing P1-068767_E55A_D102V.

[0387] (tt) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_D102V, and VL containing the amino acid sequence of VL containing P1-068767_D102V.

[0388] (uu) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_E55A and VL containing the amino acid sequence of VL containing P1-068767_E55A.

[0389] (vv) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_E30D_D52N and VL containing the amino acid sequence of VL.

[0390] (ww) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E30D_D102V and VL containing the amino acid sequence of VL containing P1-068767_E30D_D102V.

[0391] (xx) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E30D and VL containing the amino acid sequence of VL containing P1-068767_E30D.

[0392] (yy) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_E30D_E55A and VL containing the amino acid sequence of VL containing P1-068767_E30D_E55A.

[0393] (zz) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-068767_E100fF_D102V and VL containing the amino acid sequence of VL containing P1-068767_E100fF_D102V;

[0394] (aaa) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E55A_E100fF and VL containing the amino acid sequence of VL containing P1-068767_E55A_E100fF;

[0395] (bbb) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_D52N_E100fF and VL containing the amino acid sequence of VL.

[0396] (ccc) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E100fF and VL containing the amino acid sequence of VL containing P1-068767_E100fF.

[0397] (ddd) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-068767_E30D_E100fF and VL containing the amino acid sequence of VL containing P1-068767_E30D_E100fF;

[0398] (eee) VH, which contains the amino acid sequence of VH modified by K16R and / or T84A substitution of P1-061029_F100fE_V102D, and VL, which contains the amino acid sequence of VL.

[0399] (fff) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-061029_F100fE and VL containing the amino acid sequence of VL containing P1-061029_F100fE.

[0400] (ggg) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-061029_V102D and VL containing the amino acid sequence of VL containing P1-061029_V102D.

[0401] (hhh) VH, containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-061029_Y32E, and VL, containing the amino acid sequence of VL; or

[0402] (iii) VH containing the amino acid sequence of VH modified by K16R and / or T84A substitution with P1-061029_Y32E_F100fE and VL containing the amino acid sequence of VL containing P1-061029_Y32E_F100fE.

[0403] Anti-hVISTA Ab may include:

[0404] (a) VH containing VH CDR of P1-061029 and VL containing VL CDR of P1-061029, and VH and VL amino acid sequences 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 to VH and VL of P1-061029;

[0405] (b) VH containing VH CDR of P1-061015 and VL containing VL CDR of P1-061015, and VH and VL amino acid sequences 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 to VH and VL of P1-061015;

[0406] (c) VH containing VH CDR of P1-068757 and VL containing VL CDR of P1-068757, and VH and VL amino acid sequences 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 to VH and VL of P1-068757;

[0407] (d) VH containing VH CDR of P1-068759 and VL containing VL CDR of P1-068759, and VH and VL amino acid sequences 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 to VH and VL of P1-068759;

[0408] (e) VH containing VH CDR of VH of P1-068761 and VL containing VL CDR of P1-068761, and VH and VL amino acid sequences 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 to VH and VL of P1-068761;

[0409] (f) VH containing VH CDR of VH and VL containing VL CDR of P1-068763, and VH and VL amino acid sequences 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 to VH and VL of P1-068763;

[0410] (g) VH containing VH CDR of VH of P1-068765 and VL containing VL CDR of P1-068765, and VH and VL amino acid sequences 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 to VH and VL of P1-068765;

[0411] (h) VH containing VH CDR of VH of P1-068767 and VL containing VL CDR of P1-068767, and VH and VL amino acid sequences 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 to VH and VL of P1-068767;

[0412] (i) VH containing VH CDR of VH of P1-068769 and VL containing VL CDR of P1-068769, and VH and VL amino acid sequences 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 to VH and VL of P1-068769;

[0413] (j) VH containing the VH CDR of P1-068771 and VL containing the VL CDR of P1-068771, and VH and VL amino acid sequences 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 to the VH and VL of P1-068771;

[0414] (k) VH containing VH CDR of VH of P1-068773 and VL containing VL CDR of P1-068773, and VH and VL amino acid sequences 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 to VH and VL of P1-068773;

[0415] (l) VH containing VH CDR of VH of P1-068775 and VL containing VL CDR of P1-068775, and VH and VL amino acid sequences 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 to VH and VL of P1-068775;

[0416] (m) VH containing VH CDR of P1-069059 and VL containing VL CDR of P1-069059, and VH and VL amino acid sequences 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 to VH and VL of P1-069059;

[0417] (n) VH containing VH CDR of VH of P1-069061 and VL containing VL CDR of P1-069061, and VH and VL amino acid sequences 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 to VH and VL of P1-069061;

[0418] (o) VH containing VH CDR of VH of P1-069063 and VL containing VL CDR of P1-069063, and VH and VL amino acid sequences 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 to VH and VL of P1-069063;

[0419] (p) VH containing VH CDR of VH of P1-069065 and VL containing VL CDR of P1-069065, and VH and VL amino acid sequences 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 to VH and VL of P1-069065;

[0420] (q) VH containing VH CDR of VH of P1-069067 and VL containing VL CDR of P1-069067, and VH and VL amino acid sequences 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 to VH and VL of P1-069067;

[0421] (r) VH containing VH CDR of P1-069069 and VL containing VL CDR of P1-069069, and VH and VL amino acid sequences 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 to VH and VL of P1-069069;

[0422] (s) VH containing VH CDR of VH of P1-069071 and VL containing VL CDR of P1-069071, and VH and VL amino acid sequences 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 to VH and VL of P1-069071;

[0423] (t) VH containing VH CDR of P1-069073 and VL containing VL CDR of P1-069073, and VH and VL amino acid sequences 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 to VH and VL of P1-069073;

[0424] (u) VH containing VH CDR of VH of P1-069075 and VL containing VL CDR of P1-069075, and VH and VL amino acid sequences 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 to VH and VL of P1-069075;

[0425] (v) VH containing VH CDR of VH of P1-069077 and VL containing VL CDR of P1-069077, and VH and VL amino acid sequences 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 to VH and VL of P1-069077;

[0426] (w) VH containing VH CDR of VH and VL containing VL CDR of P1-068736, and VH and VL amino acid sequences 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 to VH and VL of P1-068736;

[0427] (x) VH containing VH CDR of VH and VL containing VL CDR of P1-068738, and VH and VL amino acid sequences 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 to VH and VL of P1-068738;

[0428] (y) VH containing VH CDR of VH of P1-068740 and VL containing VL CDR of P1-068740, and VH and VL amino acid sequences 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 to VH and VL of P1-068740;

[0429] (z) VH containing VH CDR of VH of P1-068742 and VL containing VL CDR of P1-068742, and VH and VL amino acid sequences 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 to VH and VL of P1-068742;

[0430] (aa) VH containing VH CDR of VH of P1-068744 and VL containing VL CDR of P1-068744, and VH and VL amino acid sequences 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 to VH and VL of P1-068744;

[0431] (bb) VH containing VH CDR of P1-068746 and VL containing VL CDR of P1-068746, and VH and VL amino acid sequences 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 to VH and VL of P1-068746;

[0432] (cc) VH containing VH CDR of VH and VL containing VL CDR of P1-068748, and VH and VL amino acid sequences 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 to VH and VL of P1-068748;

[0433] (dd) VH containing VH CDR of VH and VL containing VL CDR of P1-068750, and VH and VL amino acid sequences 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 to VH and VL of P1-068750;

[0434] (ee) VH containing VH CDR of VH of P1-068752 and VL containing VL CDR of P1-068752, and VH and VL amino acid sequences 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 to VH and VL of P1-068752;

[0435] (ff) VH containing VH CDR of VH of P1-068754 and VL containing VL CDR of P1-068754, and VH and VL amino acid sequences 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 to VH and VL of P1-068754;

[0436] (gg) VH containing VH CDR of VH of P1-068761_E55A and VL containing VL CDR of P1-068761_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E55A;

[0437] (hh) VH containing VH CDR of VH of P1-068761_H100G and VL containing VL CDR of P1-068761_H100G, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_H100G;

[0438] (ii) VH containing the VH CDR of VH of P1-068761_E56N and VL containing the VL CDR of P1-068761_E56N, and VH and VL amino acid sequences 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 to the VH and VL of P1-068761_E56N;

[0439] (jj) VH containing VH CDR of VH of P1-068761_E55A_E56N and VL containing VL CDR of P1-068761_E55A_E56N, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E55A_E56N;

[0440] (kk) VH containing VH CDR of VH with P1-068761_E30D and VL containing VL CDR of P1-068761_E30D, and VH and VL amino acid sequences 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 to VH and VL with P1-068761_E30D;

[0441] (ll) VH containing VH CDR of VH containing P1-068761_E30D_E55A and VL containing VL CDR of P1-068761_E30D_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E30D_E55A;

[0442] (mm) VH containing VH CDR of VH of P1-068761_E56N_H100G and VL containing VL CDR of P1-068761_E56NH100G, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E56N_H100G;

[0443] (nn) VH containing the VH CDR of VH of P1-068761_E30D_H100G and VL containing the VL CDR of P1-068761_E30D_H100G, and VH and VL amino acid sequences 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 to the VH and VL of P1-068761_E30D_H100G;

[0444] (oo) VH containing VH CDR of VH of P1-068761_E30D_E56N and VL containing VL CDR of P1-068761_E30D_E56N, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E30D_E56N;

[0445] (pp) VH of VH CDR containing P1-068761_E100fF and VL of VLCDR containing P1-068761_E100fF, and VH and VL amino acid sequences 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 to the VH and VL of P1-068761_E100fF;

[0446] (qq) VH containing VH CDR of VH containing P1-068761_E55A_E100fF and VL containing VL CDR of P1-068761_E55A_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E55A_E100fF;

[0447] (rr) VH containing VH CDR of VH containing P1-068761_H100G_E100fF and VL containing VL CDR of P1-068761_H100G_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_H100G_E100fF;

[0448] (ss) VH containing VH CDR of VH containing P1-068761_E30D_E100fF and VL containing VL CDR of P1-068761_E30D_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E30D_E100fF;

[0449] (tt) VH containing VH CDR of VH containing P1-068761_E56N_E100fF and VL containing VL CDR of P1-068761_E56N_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E56N_E100fF;

[0450] (uu) VH containing VH CDR of VH of P1-068761_E32Y and VL containing VL CDR of P1-068761_E32Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E32Y;

[0451] (vv) VH containing VH CDR of VH of P1-068761_E32Y_E55A and VL containing VL CDR of P1-068761_E32Y_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E32Y_E55A;

[0452] (ww) VH containing VH CDR of VH of P1-068761_E32Y_E56N and VL containing VL CDR of P1-068761_E32Y_E56N, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E32Y_E56N;

[0453] (xx) VH containing VH CDR of VH of P1-068761_E30D_E32Y and VL containing VL CDR of P1-068761_E30D_E32Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E30D_E32Y;

[0454] (yy) VH containing VH CDR of VH of P1-068761_E32Y_H100G and VL containing VL CDR of P1-068761_E32Y_H100G, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E32Y_H100G;

[0455] (zz) VH containing VH CDR of VH containing P1-068761_E32Y_E100fF and VL containing VL CDR of P1-068761_E32Y_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068761_E32Y_E100fF;

[0456] (aaa) VH containing the VH CDR of VH of P1-068767_D52N_D102V and VL containing the VL CDR of P1-068767_D52N_D102V, and VH and VL amino acid sequences 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 to the VH and VL of P1-068767_D52N_D102V;

[0457] (bbb) VH containing VH CDR of VH and VL containing VL CDR of P1-068767_D52N, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_D52N;

[0458] (ccc) VH containing VH CDR of VH containing P1-068767_D52N_E55A and VL containing VL CDR of P1-068767_D52N_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_D52N_E55A;

[0459] (ddd) VH containing the VH CDR of VH of P1-068767_E55A_D102V and VL containing the VL CDR of P1-068767_E55A_D102V, and VH and VL amino acid sequences 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 to the VH and VL of P1-068767_E55A_D102V;

[0460] (eee) VH containing VH CDR of VH of P1-068767_D102V and VL containing VL CDR of P1-068767_D102V, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_D102V;

[0461] (fff) VH containing VH CDR of VH containing P1-068767_E55A and VL containing VL CDR of P1-068767_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E55A;

[0462] (ggg) VH containing VH CDR of VH of P1-068767_E30D_D52N and VL containing VL CDR of P1-068767_E30D_D52N, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E30D_D52N;

[0463] (hhh) VH containing the VH CDR of VH of P1-068767_E30D_D102V and VL containing the VL CDR of P1-068767_E30D_D102V, and VH and VL amino acid sequences 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 to the VH and VL of P1-068767_E30D_D102V;

[0464] (iii) VH containing VH CDR of VH containing P1-068767_E30D and VL containing VL CDR of P1-068767_E30D, and VH and VL amino acid sequences 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 to VH and VL containing P1-068767_E30D;

[0465] (jjj) VH containing VH CDR of VH of P1-068767_E30D_E55A and VL containing VL CDR of P1-068767_E30D_E55A, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E30D_E55A;

[0466] (kkk) VH containing VH CDR of VH of P1-068767_E100fF_D102V and VL containing VL CDR of P1-068767_E100fF_D102V, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E100fF_D102V;

[0467] (lll) VH containing VH CDR of VH containing P1-068767_E55A_E100fF and VL containing VL CDR of P1-068767_E55A_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E55A_E100fF;

[0468] (mmm) VH containing VH CDR of VH containing P1-068767_D52N_E100fF and VL containing VL CDR of P1-068767_D52N_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_D52N_E100fF;

[0469] (nnn) VH containing VH CDR of VH of P1-068767_E100fF and VL containing VLCDR of P1-068767_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E100fF;

[0470] (ooo) VH containing VH CDR of VH containing P1-068767_E30D_E100fF and VL containing VL CDR of P1-068767_E30D_E100fF, and VH and VL amino acid sequences 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 to VH and VL of P1-068767_E30D_E100fF;

[0471] (ppp) VH containing VH CDR of VH of P1-061029_F100fE_V102D and VL containing VL CDR of P1-061029_F100fE_V102D, and VH and VL amino acid sequences 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 to VH and VL of P1-061029_F100fE_V102D;

[0472] (qqq) VH containing VH CDR of VH of P1-061029_F100fE and VL containing VLCDR of P1-061029_F100fE, and VH and VL amino acid sequences 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 to VH and VL of P1-061029_F100fE;

[0473] (rrr) VH containing VH CDR of VH of P1-061029_V102D and VL containing VL CDR of P1-061029_V102D, and VH and VL amino acid sequences 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 to VH and VL of P1-061029_V102D;

[0474] (sss) VH containing VH CDR of P1-061029_Y32E and VL containing VL CDR of P1-061029_Y32E, and VH and VL amino acid sequences 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 to VH and VL of P1-061029_Y32E;

[0475] (ttt) VH containing VH CDR of VH containing P1-061029_Y32E_F100fE and VL containing VL CDR of P1-061029_Y32E_F100fE, and VH and VL amino acid sequences 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 to VH and VL of P1-061029_Y32E_F100fE;

[0476] (uuu) VH containing VH CDR of P1-068744_E31S and VL containing VL CDR of P1-068744_E31S, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E31S;

[0477] (vvv) VH containing VH CDR of P1-068744_H50I and VL containing VL CDR of P1-068744_H50I, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_H50I;

[0478] (www) VH containing VH CDR of P1-068744_E59Y and VL containing VL CDR of P1-068744_E59Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E59Y;

[0479] (xxx) VH containing VH CDR of VH of P1-068744_E100S and VL containing VL CDR of P1-068744_E100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E100S;

[0480] (yyy) VH containing the VH CDR of VH with P1-068744_E102Y and VL containing the VL CDR of P1-068744_E102Y, and VH and VL amino acid sequences 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 to VH and VL;

[0481] (zzz) VH containing VH CDR of VH of P1-068744_E31S_H50I and VL containing VL CDR of P1-068744_E31S_H50I, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E102Y;

[0482] (aaaa) VH containing VH CDR of VH of P1-068744_H50I_E59Y and VL containing VL CDR of P1-068744_H50I_E59Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_H50I_E59Y;

[0483] (bbbb) VH containing VH CDR of P1-068744_E59Y_E100S and VL containing VL CDR of P1-068744_E59Y_E100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E59Y_E100S;

[0484] (cccc) VH containing VH CDR of VH of P1-068744_E100S_E102Y and VL containing VL CDR of P1-068744_E100S_E102Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E100S_E102Y;

[0485] (dddd) VH containing VH CDR of P1-068744_E31S_E102Y and VL containing VL CDR of P1-068744_E31S_E102Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E31S_E102Y;

[0486] (eeee) VH containing VH CDR of VH of P1-068744_E31S_E59Y and VL containing VL CDR of P1-068744_E31S_E59Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E31S_E59Y;

[0487] (ffff) VH containing VH CDR of VH of P1-068744_E31S_E100S and VL containing VL CDR of P1-068744_E31S_E100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_E31S_E100S;

[0488] (gggg) VH containing VH CDR of VH of P1-068744_H50I_E100S and VL containing VL CDR of P1-068744_H50I_E100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_H50I_E100S;

[0489] (hhhh) VH containing VH CDR of VH of P1-068744_H50I_E102Y and VL containing VL CDR of P1-068744_H50I_E102Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068744_H50I_E102Y;

[0490] (iiii) VH containing the VH CDR of VH containing P1-068744_E59Y_E102Y and VL containing the VL CDR of P1-068744_E59Y_E102Y, and VH and VL amino acid sequences 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 to the VH and VL of P1-068744_E59Y_E102Y;

[0491] (jjjj) VH containing VH CDR of P1-068748_H31S and VL containing VL CDR of P1-068748_H31S, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H31S;

[0492] (kkkk)VH containing VH CDR of VH of P1-068748_H32Y and VL containing VL CDR of P1-068748_H32Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H32Y;

[0493] (llll) VH containing the VH CDR of VH with P1-068748_D57K and VL containing the VL CDR of P1-068748_D57K, and VH and VL amino acid sequences 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 to the VH and VL of P1-068748_D57K;

[0494] (mmmm) VH containing VH CDR of P1-068748_D58Y and VL containing VLCDR1, CDR2 and CDR3 of P1-068748_D58Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_D58Y;

[0495] (nnnn) VH containing VH CDR of P1-068748_D100S and VL containing VLCDR of P1-068748_D100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_D100S;

[0496] (oooo) VH containing VH CDR of VH of P1-068748_H31S_H32Y and VL containing VL CDR of P1-068748_H31S_H32Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H31S_H32Y;

[0497] (pppp) VH containing VH CDR of VH of P1-068748_H32Y_D57K and VL containing VL CDR of P1-068748_H32Y_D57K, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H32Y_D57K;

[0498] (qqqq) VH containing VH CDR of P1-068748_D57K_D58Y and VL containing VL CDR of P1-068748_D57K_D58Y, and VH and VL amino acid sequences 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 to the VH and VL of P1-068748_D57K_D58Y;

[0499] (rrrr) VH containing VH CDR of P1-068748_D58Y_D100S and VL containing VL CDR of P1-068748_D58Y_D100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_D58Y_D100S;

[0500] (ssss)VH containing the VH CDR of VH of P1-068748_H31S_D57K and VL containing the VL CDR of P1-068748_H31S_D57K, and VH and VL amino acid sequences 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 to the VH and VL of P1-068748_H31S_D57K;

[0501] (tttt)VH containing VH CDR of VH of P1-068748_H31S_D58Y and VL containing VL CDR of P1-068748_H31S_D58Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H31S_D58Y;

[0502] (uuuu) VH containing VH CDR of VH of P1-068748_H31S_D100S and VL containing VL CDR of P1-068748_H31S_D100S, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H31S_D100S;

[0503] (vvvv) VH containing VH CDR of VH of P1-068748_H32Y_D58Y and VL containing VL CDR of P1-068748_H32Y_D58Y, and VH and VL amino acid sequences 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 to VH and VL of P1-068748_H32Y_D58Y;

[0504] (www) VH containing the VH CDR of VH of P1-068748_H32Y_D100S and VL containing the VL CDR of P1-068748_H32Y_D100S, and VH and VL amino acid sequences 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 to the VH and VL of P1-068748_H32Y_D100S; or

[0505] (xxxx) VH containing the VH CDR of VH of P1-068748_D57K_D100S and VL containing the VL CDR of P1-068748_D57K_D100S, and VH and VL amino acid sequences 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 to the VH and VL of P1-068748_D57K_D100S.

[0506] Optionally, the VH in any of (a) or (c)-(ttt) contains one or both of K16R and T84A substitutions, or the VL in any of (b) or (uuu)-(xxxx) contains T85V substitution.

[0507] In some of the above embodiments, VH and / or VL may differ from the sequences of each of classes (a) to (ttt) due to the presence of 1, 2, 3, 4, or 5 amino acid substitutions, such as 1, 2, 3, 4, or 5 conserved substitutions. In some embodiments, the VH of one of P1-061029 or its progeny antibodies may contain one or both of the K16R and T84A substitutions. In some embodiments, the VL of one of P1-061015 or its progeny antibodies may contain the T85V substitution.

[0508] Anti-hVISTA Ab may include:

[0509] (a) VH composed of the amino acid sequence of VH from P1-061029 and VL composed of VL from P1-061029;

[0510] (b) VH consisting of the amino acid sequence of VH from P1-061015 and VL consisting of VL from P1-061015;

[0511] (c) VH composed of the amino acid sequence of VH from P1-068757 and VL composed of VL from P1-068757;

[0512] (d) VH composed of the amino acid sequence of VH from P1-068759 and VL composed of VL from P1-068759;

[0513] (e) VH composed of the amino acid sequence of VH from P1-068761 and VL composed of VL from P1-068761;

[0514] (f) VH composed of the amino acid sequence of VH from P1-068763 and VL composed of VL from P1-068763;

[0515] (g) VH composed of the amino acid sequence of VH from P1-068765 and VL composed of VL from P1-068765;

[0516] (h) VH composed of the amino acid sequence of VH from P1-068767 and VL composed of VL from P1-068767;

[0517] (i) VH composed of the amino acid sequence of VH from P1-068769 and VL composed of VL from P1-068769;

[0518] (j) VH composed of the amino acid sequence of VH from P1-068771 and VL composed of VL from P1-068771;

[0519] (k) VH composed of the amino acid sequence of VH from P1-068773 and VL composed of VL from P1-068773;

[0520] (l) VH composed of the amino acid sequence of VH from P1-068775 and VL composed of VL from P1-068775;

[0521] (m) VH, which is composed of the amino acid sequence of VH from P1-069059, and VL, which is composed of VL from P1-069059;

[0522] (n) VH, which is composed of the amino acid sequence of VH from P1-069061, and VL, which is composed of VL from P1-069061;

[0523] (o) VH composed of the amino acid sequence of VH from P1-069063 and VL composed of VL from P1-069063;

[0524] (p) VH, which is composed of the amino acid sequence of VH from P1-069065, and VL, which is composed of VL from P1-069065;

[0525] (q) VH composed of the amino acid sequence of VH from P1-069067 and VL composed of VL from P1-069067;

[0526] (r) VH composed of the amino acid sequence of VH from P1-069069 and VL composed of VL from P1-069069;

[0527] (s) VH, which is composed of the amino acid sequence of VH from P1-069071, and VL, which is composed of VL from P1-069071;

[0528] (t) VH, which is composed of the amino acid sequence of VH from P1-069073, and VL, which is composed of VL from P1-069073;

[0529] (u) VH composed of the amino acid sequence of VH from P1-069075 and VL composed of VL from P1-069075;

[0530] (v) VH composed of the amino acid sequence of VH from P1-069077 and VL composed of VL from P1-069077;

[0531] (w) VH composed of the amino acid sequence of VH from P1-068736 and VL composed of VL from P1-068736;

[0532] (x) VH composed of the amino acid sequence of VH from P1-068738 and VL composed of VL from P1-068738;

[0533] (y) VH composed of the amino acid sequence of VH from P1-068740 and VL composed of VL from P1-068740;

[0534] (z) VH composed of the amino acid sequence of VH from P1-068742 and VL composed of VL from P1-068742;

[0535] (aa) VH is composed of the amino acid sequence of VH from P1-068744 and VL is composed of VL from P1-068744;

[0536] (bb) VH is composed of the amino acid sequence of VH from P1-068746 and VL is composed of VL from P1-068746;

[0537] (cc) VH, which is composed of the amino acid sequence of VH from P1-068748, and VL, which is composed of VL from P1-068748;

[0538] (dd) VH is composed of the amino acid sequence of VH from P1-068750 and VL is composed of VL from P1-068750;

[0539] (ee) VH is composed of the amino acid sequence of VH from P1-068752 and VL is composed of VL from P1-068752;

[0540] (ff) VH is composed of the amino acid sequence of VH from P1-068754 and VL is composed of VL from P1-068754;

[0541] (gg) VH, which is composed of the amino acid sequence of VH from P1-068761_E55A, and VL, which is composed of the amino acid sequence of VL from P1-068761_E55A.

[0542] (hh) VH is composed of the amino acid sequence of VH from P1-068761_H100G and VL is composed of the amino acid sequence of VL from P1-068761_H100G.

[0543] (ii) VH consisting of the amino acid sequence of VH from P1-068761_E56N and VL consisting of the amino acid sequence of VL from P1-068761_E56N;

[0544] (jj) VH is composed of the amino acid sequence of VH from P1-068761_E55A_E56N and VL is composed of the amino acid sequence of VL from P1-068761_E55A_E56N;

[0545] (kk) VH is composed of the amino acid sequence of VH from P1-068761_E30D and VL is composed of the amino acid sequence of VL from P1-068761_E30D;

[0546] (ll) VH, which is composed of the amino acid sequence of VH from P1-068761_E30D_E55A, and VL, which is composed of the amino acid sequence of VL from P1-068761_E30D_E55A;

[0547] (mm) VH, which is composed of the amino acid sequence of VH from P1-068761_E56N_H100G, and VL, which is composed of the amino acid sequence of VL from P1-068761_E56N_H100G;

[0548] (nn) VH, which is composed of the amino acid sequence of VH from P1-068761_E30D_H100G, and VL, which is composed of the amino acid sequence of VL from P1-068761_E30D_H100G;

[0549] (oo) VH, which is composed of the amino acid sequence of VH from P1-068761_E30D_E56N, and VL, which is composed of the amino acid sequence of VL from P1-068761_E30D_E56N;

[0550] (pp) VH, which is composed of the amino acid sequence of VH from P1-068761_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068761E100fF;

[0551] (qq) VH is composed of the amino acid sequence of VH from P1-068761_E55A_E100fF and VL is composed of the amino acid sequence of VL from P1-068761_E55A_E100fF;

[0552] (rr) VH, which is composed of the amino acid sequence of VH from P1-068761_H100G_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068761_H100G_E100fF;

[0553] (ss) VH, which is composed of the amino acid sequence of VH from P1-068761_E30D_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068761_E30D_E100fF;

[0554] (tt) VH, which is composed of the amino acid sequence of VH from P1-068761_E56N_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068761_E56N_E100fF;

[0555] (uu) VH is composed of the amino acid sequence of VH of P1-068761_E32Y and VL is composed of the amino acid sequence of VL of P1-068761_E32Y;

[0556] (vv) VH, which is composed of the amino acid sequence of VH from P1-068761_E32Y_E55A, and VL, which is composed of the amino acid sequence of VL from P1-068761_E32Y_E55A;

[0557] (ww) VH, which is composed of the amino acid sequence of VH from P1-068761_E32Y_E56N, and VL, which is composed of the amino acid sequence of VL from P1-068761_E32Y_E56N;

[0558] (xx) VH, which is composed of the amino acid sequence of VH of P1-068761_E30D_E32Y, and VL, which is composed of the amino acid sequence of VL of P1-068761_E30D_E32Y;

[0559] (yy) VH is composed of the amino acid sequence of VH from P1-068761_E32Y_H100G and VL is composed of the amino acid sequence of VL from P1-068761_E32Y_H100G;

[0560] (zz) VH, which is composed of the amino acid sequence of VH from P1-068761_E32Y_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068761_E32Y_E100fF;

[0561] (aaa) VH is composed of the amino acid sequence of VH from P1-068767_D52N_D102V and VL is composed of the amino acid sequence of VL from P1-068767_D52N_D102V;

[0562] (bbb) VH, which is composed of the amino acid sequence of VH from P1-068767_D52N, and VL, which is composed of the amino acid sequence of VL from P1-068767_D52N;

[0563] (ccc) VH, which is composed of the amino acid sequence of VH from P1-068767_D52N_E55A, and VL, which is composed of the amino acid sequence of VL from P1-068767_D52N_E55A;

[0564] (ddd) VH is composed of the amino acid sequence of VH from P1-068767_E55A_D102V and VL is composed of the amino acid sequence of VL from P1-068767_E55A_D102V;

[0565] (eee) VH is composed of the amino acid sequence of VH of P1-068767_D102V and VL is composed of the amino acid sequence of VL of P1-068767_D102V;

[0566] (fff) VH is composed of the amino acid sequence of VH from P1-068767_E55A and VL is composed of the amino acid sequence of VL from P1-068767_E55A.

[0567] (ggg) VH is composed of the amino acid sequence of VH from P1-068767_E30D_D52N and VL is composed of the amino acid sequence of VL from P1-068767_E30D_D52N;

[0568] (hhh) VH is composed of the amino acid sequence of VH from P1-068767_E30D_D102V and VL is composed of the amino acid sequence of VL from P1-068767_E30D_D102V;

[0569] (iii) VH consisting of the amino acid sequence of VH from P1-068767_E30D and VL consisting of the amino acid sequence of VL from P1-068767_E30D;

[0570] (jjj) VH is composed of the amino acid sequence of VH from P1-068767_E30D_E55A and VL is composed of the amino acid sequence of VL from P1-068767_E30D_E55A;

[0571] (kkk) VH is composed of the amino acid sequence of VH from P1-068767_E100fF_D102V and VL is composed of the amino acid sequence of VL from P1-068767_E100fF_D102V;

[0572] (lll) VH, which is composed of the amino acid sequence of VH from P1-068767_E55A_E100fF, and VL, which is composed of the amino acid sequence of VL from P1-068767_E55A_E100fF;

[0573] (mmm) VH is composed of the amino acid sequence of VH from P1-068767_D52N_E100fF and VL is composed of the amino acid sequence of VL from P1-068767_D52N_E100fF;

[0574] (nnn) VH is composed of the amino acid sequence of VH from P1-068767_E100fF and VL is composed of the amino acid sequence of VL from P1-068767_E100fF;

[0575] (ooo) VH is composed of the amino acid sequence of VH from P1-068767_E30D_E100fF and VL is composed of the amino acid sequence of VL from P1-068767_E30D_E100fF;

[0576] (ppp) VH, which is composed of the amino acid sequence of VH from P1-061029_F100fE_V102D, and VL, which is composed of the amino acid sequence of VL from P1-061029_F100fE_V102D;

[0577] (qqq) VH is composed of the amino acid sequence of VH from P1-061029_F100fE and VL is composed of the amino acid sequence of VL from P1-061029_F100fE;

[0578] (rrr) VH is composed of the amino acid sequence of VH from P1-061029_V102D and VL is composed of the amino acid sequence of VL from P1-061029_V102D;

[0579] (sss) VH is composed of the amino acid sequence of VH from P1-061029_Y32E and VL is composed of the amino acid sequence of VL from P1-061029_Y32E;

[0580] (ttt)VH is composed of the amino acid sequence of VH from P1-061029_Y32E_F100fE and VL is composed of the amino acid sequence of VL from P1-061029_Y32E_F100fE;

[0581] (uuu) VH is composed of the amino acid sequence of VH from P1-068744_E31S and VL is composed of the amino acid sequence of VL from P1-068744_E31S.

[0582] (vvv) VH is composed of the amino acid sequence of VH from P1-068744_H50I and VL is composed of the amino acid sequence of VL from P1-068744_H50I.

[0583] (www) VH, which is composed of the amino acid sequence of VH of P1-068744_E59Y, and VL, which is composed of the amino acid sequence of VL of P1-068744_E59Y;

[0584] (xxx) VH is composed of the amino acid sequence of VH from P1-068744_E100S and VL is composed of the amino acid sequence of VL from P1-068744_E100S;

[0585] (yyy) VH is composed of the amino acid sequence of VH of P1-068744_E102Y and VL is composed of the amino acid sequence of VL of P1-068744_E102Y;

[0586] (zzz) VH consisting of the amino acid sequence of VH of P1-068744_E31S_H50I and VL containing P1-068744_E31S_H50I;

[0587] (aaaa) VH containing the amino acid sequence of VH of P1-068744_H50I_E59Y and VL consisting of the amino acid sequence of VL of P1-068744_H50I_E59Y;

[0588] (bbbb) VH is composed of the amino acid sequence of VH from P1-068744_E59Y_E100S and VL is composed of the amino acid sequence of VL from P1-068744_E5;9Y_E100S;

[0589] (cccc) VH is composed of the amino acid sequence of VH of P1-068744_E100S_E102Y and VL is composed of the amino acid sequence of VL of P1-068744_E100S_E102Y;

[0590] (dddd) VH is composed of the amino acid sequence of VH of P1-068744_E31S_E102Y and VL is composed of the amino acid sequence of VL of P1-068744_E31S_E102Y;

[0591] (eeee) VH is composed of the amino acid sequence of VH of P1-068744_E31S_E59Y and VL is composed of the amino acid sequence of VL of P1-068744_E31S_E59Y.

[0592] (ffff) VH is composed of the amino acid sequence of VH from P1-068744_E31S_E100S and VL is composed of the amino acid sequence of VL from P1-068744_E31S_E100S.

[0593] (gggg) VH is composed of the amino acid sequence of VH from P1-068744_H50I_E100S and VL is composed of the amino acid sequence of VL from P1-068744_H50I_E100S.

[0594] (hhhh) VH is composed of the amino acid sequence of VH from P1-068744_H50I_E102Y and VL is composed of the amino acid sequence of VL from P1-068744_H50I_E102Y;

[0595] (iiii) VH consisting of the amino acid sequence of VH of P1-068744_E59Y_E102Y and VL consisting of the amino acid sequence of VL of P1-068744_E59Y_E102Y;

[0596] (jjjj) VH is composed of the amino acid sequence of VH from P1-068748_H31S and VL is composed of the amino acid sequence of VL from P1-068748_H31S.

[0597] (kkkk) VH is composed of the amino acid sequence of VH of P1-068748_H32Y and VL is composed of the amino acid sequence of VL of P1-068748_H32Y;

[0598] (llll) VH is composed of the amino acid sequence of VH from P1-068748_D57K and VL is composed of the amino acid sequence of VL from P1-068748_D57K.

[0599] (mmmm) VH is composed of the amino acid sequence of VH of P1-068748_D58Y and VL is composed of the amino acid sequence of VL of P1-068748_D58Y.

[0600] (nnnn) VH is composed of the amino acid sequence of VH from P1-068748_D100S and VL is composed of the amino acid sequence of VL from P1-068748_D100S.

[0601] (oooo) VH is composed of the amino acid sequence of VH of P1-068748_H31S_H32Y and VL is composed of the amino acid sequence of VL of P1-068748_H31S_H32Y;

[0602] (pppp) VH is composed of the amino acid sequence of VH from P1-068748_H32Y_D57K and VL is composed of the amino acid sequence of VL from P1-068748_H32Y_D57K;

[0603] (qqqq) VH is composed of the amino acid sequence of VH of P1-068748_D57K_D58Y and VL is composed of the amino acid sequence of VL of P1-068748_D57K_D58Y;

[0604] (rrrr) VH is composed of the amino acid sequence of VH from P1-068748_D58Y_D100S and VL is composed of the amino acid sequence of VL from P1-068748_D58Y_D100S;

[0605] (ssss) VH is composed of the amino acid sequence of VH from P1-068748_H31S_D57K and VL is composed of the amino acid sequence of VL from P1-068748_H31S_D57K.

[0606] (tttt)VH is composed of the amino acid sequence of VH of P1-068748_H31S_D58Y and VL is composed of the amino acid sequence of VL of P1-068748_H31S_D58Y;

[0607] (uuuu) VH is composed of the amino acid sequence of VH from P1-068748_H31S_D100S and VL is composed of the amino acid sequence of VL from P1-068748_H31S_D100S.

[0608] (vvvv) VH is composed of the amino acid sequence of VH of P1-068748_H32Y_D58Y and VL is composed of the amino acid sequence of VL of P1-068748_H32Y_D58Y;

[0609] (www) VH, composed of the amino acid sequence of VH from P1-068748_H32Y_D100S, and VL, composed of the amino acid sequence of VL from P1-068748_H32Y_D100S; or

[0610] (xxxx) VH, composed of the amino acid sequence of VH from P1-068748_D57K_D100S, and VL, composed of the amino acid sequence of VL from P1-068748_D57K_D100S.

[0611] Optionally, the VH in any of (a) or (c)-(ttt) contains one or both of heavy chain K16R and T84A substitutions, and optionally, the VL in any of (b) or (uuu)-(xxxx) contains T85V substitutions.

[0612] In some embodiments, anti-VISTA Ab includes any of the variable regions and / or variable regions CDR 1-3 of the antibodies described above and elsewhere herein, such as:

[0613] (1) One or more of the following VH CDR1, CDR2 and CDR3:

[0614] (2) VH CDR1, CDR2 and CDR3 for the following items:

[0615] (3) VH for the following items:

[0616] (4) One or more of the following VH CDR1, CDR2 and CDR3 and one or more of the following VL CDR1, CDR2 and CDR3:

[0617] (5) VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 for the following items:

[0618] (6) VH and VL for the following items:

[0619] or

[0620] (7) The following VL (optionally, except for the T85V substitution in the VL of P1-061015 or its offspring) and VH (optionally, except for one or both of the K16R and T84A substitutions in the VH in the case of P1-061029 or its offspring):

[0621] 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_Y 32E, 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_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_H3 2Y, 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; and the anti-VISTA Ab is also an IgG antibody, such as IgG1, IgG2, IgG3, or IgG4 antibody, or its modified form as described in the following sections. In some embodiments, the constant region has effector functionality, and in some embodiments, the constant region is effector-free. In some implementations, the constant region is the constant region of IgG1.3.

[0622] In some embodiments, anti-VISTA Ab includes any of the variable regions and / or variable regions CDR 1-3 of the antibodies described above and elsewhere herein, such as:

[0623] (1) One or more of the following VH CDR1, CDR2 and CDR3:

[0624] (2) VH CDR1, CDR2 and CDR3 for the following items:

[0625] (3) VH for the following items:

[0626] (4) One or more of the following VH CDR1, CDR2 and CDR3 and one or more of the following VL CDR1, CDR2 and CDR3:

[0627] (5) VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 for the following items:

[0628] (6) VH and VL for the following items:

[0629] or

[0630] (7) VL and VH of the following, except (in the case of P1-061029 and its offspring) one or both of the K16R and T84A substitutions in VH:

[0631] 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_E32YE56N、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_E100fFD102V、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、P1-68744E59Y、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_H32Y、P1-068748_H32Y_D57K、P1-068748_D57K_D58Y、P1-068748_D58Y_D100S、P1-068748_H31S_D57K、P1-068748H31S D58Y、P1-068748_H31SD100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S or P1-068748_D57K_D100S

[0632] It also includes one or more of the following characteristics:

[0633] -Specifically binds to hVISTA, such as the histidine-rich region of ECD or a polypeptide containing amino acid residues 35-127 of SEQ ID NO: 2, at acidic pH such as pH 6.0 or pH 6.5;

[0634] - Lack of significant binding to histidine-rich regions of hVISTA, such as ECD, or peptides containing amino acid residues 35-127 of SEQ ID NO: 2 at physiological or neutral pH, such as pH 7.4 or pH 7.0;

[0635] - It specifically binds to histidine-rich regions of cynomolgus monkey VISTA, such as ECD, at acidic pH levels, such as pH 6.0 or pH 6.5;

[0636] - Lack of significant binding to histidine-rich regions of cynomolgus monkey VISTA, such as ECD, at physiological or neutral pH levels, such as pH 7.4 or pH 7.0;

[0637] - Compared to hVISTA ECD having SEQ ID NO: 2, it has reduced binding with hVISTA-ECD having substitutions 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;

[0638] - Cross-competing with P1-061029, P1-068761, P1-068767, P1-061015, P1-068744 and / or P1-068748 for combination with hVISTA;

[0639] - Inhibits the binding of hVISTA to human T cells (e.g., primordial or activated T cells) that express VISTA at acidic pH levels such as pH 6.0 or pH 6.5;

[0640] - Inhibit the binding of hVISTA to PSGL-1 at an acidic pH such as pH 6.0 or pH 6.5 (e.g., inhibit the interaction between H153 and H154 of hVISTA having SEQ ID NO: 1 and tyrosines Y46 and Y48 of PSGL-1), wherein PSGL-1 has or does not have sialylated Lewis X, and wherein the tyrosine is preferably sulfotyrosine;

[0641] -Inhibits the binding of hVISTA to VSIG-3;

[0642] - For example, the average dwell time (MRT) measured in cynomolgus monkeys for at least 100, 200, 300, 350, 400, 450, 500, 600 or 700 hours (e.g. at least 350 hours) as described in the embodiments;

[0643] - Stimulate T cell activation by, for example, enhancing T cell proliferation; enhancing IFN-γ production from T cells; and / or stimulating T cell receptor-mediated NF-κB signaling;

[0644] - Inhibits VISTA-mediated cell adhesion;

[0645] -Specifically binds to hVISTA in human tumor cell samples or inflamed human tissue samples that express VISTA;

[0646] - As determined, for example, by yeast surface display and NGS assays as described in the examples, hVISTA is contacted by one or more (e.g., at least 1-3, 1-5, 1-10, 5-10, 5-15 or all) energy-important contact residues Y37, T39, R54, F62, H66, V117, I119 or S124 (see Table 21); and wherein the number is the number of a mature hVISTA (see SEQ ID NO: 2);

[0647] - As determined by MS-HDX as described in the examples, binding to region 1 of hVISTA having SEQ ID NO: 1: 57 LGPVDKGHDVTF 68 (SEQ ID NO: 566); Region 2: 86 RRPIRNLTFQDL 97 (SEQ ID NO: 567); and region 3: 148 VVEIRHHHSEHRVHGAME 165 (SEQ ID NO: 568), and optionally, the binding with region 2 is the strongest;

[0648] - As determined, for example, by lentigraphy as described in the embodiments, binding to the histidine-rich β-sheet extension of hVISTA;

[0649] - As determined, for example, by crystalline radiography as described in the embodiments, such as by hydrogen bonding contact of mature hVISTA's H121, H122, and / or H123 (4.0 Å) (or even a smaller distance);

[0650] - Contact hVISTA via at least one or more glutamic acid, aspartic acid or histidine residues located in VH CDR1, CDR2 or CDR3;

[0651] - As described in the examples, it accumulates better in tumor tissues of human VISTA knock-in mice with MC38 tumors than myeloid cells, blood, and / or liver, lung, and spleen cells;

[0652] - It accumulates better in the patient's tumor than myeloid cells, blood, and / or liver, lung, and spleen cells (e.g., by administration of PET-traceable Abs for detection).

[0653] -As determined by surface plasmon resonance (SPR) as described in Example 30 herein, compared to its binding K with hVISTA at neutral or physiological pH. D Or K dissociates, under acidic conditions (e.g., at pH 6.5) at most 1 / 10, 1 / 100, or 1 / 1000 of K. D (and / or k dissociation) specifically binds to hVISTA;

[0654] - Inhibit the binding of hVISTA to cells that bind to hVISTA (e.g., cells expressing PSGL-1), wherein the binding is determined according to the protocol described in Example 31 herein (optionally, wherein the cells are human leukocytes, PBMCs, or T cells).

[0655] - Blocking or cross-blocking the binding of any of the antibodies described herein, as determined using competitive SPR epitope segmentation as described in Example 32 herein; and

[0656] - Any additional features set forth in the claims and / or embodiments.

[0657] In some embodiments, the anti-hVISTA Ab comprises a heavy chain (HC) containing the amino acid sequence of any of the heavy chains of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a heavy chain containing the amino acid sequence of a heavy chain containing: P1-061029 or P1-061015 or their progeny, such as P1-061029.IgG1.3 (SEQ ID NO. 1.3), which contains the constant region of the IgG1.3 heavy chain. NO: 69), P1-068757.IgG1.3, P1-068759.IgG1.3, P1-068761.IgG1.3, P1-068763.IgG1.3, P1-068765.IgG1.3, P1-068767 .IgG1.3, P1-068769.IgG1.3, P1-068771.IgG1.3, P1-068773.IgG1.3, P1-068775.IgG1.3, P1-069059.IgG1.3, P1-06906 1.IgG1.3, P1-069063.IgG1.3, P1-069065.IgG1.3, P1-069067.IgG1.3, P1-069069.IgG1.3, P1-069071.IgG1.3, P1-0690 73.IgG1.3, P1-069075.IgG1.3, P1-069077.IgG1.3, P1-061015.IgG1.3, P1-068736.IgG1.3, P1-068738.IgG1.3, P1-0687 40.IgG1.3, P1-068742.IgG1.3, P1-068744.IgG1.3, P1-068766.IgG1.3, P1-068748.IgG1.3, P1-068750.IgG1.3, P1-068 752.IgG1.3, P1-068754.IgG1.3, P1-068761_E55A.IgG1.3, P1-068761_H100G.IgG1.3, P1-068761_E56N.IgG1.3, P1-0687 61_E55A_E56N.IgG1.3, P1-068761_E30D.IgG1.3, P1-068761_E30D_E55A.IgG1.3, P1-068761_E56N_H100G.IgG1.3, P1-0 68761_E30D_H100G.IgG1.3, P1-068761_E30D_E56N.IgG1.3, P1-068761_E100fF.IgG1.3, P1-068761_E55A_E100fF.IgG1.3、P1-068761_H100G_E100fF.IgG1.3、P1-068761_E30D_E100fF.IgG1.3、P1-068761_E56N_E100fF.IgG1.3、P1-068761_E32Y.IgG1.3、P1-068761_E32Y_E55A.IgG1.3、P1-068761_E32Y_E56N.IgG1.3、P1-068761_E30D_E32Y.IgG1.3、P1-068761_E32Y_H100G.IgG1.3、P1-068761_E32Y_E100fF.IgG1.3、P1-068767_D52N_D102V.IgG1.3、P1-068767_D52N.IgG1.3、P1-068767_D52N_E55A.IgG1.3、P1-068767_E55A_D102V.IgG1.3、P1-068767_D102V.IgG1.3、P1-068767_E55A.IgG1.3、P1-068767_E30D_D52N.IgG1.3、P1-068767_E30D_D102V.IgG1.3、P1-068767_E30D.IgG1.3、P1-068767_E30D_E55A.IgG1.3、P1-068767_E100fF_D102V.IgG1.3、P1-068767_E55A_E100fF.IgG1.3、P1-068767_D52N_E100fF.IgG1.3、P1-068767_E100fF.IgG1.3、P1-068767_E30D_E100fF.IgG1.3、P1-061029_F100fE_V102D.IgG1.3、P1-061029_F100fE.IgG1.3、P1-061029_V102D.IgG1.3、P1-061029_Y32E.IgG1.3、P1-061029_Y32E_F100fE.IgG1.3. P1-068744_E3 1S, 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, P1-068744E31S_E100S, P1-068744_H50I_E100S, P1-068744_H50I_E1 02Y, 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_D 58Y, P1-068748_D58Y_D100S, P1-068748_H31S_D57K, P1-068748_H31S_D58Y, P1-068748_H31S The sequence is D100S, P1-068748_H32Y_D58Y, P1-068748_H32Y_D100S, or P1-068748_D57K_D100S. Optionally, for the P1-061029 offspring, VH contains one or both of K16R and T84A substitutions, and for the P1-061015 offspring, VL contains T85V substitution.

[0658] In some embodiments, the anti-hVISTA Ab comprises a heavy chain containing the amino acid sequence of the heavy chain of any anti-hVISTA Ab provided herein, which includes the IgG1.3 heavy chain constant region, and the amino acid sequence of the light chain of any anti-hVISTA Ab provided herein. In some embodiments, the anti-hVISTA Ab comprises a heavy chain containing the amino acid sequence of VH of: P1-061029 or P1-061015 or their progeny, such as P1-061029.IgG1.3 (SEQ ID NO: 1.3). NO: 69), P1-068757.IgG1.3, P1-068759.IgG1.3, P1-068761.IgG1.3, P1-068763.IgG1.3, P1-068765.IgG1.3, P1-06 8767.IgG1.3, P1-068769.IgG1.3, P1-068771.IgG1.3, P1-068773.IgG1.3, P1-068775.IgG1.3, P1-069059.IgG1.3, P 1-069061.IgG1.3, P1-069063.IgG1.3, P1-069065.IgG1.3, P1-069067.IgG1.3, P1-069069.IgG1.3, P1-069071.IgG 1.3, P1-069073.IgG1.3, P1-069075.IgG1.3, P1-069077.IgG1.3, P1-061015.IgG1.3, P1-068736.IgG1.3, P1-068738 .IgG1.3, P1-068740.IgG1.3, P1-068742.IgG1.3, P1-068744.IgG1.3, P1-068766.IgG1.3, P1-068748.IgG1.3, P1-0 68750.IgG1.3, P1-068752.IgG1.3, P1-068754.IgG1.3, P1-068761_E55A.IgG1.3, P1-068761_H100G.IgG1.3, P1-068 761_E56N.IgG1.3, P1-068761_E55A_E56N.IgG1.3, P1-068761_E30D.IgG1.3, P1-068761_E30D_E55A.IgG1.3, P1-068 761_E56N_H100G.IgG1.3, P1-068761_E30D_H100G.IgG1.3, P1-068761_E30D_E56N.IgG1.3, P1-068761E100fF.IgG1.3、P1-068761_E55A_E100fF.IgG1.3、P1-068761_H100G_E100fF.IgG1.3、P1-068761_E30D_E100fF.IgG1.3、P1-068761_E56N_E100fF.IgG1.3、P1-068761_E32Y.IgG1.3、P1-068761_E32Y_E55A.IgG1.3、P1-068761_E32Y_E56N.IgG1.3、P1-068761_E30D_E32Y.IgG1.3、P1-068761_E32Y_H100G.IgG1.3、P1-068761_E32Y_E100fF.IgG1.3、P1-068767_D52N_D102V.IgG1.3、P1-068767_D52N.IgG1.3、P1-068767_D52N_E55A.IgG1.3、P1-068767_E55A_D102V.IgG1.3、P1-068767_D102V.IgG1.3、P1-068767_E55A.IgG1.3、P1-068767_E30D_D52N.IgG1.3、P1-068767_E30D_D102V.IgG1.3、P1-068767_E30D.IgG1.3、P1-068767_E30D_E55A.IgG1.3、P1-068767_E100fF_D102V.IgG1.3、P1-068767_E55A_E100fF.IgG1.3、P1-068767_D52N_E100fF.IgG1.3、P1-068767_E100fF.IgG1.3、P1-068767_E30D_E100fF.IgG1.3、P1-061029_F100fE_V102D.IgG1.3、P1-061029_F100fE.IgG1.3、P1-061029_V102D.IgG1.3、P1-061029_Y32E.IgG1.3、P1-061029_Y32E_F100fE.IgG1.3、P1-068744_E31S.IgG1.3、P1-68744_H50I.IgG1.3、P1-68744_E59Y.IgG1.3、P1-068744_E100S.IgG1.3、P1-068744_E102Y.IgG1.3、P1-068744_E31S_H50I.IgG1.3、P1-068744_H50I_E59Y.IgG1.3、P1-068744_E59Y_E100S.IgG1.3. P1-068744_E100S_E102Y.IgG1.3, P1-068744_E31S_E102Y.IgG1.3, P1-068744_E31S_E59Y.IgG1.3, P1-068744_E31S_E100S.IgG1.3, P1-068744_H50I_E100S.IgG1.3, P1-068744_H50I_E102Y.IgG1.3, P1- 068744_E59Y_E102Y.IgG1.3, P1-068748_H31S, P1-068748_H32Y.IgG1.3, P1-068748_D57K.IgG1.3, P1 -068748_D58Y.IgG1.3, P1-068748_D100S.IgG1.3, P1-068748_H31S_H32Y.IgG1.3, P1-068748_H32Y_D5 7K.IgG1.3, P1-068748_D57K_D58Y.IgG1.3, P1-068748_D58Y_D100S.IgG1.3, P1-068748_H31S_D57K.I gG1.3, P1-068748_H31S_D58Y.IgG1.3, P1-068748_H31S_D100S.IgG1.3, P1-068748_H32Y_D58Y.IgG1.3 The light chain contains either P1-068748_H32Y_D100S.IgG1.3 or P1-068748_D57K_D100S.IgG1.3, and optionally, for the P1-061029 progeny, the VH sequence contains one or both of the K16R and T84A substitutions; and a light chain containing the amino acid sequence of the light chain of P1-061029 or P1-061015, and optionally, for the P1-061015 progeny, the VL sequence contains the T85V substitution.

[0659] Anti-hVISTA Ab may include:

[0660] (a) The heavy chain containing the amino acid sequence of the heavy chain of P1-061029.IgG1.3 (SEQ ID NO: 69) and the light chain containing the amino acid sequence of the light chain of P1-061029 (SEQ ID NO: 70);

[0661] (b) The heavy chain containing the amino acid sequence of the heavy chain of P1-061015.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-061015.

[0662] (c) Heavy chain containing the amino acid sequence of the heavy chain of P1-068757.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068757.

[0663] (d) Heavy chain containing the amino acid sequence of the heavy chain of P1-068759.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068759.

[0664] (e) Heavy chains containing the amino acid sequence of the heavy chain of P1-068761.IgG1.3 and light chains containing the amino acid sequence of the light chain of P1-068761.

[0665] (f) Heavy chains containing the amino acid sequence of the heavy chain of P1-068763.IgG1.3 and light chains containing the amino acid sequence of the light chain of P1-068763.

[0666] (g) Heavy chain containing the amino acid sequence of the heavy chain of P1-068765.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068765.

[0667] (h) Heavy chain containing the amino acid sequence of the heavy chain of P1-068767.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068767.

[0668] (i) The heavy chain containing the amino acid sequence of the heavy chain of P1-068769.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068769.

[0669] (j) Heavy chain containing the amino acid sequence of the heavy chain of P1-068771.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068771.

[0670] (k) Heavy chain containing the amino acid sequence of the heavy chain of P1-068773.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068773.

[0671] (l) The heavy chain containing the amino acid sequence of the heavy chain of P1-068775.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068775.

[0672] (m) The heavy chain containing the amino acid sequence of the heavy chain of P1-069059.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-069059.

[0673] (n) Heavy chain containing the amino acid sequence of the heavy chain of P1-069061.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069061.

[0674] (o) Heavy chain containing the amino acid sequence of the heavy chain of P1-069063.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069063.

[0675] (p) Heavy chain containing the amino acid sequence of the heavy chain of P1-069065.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069065.

[0676] (q) Heavy chain containing the amino acid sequence of the heavy chain of P1-069067.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069067.

[0677] (r) Heavy chain containing the amino acid sequence of the heavy chain of P1-069069.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069069.

[0678] (s) The heavy chain containing the amino acid sequence of the heavy chain of P1-069071.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-069071.

[0679] (t) Heavy chain containing the amino acid sequence of the heavy chain of P1-069073.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069073.

[0680] (u) The heavy chain containing the amino acid sequence of the heavy chain of P1-069075.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-069075.

[0681] (v) Heavy chain containing the amino acid sequence of the heavy chain of P1-069077.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-069077.

[0682] (w) Heavy chain containing the amino acid sequence of the heavy chain of P1-068736.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068736.

[0683] (x) Heavy chain containing the amino acid sequence of the heavy chain of P1-068738.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068738.

[0684] (y) Heavy chain containing the amino acid sequence of the heavy chain of P1-068740.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068740.

[0685] (z) Heavy chain containing the amino acid sequence of the heavy chain of P1-068742.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068742.

[0686] (aa) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068744.

[0687] (bb) Heavy chain containing the amino acid sequence of the heavy chain of P1-068746.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068746.

[0688] (cc) Heavy chain containing the amino acid sequence of the heavy chain of P1-068748.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068748.

[0689] (dd) Heavy chain containing the amino acid sequence of the heavy chain of P1-068750.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068750.

[0690] (ee) The heavy chain containing the amino acid sequence of the heavy chain of P1-068752.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068752.

[0691] (ff) Heavy chain containing the amino acid sequence of the heavy chain of P1-068754.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068754.

[0692] (gg) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E55A.

[0693] (hh) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_H100G.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_H100G.

[0694] (ii) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E56N.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E56N.

[0695] (jj) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E55A_E56N.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E55A_E56N;

[0696] (kk) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E30D.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E30D.

[0697] (ll) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E30D_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E30D_E55A.

[0698] (mm) Heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E56N_H100G.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068761_E56N_H100G; (nn) Heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E30D_H100G.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068761_E30D_H100G;

[0699] (oo) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E30D_E56N.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E30D_E56N.

[0700] (pp) Heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E100fF.IgG1.3 and light chain containing the amino acid sequence of the light chain of P1-068761E100fF.

[0701] (qq) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E55A_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E55A_E100fF;

[0702] (rr) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_H100G_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_H100G_E100fF;

[0703] (ss) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E30D_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E30D_E100fF;

[0704] (tt) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E56N_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E56N_E100fF;

[0705] (uu) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E32Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E32Y.

[0706] (vv) The heavy chain containing the amino acid sequence of the heavy chain of P1-068761_E32Y_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068761_E32Y_E55A;

[0707] (ww) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E32Y_E56N.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E32Y_E56N;

[0708] (xx) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E30D_E32Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E30D_E32Y;

[0709] (yy) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E32Y_H100G.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E32Y_H100G;

[0710] (zz) The heavy chain containing the amino acid sequence of the heavy chain P1-068761_E32Y_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068761_E32Y_E100fF;

[0711] (aaa) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_D52N_D102V.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_D52N_D102V.

[0712] (bbb) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_D52N.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_D52N.

[0713] (ccc) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_D52N_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_D52N_E55A.

[0714] (ddd) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E55A_D102V.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E55A_D102V;

[0715] (eee) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_D102V.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_D102V.

[0716] (fff) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E55A.

[0717] (ggg) The heavy chain containing the amino acid sequence of the heavy chain P1-068767m30D_D52N.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068767_E30D_D52N;

[0718] (hhh) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E30D_D102V.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E30D_D102V;

[0719] (iii) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E30D.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E30D.

[0720] (jjj) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E30D_E55A.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E30D_E55A;

[0721] (kkk) The heavy chain containing the amino acid sequence of the heavy chain P1-068767_E100fF_D102V.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068767_E100fF D102V;

[0722] (lll) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_E55A_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_E55A_E100fF;

[0723] (mmm) The heavy chain containing the amino acid sequence of the heavy chain of P1-068767_D52N_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068767_D52N_E100fF;

[0724] (nnn) The heavy chain containing the amino acid sequence of the heavy chain P1-068767_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068767_E100fF;

[0725] (ooo) The heavy chain containing the amino acid sequence of the heavy chain P1-068767_E30D_E100fF.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068767_E30D_E100fF.

[0726] (ppp) The heavy chain containing the amino acid sequence of the heavy chain P1-061029_F100fE_V102D.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-061029_F100fE_V102D;

[0727] (qqq) The heavy chain containing the amino acid sequence of the heavy chain P1-061029_F100fE.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-061029_F100fE;

[0728] (rrr) The heavy chain containing the amino acid sequence of the heavy chain of P1-061029_V102D.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-061029_V102D;

[0729] (sss) The heavy chain containing the amino acid sequence of the heavy chain of P1-061029_Y32E.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-061029_Y32E;

[0730] (ttt) The heavy chain containing the amino acid sequence of the heavy chain P1-061029_Y32E_F100fE.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-061029_Y32E_F100fE;

[0731] (uuu) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744_E31S.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068744_E31S.

[0732] (vvv) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744_H50I.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068744_H50I.

[0733] (www) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E59Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E59Y.

[0734] (xxx) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E100S.

[0735] (yyy) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744_E102Y.IgG1.3 and the light chain containing the amino acid sequence of P1-068744_E102Y;

[0736] (zzz) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E31S_H50I.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E31S_H50I.

[0737] (aaaa) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744_H50I_E59Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068744_H50I_E59Y.

[0738] (bbbb) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E59Y_E100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E59Y_E100S.

[0739] (cCcc) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E100S_E102Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E100S_E102Y.

[0740] (dddd) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E31S_E102Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E31S_E102Y;

[0741] (eeee) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E31S_E59Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E31S_E59Y.

[0742] (ffff) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_E31S_E100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_E31S_E100S.

[0743] (gggg) The heavy chain containing the amino acid sequence of the heavy chain of P1-068744_H50I_E100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068744_H50I_E100S.

[0744] (hhhh) The heavy chain containing the amino acid sequence of the heavy chain P1-068744_H50I_E102Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068744_H50I_E102Y;

[0745] (iiii) Heavy chain containing the amino acid sequence of P1-068744_E59Y_E102Y.IgG1.3 and VL and light chain containing the amino acid sequence of P1-068744_E59Y_E102Y;

[0746] (jjjj) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H31S.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H31S.

[0747] (kkkk) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_H32Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_H32Y;

[0748] (llll) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_D57K.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_D57K.

[0749] (mmmm) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_D58Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_D58Y.

[0750] (nnnn) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_D100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_D100S.

[0751] (oooo) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H31S_H32Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H31S_H32Y;

[0752] (pPPP) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_H32Y_D57K.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_H32Y_D57K;

[0753] (qqqq) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_D57K_D58Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_D57K_D58Y;

[0754] (rrrr) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_D58Y_D100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_D58Y_D100S.

[0755] (ssss) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H31S_D57K.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H31S_D57K.

[0756] (tttt) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H31S_D58Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H31S_D58Y.

[0757] (uuuu) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H31S_D100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H31S_D100S.

[0758] (vvvv) The heavy chain containing the amino acid sequence of the heavy chain of P1-068748_H32Y_D58Y.IgG1.3 and the light chain containing the amino acid sequence of the light chain of P1-068748_H32Y_D58Y;

[0759] (www) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_H32Y_D100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_H32Y_D100S; or

[0760] (xxxx) The heavy chain containing the amino acid sequence of the heavy chain P1-068748_D57K_D100S.IgG1.3 and the light chain containing the amino acid sequence of the light chain P1-068748_D57K_D100S.

[0761] Optionally, the VH in any of (a) or (c) to (ttt) contains one or both of K16R and T84A substitutions, and optionally, the VL in any of (b) or (uuu) to (xxxx) contains T85V substitutions.

[0762] Anti-hVISTA Ab may include:

[0763] (a) The heavy chain (HC) of the HC CDR containing P1-061029 and the light chain (LC) of the LC CDR containing P1-061029, and the HC and LC amino acid sequences 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 to the HC and LC of P1-061029.IgG1.3, respectively;

[0764] (b) HC containing HC CDR of P1-061015 and LC containing LC CDR of P1-061015, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-061015.IgG1.3, respectively.

[0765] (c) HC containing HC CDR of P1-068757 and LC containing LC CDR of P1-068757, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068757.IgG1.3, respectively.

[0766] (d) HC containing HC CDR of P1-068759 and LC containing LC CDR of P1-068759, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068759.IgG1.3, respectively.

[0767] (e) HC containing HC CDR of P1-068761 and LC containing LC CDR of P1-068761, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761.IgG1.3, respectively.

[0768] (f) HC containing HC CDR of P1-068763 and LC containing LC CDR of P1-068763, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068763.IgG1.3, respectively.

[0769] (g) HC containing HC CDR of P1-068765 and LC containing LC CDR of P1-068765, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068765.IgG1.3, respectively.

[0770] (h) HC containing HC CDR of P1-068767 and LC containing LC CDR of P1-068767, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068767.IgG1.3, respectively.

[0771] (i) HC containing HC CDR of P1-068769 and LC containing LC CDR of P1-068769, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068769.IgG1.3, respectively;

[0772] (j) HC containing HC CDR of P1-068771 and LC containing LC CDR of P1-068771, and HC and LC amino acid sequences 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 to the amino acid sequences of P1-068771.IgG1.3, respectively.

[0773] (k) HC containing HC CDR of P1-068773 and LC containing LC CDR of P1-068773, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068773.IgG1.3, respectively.

[0774] (l) HC containing HC CDR of P1-068775 and LC containing LC CDR of P1-068775, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068775.IgG1.3, respectively;

[0775] (m) HC containing HC CDR of P1-069059 and LC containing LC CDR of P1-069059, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069059.IgG1.3, respectively.

[0776] (n) HC containing HC CDR of P1-069061 and LC containing LC CDR of P1-069061, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC of P1-069061.IgG1.3, respectively.

[0777] (o) HC containing HC CDR of P1-069063 and LC containing LC CDR of P1-069063, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069063.IgG1.3, respectively.

[0778] (p) HC containing HC CDR of P1-069065 and LC containing LC CDR of P1-069065, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069065.IgG1.3, respectively.

[0779] (q) HC containing HC CDR of P1-069067 and LC containing LC CDR of P1-069067, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069067.IgG1.3, respectively.

[0780] (r) HC containing HC CDR of P1-069069 and LC containing LC CDR of P1-069069, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069069.IgG1.3, respectively.

[0781] (s) HC containing HC CDR of P1-069071 and LC containing LC CDR of P1-069071, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC of P1-069071.IgG1.3, respectively.

[0782] (t) HC containing HC CDR of P1-069073 and LC containing LC CDR of P1-069073, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069073.IgG1.3, respectively.

[0783] (u) HC containing HC CDR of P1-069075 and LC containing LC CDR of P1-069075, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069075.IgG1.3, respectively.

[0784] (v) HC containing HC CDR of P1-069077 and LC containing LC CDR of P1-069077, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-069077.IgG1.3, respectively.

[0785] (w) HC containing HC CDR of P1-068736 and LC containing LC CDR of P1-068736, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068736.IgG1.3, respectively.

[0786] (x) HC containing HC CDR of P1-068738 and LC containing LC CDR of P1-068738, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068738.IgG1.3, respectively.

[0787] (y) HC containing HC CDR of P1-068740 and LC containing LC CDR of P1-068740, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068740.IgG1.3, respectively.

[0788] (z) HC containing HC CDR of P1-068742 and LC containing LC CDR of P1-068742, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068742.IgG1.3, respectively.

[0789] (aa) HC containing HC CDR of P1-068744 and LC containing LC CDR of P1-068744, and HC and LC amino acid sequences 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 to the amino acid sequences of P1-068744.IgG1.3, respectively;

[0790] (bb) HC containing HC CDR of P1-068746 and LC containing LC CDR of P1-068746, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068746.IgG1.3, respectively.

[0791] (cc) HC containing HC CDR of P1-068748 and LC containing LC CDR of P1-068748, and HC and LC amino acid sequences 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 to the amino acid sequences of P1-068748.IgG1.3, respectively.

[0792] (dd) HC containing HC CDR of P1-068750 and LC containing LC CDR of P1-068750, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068750.IgG1.3, respectively.

[0793] (ee) HC containing HC CDR of P1-068752 and LC containing LC CDR of P1-068752, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068752.IgG1.3, respectively.

[0794] (ff) HC containing HC CDR of P1-068754 and LC containing LC CDR of P1-068754, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068754.IgG1.3, respectively.

[0795] (gg) HC containing HC CDR of P1-068761_E55A.IgG1.3 and LC containing LCCDR of P1-068761_E55A, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E55A.IgG1.3, respectively.

[0796] (hh) HC containing HC CDR of P1-068761_H100G.IgG1.3 and LC containing LC CDR of P1-068761_H100G, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_H100G.IgG1.3, respectively;

[0797] (ii) HC containing HC CDR of P1-068761_E56N.IgG1.3 and LC containing LCCDR of P1-068761_E56N, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E56N.IgG1.3, respectively;

[0798] (jj) HC containing HC CDR of P1-068761_E55A_E56N.IgG1.3 and LC containing LC CDR of P1-068761_E55A_E56N, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E55A_E56N.IgG1.3, respectively;

[0799] (kk) HC containing HC CDR of P1-068761_E30D.IgG1.3 and LC containing LCCDR of P1-068761_E30D, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E30D.IgG1.3, respectively;

[0800] (l1) HC containing HC CDR of P1-068761_E30D_E55A.IgG1.3 and LC containing LC CDR of P1-068761_E30D_E55A, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E30D_E55A.IgG1.3, respectively;

[0801] (mm) HC containing HC CDR of P1-068761_E56N_H100G.IgG1.3 and LC containing LC CDR of P1-068761_E56N_H100G, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E56N_H100G.IgG1.3, respectively.

[0802] (nn) HC containing HC CDR of P1-068761_E30D_H100G.IgG1.3 and LC containing LC CDR of P1-068761_E30D_H100G, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E30D_H100G.IgG1.3, respectively;

[0803] (oo) HC containing HC CDR of P1-068761_E30D_E56N.IgG1.3 and LC containing LC CDR of P1-068761_E30D_E56N, and HC and LC amino acid sequences 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 to the amino acid sequences of HC and LC containing P1-068761_E30D_E56N.IgG1.3, respectively;

[0804] (pp) HC containing HC CDR of P1-068761_E100fF.IgG1.3 and LC containing LC CDR of P1-068761_E100fF, and HC and LC amino acid sequences 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 to the amino acid sequences ...

Claims

1. An antibody that specifically binds to human VISTA (hVISTA), wherein the antibody comprises heavy chain variable regions (VH) CDR1, CDR2 and CDR3 and light chain variable regions (VL) CDR1, CDR2 and CDR3, wherein the heavy chain variable regions (VH) CDR1, CDR2 and CDR3 are composed of amino acid sequences of residues 26-35, 50-66 and 99-110 of SEQ ID NO: 586, respectively, and the light chain variable regions (VL) CDR1, CDR2 and CDR3 are composed of amino acid sequences of residues 24-35, 51-57 and 90-98 of SEQ ID NO: 96, respectively.

2. The antibody of claim 1, wherein the antibody comprises a VH containing at least 90%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 586; or VH contains an amino acid sequence modified by substitution of 1, 2, 3, 4 or 5 amino acids, which is SEQ ID NO:

586.

3. The antibody of claim 1, wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO:

586.

4. The antibody of claim 1, wherein the antibody comprises a VL containing at least 90%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

96.

5. The antibody of claim 1, wherein the antibody comprises a VH containing an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 586 modified by substitution of 1, 2, 3, 4, or 5 amino acids; and wherein the antibody comprises a VL containing an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

96.

6. The antibody of claim 1, wherein the antibody comprises a VL containing the amino acid sequence of SEQ ID NO:

96.

7. The antibody of claim 1, wherein the antibody comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 586, and wherein the antibody comprises VL comprising the amino acid sequence of SEQ ID NO:

96.

8. The antibody of claim 4, wherein the antibody comprises a VL containing the amino acid sequence modified by T85V substitution of SEQ ID NO:

96.

9. The antibody according to any one of claims 1-7, wherein the antibody comprises a heavy chain constant region containing an amino acid sequence of SEQ ID NO: 182, 183 or 184.

10. The antibody according to any one of claims 1-7, wherein the antibody comprises a light chain (LC) containing an amino acid sequence of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO: 98 modified by T85V substitution.

11. The antibody according to any one of claims 1-7, wherein the antibody binds at or near a histidine-rich region of hVISTA.

12. The antibody of claim 11, wherein the antibody binds at or near the histidine-rich β-sheet extension of hVISTA at a pH of 6.0-6.

5.

13. The antibody of any one of claims 1-7, wherein the antibody competes or cross-competes with an antibody comprising the following VH and VL amino acid sequences: VH comprising the amino acid sequence of SEQ ID NO: 95 and VL comprising the amino acid sequence of SEQ ID NO: 96; VH comprising the amino acid sequence of SEQ ID NO: 103 and VL comprising the amino acid sequence of SEQ ID NO: 104; VH comprising the amino acid sequence of SEQ ID NO: 99 and VL comprising the amino acid sequence of SEQ ID NO: 100; VH comprising the amino acid sequence of SEQ ID NO: 570 and VL comprising the amino acid sequence of SEQ ID NO: 572; VH comprising the amino acid sequence of SEQ ID NO: 574 and VL comprising the amino acid sequence of SEQ ID NO: 576; VH comprising the amino acid sequence of SEQ ID NO: 578 and VL comprising the amino acid sequence of SEQ ID NO: 580; VH comprising the amino acid sequence of SEQ ID NO: 582 and VL ...5; VH comprising the amino acid sequence of SEQ ID NO: 576 and VL comprising the amino acid sequence of SEQ ID NO: 578; VH comprising the amino acid sequence of SEQ ID NO: 579 and VL comprising the amino acid sequence of SEQ ID NO: 579; VH comprising the amino acid sequence of SEQ ID NO: 579 and VL comprising the amino acid sequence of SEQ ID NO: 579; VH comprising the VL containing the amino acid sequence of SEQ ID NO: 584; VH containing the amino acid sequence of SEQ ID NO: 586 and VL containing the amino acid sequence of SEQ ID NO: 588; VH containing the amino acid sequence of SEQ ID NO: 590 and VL containing the amino acid sequence of SEQ ID NO: 592; VH containing the amino acid sequence of SEQ ID NO: 594 and VL containing the amino acid sequence of SEQ ID NO: 596; VH containing the amino acid sequence of SEQ ID NO: 598 and VL containing the amino acid sequence of SEQ ID NO: 600; VH containing the amino acid sequence of SEQ ID NO: 602 and VL containing the amino acid sequence of SEQ ID NO: 604; VH containing the amino acid sequence of SEQ ID NO: 606 and VL containing the amino acid sequence of SEQ ID NO: 608; VH containing the amino acid sequence of SEQ ID NO: 610 and VL containing the amino acid sequence of SEQ ID NO: 612; VL containing the amino acid sequence of SEQ ID NO: VH containing the amino acid sequence of SEQ ID NO: 614 and VL containing the amino acid sequence of SEQ ID NO: 616; VH containing the amino acid sequence of SEQ ID NO: 618 and VL containing the amino acid sequence of SEQ ID NO: 620; VH containing the amino acid sequence of SEQ ID NO: 622 and VL containing the amino acid sequence of SEQ ID NO: 624; VH containing the amino acid sequence of SEQ ID NO: 626 and VL containing the amino acid sequence of SEQ ID NO: 628;VH containing the amino acid sequence of SEQ ID NO: 630 and VL containing the amino acid sequence of SEQ ID NO: 632; VH containing the amino acid sequence of SEQ ID NO: 634 and VL containing the amino acid sequence of SEQ ID NO: 636; VH containing the amino acid sequence of SEQ ID NO: 638 and VL containing the amino acid sequence of SEQ ID NO: 640; VH containing the amino acid sequence of SEQ ID NO: 642 and VL containing the amino acid sequence of SEQ ID NO: 644; VH containing the amino acid sequence of SEQ ID NO: 646 and VL containing the amino acid sequence of SEQ ID NO: 648; VH containing the amino acid sequence of SEQ ID NO: 650 and VL containing the amino acid sequence of SEQ ID NO: 652; VH containing the amino acid sequence of SEQ ID NO: 654 and VL containing the amino acid sequence of SEQ ID NO: 656; VH containing the amino acid sequence of SEQ ID NO: 658 and VL containing the amino acid sequence of SEQ ID NO: 652; VH containing the amino acid sequence of SEQ ID NO: 654 and VL containing the amino acid sequence of SEQ ID NO: 656; VH containing the amino acid sequence of SEQ ID NO: 658 and VL containing the amino acid sequence of SEQ ID NO: 652; VH containing the amino acid sequence of SEQ ID NO: 652 ... VL containing the amino acid sequence of SEQ ID NO: 660; VH containing the amino acid sequence of SEQ ID NO: 662 and VL containing the amino acid sequence of SEQ ID NO: 664; VH containing the amino acid sequence of SEQ ID NO: 666 and VL containing the amino acid sequence of SEQ ID NO: 668; VH containing the amino acid sequence of SEQ ID NO: 670 and VL containing the amino acid sequence of SEQ ID NO:

672.

14. The antibody according to any one of claims 1-3, wherein the antibody reduces binding to hVISTA modified by mutation of one or more 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, wherein the number is the number of a mature hVISTA having SEQ ID NO:

2.

15. The antibody according to any one of claims 1-7, wherein the antibody specifically binds to hVISTA under acidic conditions, wherein the antibody: a. Inhibit the interaction between hVISTA and T cells; PSGL-1; and / or VSIG-3; b. Enhance T cell activation by increasing T cell proliferation; enhancing IFN-γ production from T cells; and / or stimulating T cell receptor-mediated NF-κB signaling. c. HVISTA contacted by one or more of residues Y37, T39, R54, F62, H66, V117, I119 or S124, as determined by yeast surface display and NGS assay; and wherein the number is the number of a mature hVISTA having SEQ ID NO: 2; d. As determined by lentigraphy, the histidine-rich β-sheet extension bound to hVISTA; e. As determined by crystalline radiography, mature hVISTA H121, H122 and / or H123 were contacted at a distance of 4.0 Å or less; f. SEQ ID NO: 566; SEQ ID NO: 567; and SEQ ID NO: 568 of hVISTA having SEQ ID NO: 1; g. Competitively binds to hVISTA via bidirectional competition with antibodies containing the following VH and VL amino acid sequences: VH containing the amino acid sequence of SEQ ID NO: 95 and VL containing the amino acid sequence of SEQ ID NO: 96; VH containing the amino acid sequence of SEQ ID NO: 103 and VL containing the amino acid sequence of SEQ ID NO: 104; VH containing the amino acid sequence of SEQ ID NO: 99 and VL containing the amino acid sequence of SEQ ID NO: 100; VH containing the amino acid sequence of SEQ ID NO: 67 and VL containing the amino acid sequence of SEQ ID NO: 68; VH containing the amino acid sequence of SEQ ID NO: 53 and VL containing the amino acid sequence of SEQ ID NO: 54; VH containing the amino acid sequence of SEQ ID NO: 55 and VL containing the amino acid sequence of SEQ ID NO: 56; and / or VH containing the amino acid sequence of SEQ ID NO: 551 and VL containing the amino acid sequence of SEQ ID NO: 552; h. Contact hVISTA via at least one or more glutamic acid, aspartic acid, or histidine residues located in VH CDR1, CDR2, or CDR3; i. Drug treatment with low target-mediated efficacy, resulting in a mean residence time (MRT) of at least 100, 200, 300, 400, 500, 600 or 700 hours; j. In hVISTA knock-in mice with MC38 tumors, it accumulated in greater quantities in tumor tissues compared to the lungs, liver, and spleen; and / or k. In subjects who were given the antibody, it accumulated in greater quantities in tumor tissues compared to the lungs, liver, and spleen.

16. The antibody according to any one of claims 1-7, compared to its binding K to hVISTA at neutral or physiological pH. D Or koff, the antibody under acidic conditions at a K value of at most 1 / 10, 1 / 100, or 1 / 1000. D and / or koff specifically bind to hVISTA, wherein the antibody: a. Specifically binds to hVISTA at acidic pH; b. Lack of significant binding to hVISTA at physiological or neutral pH; c. Specifically binds to VISTA, a cynomolgus monkey, at acidic pH; d. Lack of significant binding to VISTA in cynomolgus monkeys at physiological or neutral pH; e. Compared to hVISTA ECD having SEQ ID NO: 2, it has reduced binding to hVISTA-ECD having substitutions 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; f. Cross-competitive binding to hVISTA with antibodies containing the following VH and VL amino acid sequences: VH containing the amino acid sequence of SEQ ID NO: 95 and VL containing the amino acid sequence of SEQ ID NO: 96; VH containing the amino acid sequence of SEQ ID NO: 103 and VL containing the amino acid sequence of SEQ ID NO: 104; and / or VH containing the amino acid sequence of SEQ ID NO: 99 and VL containing the amino acid sequence of SEQ ID NO: 100; g. Inhibits the binding of hVISTA to VISTA-expressing human T cells under acidic pH conditions; h. Inhibiting the binding of hVISTA to PSGL-1 at acidic pH, wherein PSGL-1 has or does not have sialylated Lewis X, and wherein the tyrosine is sulfotyrosine; i. Having an average residence time of at least 100, 200, 300, 350, 400, 450, 500, 600 or 700 hours in cynomolgus monkeys; j. Stimulating T cell activation by enhancing T cell proliferation; enhancing IFN-γ production from T cells; and / or stimulating T cell receptor-mediated NF-κB signaling; k. Inhibits VISTA-mediated cell adhesion; l. Specifically binds to hVISTA in human tumor cell samples or inflamed human tissue samples expressing VISTA; m. As determined by yeast surface display and NGS assay, hVISTA is contacted via one or more energy-important contact residues Y37, T39, R54, F62, H66, V117, I119, or S124; and wherein the number is the number of a mature hVISTA having SEQ ID NO: 2; n. SEQ ID NO: 566; SEQ ID NO: 567; and SEQ ID NO: 568 of hVISTA having SEQ ID NO: 1; o. As determined by lentigraphy, the histidine-rich β-sheet extension bound to hVISTA; p. As determined by lentigraphy, mature hVISTA H121, H122, and / or H123 were contacted at a distance of 4.0 Å or less; and / or q. Contact hVISTA via at least one or more glutamic acid, aspartic acid, or histidine residues located in VH CDR1, CDR2, or CDR3.

17. The antibody according to any one of claims 1-7, wherein the antibody has an isoelectric point (pI) between 6.5 and 6.8 as measured by icIEF.

18. The antibody according to any one of claims 1-7, wherein the antibody is an IgG antibody.

19. The antibody of claim 18, wherein the antibody is an IgG1, IgG2, or IgG4 antibody.

20. The antibody of claim 19, wherein the antibody is an IgG4 antibody with an S228P mutation.

21. The antibody of any one of claims 1-7, wherein the antibody is an effectorless antibody and / or an antibody that does not bind to one or more FcγRs.

22. The antibody of claim 21, wherein the constant region comprises 1-5 mutations in the wild-type heavy chain constant region that reduce the effector function and / or binding ability to one or more FcγRs relative to the effector function and / or binding ability to one or more FcγRs of the corresponding wild-type heavy chain constant region.

23. The antibody according to any one of claims 1-7, wherein the constant region of the antibody is IgG1.3, IgG1.1, or IgG1 with P238K substitution.

24. The antibody of any one of claims 1-7, wherein the antibody has an effector function and / or binds to one or more FcγRs.

25. The antibody of claim 24, wherein the antibody is unfucosylated.

26. The antibody of claim 24, wherein, relative to the corresponding wild-type constant region, the constant region comprises 1-5 mutations that enhance the effector function of the antibody and / or its ability to bind to one or more FcγRs.

27. The antibody according to any one of claims 1-7, wherein the antibody is a full-length antibody or an antibody comprising a full-length heavy chain and a full-length light chain, wherein the full-length heavy chain may or may not contain a C-terminal lysine.

28. The antibody according to any one of claims 1-7, wherein the antibody is an antigen-binding fragment of the antibody.

29. The antibody according to any one of claims 1-7, wherein the antibody is a polyantibody.

30. The antibody according to any one of claims 1-7, wherein the antibody is linked to another molecule.

31. The antibody of claim 30, wherein the other molecule is a label.

32. The antibody of claim 30, wherein the other molecule is a peptide.

33. The antibody according to any one of claims 1-7, wherein the antibody is an activatable antibody.

34. A nucleic acid that encodes an antibody according to any one of claims 1-33.

35. A nucleic acid that encodes the heavy and light chains of the antibody according to any one of claims 1-33.

36. A composition comprising an isolated nucleic acid encoding the heavy chain of an antibody according to any one of claims 1-33 and a nucleic acid encoding the light chain of an antibody according to any one of claims 1-33.

37. A cell comprising isolated nucleic acid encoding the heavy and light chains of any one of claims 1-33.

38. A method for preparing an antibody, the method comprising culturing the cells of claim 37 under conditions expressing the antibody.

39. A composition comprising an antibody as claimed in any one of claims 1-33, a nucleic acid as claimed in claim 34 or 35, a composition as claimed in claim 36, or a cell as claimed in claim 37, and a pharmaceutically acceptable carrier.

40. The composition of claim 39, wherein the composition comprises a second therapeutic agent.

41. The composition of claim 40, wherein the second therapeutic agent is an immunostimulant or a chemotherapeutic agent.

42. The composition of claim 41, wherein the second therapeutic agent is an antagonist of PD-1 / PD-L1, CTLA-4 and / or LAG-3 or an agonist of GITR and / or OX40.

43. Use of the antibody of any one of claims 1-33 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is colorectal adenocarcinoma.

44. The use as claimed in claim 43, wherein the colorectal adenocarcinoma comprises VISTA-positive cells.

45. The use as described in claim 44, wherein the VISTA-positive cells are VISTA-positive infiltrative lymphocytes or myeloid mononuclear cells.

46. ​​The use as described in claim 43, wherein the subject is first tested for the presence of VISTA-positive cells in the tumor.

47. The use as described in claim 43, wherein the second therapy is administered to the subject.

48. The use as described in claim 47, wherein the second therapy is chemotherapy, radiotherapy, surgery, or administration of a second agent.

49. The use as described in claim 48, wherein the second therapy is a second agent, and the second agent is an immunostimulant or a chemotherapeutic agent.

50. The use as claimed in claim 48, wherein the second therapeutic agent is an immunostimulant, said immunostimulant being an antagonist of PD-1 / PD-L1, CTLA-4 and / or LAG-3 or an agonist of GITR and / or OX40.

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