EA046477B1 - ANTIBODIES BINDING TO VISTA AT ACID pH - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to antibodies that specifically bind to immunoglobulin V domain-containing suppressor of T-cell activation (VISTA) at acidic pH, and their use for the treatment of malignant tumors.

State of the art and essence of the invention

V-domain Ig suppressor of T cell activation or VISTA is a coinhibitory member of the B7 family of immunoreceptors expressed by myelomonocytic cells and other leukocytes. However, the mechanism by which VISTA suppresses immune responses is poorly understood.

The inventors discovered that, unlike other known immunoreceptors, VISTA binds its counterreceptors and acts selectively at acidic pH, with low activity at physiological pH (eg, 7.3-7.4). Thus, VISTA can suppress immune responses in acidic microenvironments, such as tumor beds or sites of inflammation, without affecting cells circulating in the blood or present in non-inflamed, non-acidic tissues. In addition, we have discovered that anti-VISTA antibodies can be engineered to selectively bind to VISTA at acidic pH, with minimal binding at physiological pH, reflecting VISTA's intrinsic selectivity for acidic pH. Such acidic pH-selective antibodies may provide desirable properties for the treatment of diseases such as cancer, compared with antibodies that bind VISTA at physiological pH.

The present invention relates to antibodies that specifically bind to the extracellular domain (ECD) of a VISTA, such as human VISTA (hVISTA or huVISTA), at acidic pH (eg, under acidic conditions). The present invention also provides antibodies that specifically bind to the extracellular domain (ECD) of a VISTA, such as hVISTA, at acidic pH, with little or no binding at neutral or physiological pH. The inventors herein have noted that the amino acid sequence of hVISTA-ECD includes a number of both conserved and non-conserved histidine residues, and that the frequency of histidine residues in the VISTA ECD is exceptionally high compared to other members of the B7 family and other members of the immunoglobulin superfamily (see FIG. 1A and 1B). In solution, the amino acid histidine has a pK _a of approximately 6.5, meaning that at pH 6.5 or lower, histidine residues in proteins are often protonated and thus positively charged, whereas at pH above pH 6.5 they become increasingly less protonated and charge neutral. Tumor microenvironments and inflamed tissues are often acidic, and thus VISTA proteins found in these microenvironments may be at least partially protonated at their histidine residues. The inventors, as discussed herein, hypothesized that histidine protonation may influence the conformation, surface structure, and/or charge density of VISTA, which in turn may create pH-specific or pH-selective epitopes for receptor interaction(s) -ligand and antibody binding. Targeting VISTA with antibodies that bind at acidic pH but not at neutral or physiological pH may prevent target-mediated distribution of drug by circulating and lymphoid organ-resident myelomonocytic cells, improving antibody PK, receptor occupancy, and activity in tumor microenvironment. Acid pH-selective antibodies can also improve the specificity of anti-VISTA antibodies for intratumoral rather than circulating target cells in cases of therapeutic modalities such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and delivery of payload (antibody-drug conjugates).

Brief description of the figures

The US priority provisional patent applications for which this application claims priority contain at least one color drawing. If provisional applications are later published in the public domain, copies of the color drawings must be furnished to the United States Patent and Trademark Office upon request and upon payment of the required fee.

In fig. 1A-C show that the extracellular domain of VISTA contains an extremely high frequency of histidine residues, that many of these histidine residues are conserved, and that at least some of these histidine residues may be involved in receptor-ligand binding. In fig. 1A shows a diagram of proteins containing an immunoglobulin domain, with the number of extracellular domain amino acid residues for 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. In fig. Figure 1B shows the aligned amino acid sequences of human, cynomolgus and mouse extracellular domains of VISTA. The positions of the signal peptide (Sig) and transmembrane domain (TMD) sequences are indicated. Histidine residues conserved in all three species are shown in bold and underlined; histidine residues conserved in humans and cynomolgus monkeys are isolated

- 1 046477 only in bold. In fig. Figure 1C shows a model of the three-dimensional structure of the human VISTA immunoglobulin domain. Histidine residues are depicted as ball-and-stick outlines.

In fig. 2A,B shows a model in which histidine residues in the extracellular domain of VISTA confer counterreceptor selectivity for acidic pH rather than physiological pH. In fig. Figure 2A shows the equilibrium between the absence and presence of protonation of the pyrrol ammonium group (NH) on the histidine residue. The pKa value of histidine in solution is 6.5, indicating that histidine residues are more likely to be protonated at pH 6.5 and below and thus be positively charged than at higher pH. In fig. 2B shows a model in which VISTA binds to P-selectin glycoprotein ligand 1 (PSGL-1) or other counter-receptors and ligands (VISTA-R) at acidic pH. Thus, antibody binding to the extracellular domain of VISTA at acidic pH rather than at physiological pH may be critical for inhibition or modulation of VISTA activity.

In fig. Figure 3 shows the level of surface expression of VISTA (mean fluorescence intensity (MFI) when stained with anti-VISTA antibodies) on tumor-infiltrating macrophages, dendritic cells, neutrophils, CD4+ effector T cells, CD4+ regulatory T cells, CD8+ T cells, natural killer (NK) cells. and B cells. VISTA is expressed on many tumor-infiltrating leukocytes, particularly myeloid cells. The tumor microenvironment is often acidic, allowing VISTA to bind counterreceptors and ligands.

In fig. 4A-G show that VISTA selectively binds to leukocytes and to PSGL-1 at acidic pH with minimal binding at neutral pH, and that such binding can be blocked by anti-VISTA antibody. On the left in Fig. 4A shows representative histograms of binding of fluorescently conjugated recombinant VISTA multimer to activated human CD4+ T cells. The shaded histograms, from dark gray to lighter, show binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some histograms indicate corresponding pH values. Binding of VISTA-free control multimers at pH 6.0 is shown as an open histogram. Shown on the right is the average MFI of binding of VISTA (circles) and control (triangles) multimer to activated human CD4+ T cells from two donors at different pH. In fig. 4B shows typical binding histograms of recombinant VISTA multimer to peripheral blood mononuclear cells (PBMCs) at pH 6.0 and pH 7.4. The shaded histograms, from dark gray to lighter, show binding at pH 6.0 to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes. Open, solid and dotted histograms show binding at pH 7.4 to total lymphocyte and monocyte PBMCs, respectively. In fig. 4C shows typical binding of recombinant VISTA multimer to activated human CD4+ T cells in the presence of a blocking anti-VISTA antibody (squares) or a control non-VISTA isotype-matched antibody (circles). Antibody concentrations are plotted on a logarithmic scale. Nonlinear regressions are also shown. The triangle indicates the background signal from activated human CD4+ T cells that were not stained with recombinant VISTA multimers. In fig. 4D shows representative two-dimensional flow cytometry plots of the binding of recombinant VISTA multimer at pH 6.0 to heparan sulfate-deficient Chinese hamster ovary (CHO) cells (line pGSD-677, American Type Culture Collection) that were transfected to express human PSGL-1. Multimer binding was performed in the presence and absence of the anti-VISTA blocking antibody shown in FIG. 4C. Cells left unstained with recombinant VISTA multimers are shown as a control. PSGL-1 antibody staining is plotted on the ordinate, and VISTA multimer staining is plotted on the x-axis. In fig. 4E shows representative histograms of binding of recombinant mouse VISTA-Fc fusion protein to mouse splenocytes at pH 6.0 and pH 7.4. The shaded histograms, from dark gray to lighter, show binding at pH 6.0 to CD8+ T cells, CD11b+ myeloid cells and CD4+ T cells. The open histogram shows binding at pH 7.4 to total splenocytes. In fig. 4F and G show that the VISTA multimer binds to monocytes and neutrophils, respectively, with stronger binding at pH 6.0 than at pH 7.4.

In fig. 5A-D show that VISTA mediates T cell suppression and cell-cell adhesion selectively at acidic pH, and that both effects can be reversed by a blocking anti-VISTA antibody. In fig. 5A shows the formation of a representative cell:cell conjugate at pH 6.0 and 7.0 between 293T cells expressing hVISTA or control vector (shown on the Y axes) and CHO cells endogenously expressing cell surface heparan sulfate on the X axes. FIG. 5B is a graph of the frequency of cell conjugates formed at pH 6.0 between the same cells in the presence of blocking anti-VISTA antibody, non-blocking anti-VISTA antibody, or isotype-matched non-VISTA-specific control antibodies. In fig. 5C shows representative plots of luciferase activity generated by Jurkat cells (a human T cell line) expressing NFκB with a luciferase reporter after coculture at various pH with 293T cells expressing hVISTA and single chain variable

- 2 046477 fragment of an agonistic antibody against the human T-cell receptor OKT3 (artificial antigen-presenting cells). Anti-VISTA blocking antibody (squares) or an isotype-matched non-VISTA-specific control antibody (circles) was added to the cocultured cells. In fig. 5D data shown in FIG. 5A are plotted as the fold increase in luciferase signal upon treatment with anti-VISTA antibodies compared to the control (effect size).

In fig. 6A-G show that VISTA can be found in intracellular endosomes, especially in Rab11+ recycling endosomes, and can return to and from the cell surface via endosomal transport. In fig. 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. In fig. 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes. A control non-VISTA-binding antibody, the same isotype as the anti-VISTA antibody (cAb), does not bind to monocytes. In fig. 6C shows the binding of three anti-VISTA antibodies to recombinant VISTA at pH 7.4 (black), 6.7 (dark gray) and 6. (light gray). In fig. 6D shows the sensitivity of an acute myeloid leukemia (AML) cell line expressing VISTA to killing by the same anti-VISTA antibodies 1 (inverted triangles), 2 (circles), 3 (squares) or non-VISTA-specific control antibodies (triangles) carrying cathepsin B-sensitive linkers and cytotoxic payloads. Cell viability (CellTiter-Glo LU) is plotted on the Y-axis and antibody concentrations are plotted on the X-axis. In FIG. 6E compares the binding of hVISTA anti-VISTA antibody 3 to an engineered variant (VISTA mAb 3c), which does not exhibit binding impairment at acidic pH. In fig. 6F shows an antibody-drug conjugate assay comparing the potency of the antibody against VISTA 3 (squares) and 3c (diamonds). In fig. Figure 6G shows a diagram of endosomal transport with recycling of VISTA to and from the cell surface via early endosomes and recycling endosomes.

In fig. 7A-F show how anti-VISTA antibody variant libraries were constructed and screened to produce acidic pH selective antibodies. In fig. 7A shows the amino acid substitutions that were made in the CDR 3 VH of anti-human VISTA antibody clone P1-061029 (abbreviated '029) to create the '029 screening library. To potentially improve binding to the histidine-rich region of VISTA at acidic pH, the libraries allowed substitution of the negatively charged amino acids aspartate and glutamate, as well as the pH-sensitive histidine. X=H, D, or E. Sequences in square brackets were excluded from the synthesis to prevent the introduction of biases. A total of 647 unique sequences of P1-061029 HCDR3 with 1-2 mutations were synthesized. In fig. 7B shows the procedure by which the '029 library was repeatedly screened and selected for acid pH selective antibody variants. R denotes selection round. In fig. Figure 7C shows representative data from 2D flow cytometry plots 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 are shown. In fig. 7D shows a diagram of the binding of P1-061029 and its daughter clones to human VISTA at pH 6.0 and 7.4. In fig. 7E shows a plot of the dissociation rate of P1-061029 and its daughter clones with human VISTA at pH 6.0. In fig. 7F shows SPR data for the binding of antibodies P1-068761, P1-068767 and P1-061029 to human VISTA at pH 6.0 and pH 7.4.

In fig. 8A-F show the acid pH selective cell binding activity, blocking and effector activity of anti-VISTA antibodies, P1-068761 and P1-068767. In fig. 8A and FIG. 8B shows the average fluorescence intensity of acid pH selective antibodies P1-068761 (FIG. 8A) and P1-068767 (FIG. 8B) binding to Raji cells ectopically expressing human VISTA. Cells were stained at approximately pH 6.0 (circles; top curve in Fig. 8A), 6.1 (squares; third curve from top), 6.2 (triangles; second curve from top), 6.4 (inverted triangles; fourth curve top, close to the curve at pH 6.1), 6.6 (diamonds; fourth curve from the bottom), 7.0 (circles; third curve from the bottom), 7.2 (squares; second curve from the bottom) and 8.1 (unshaded triangles; lower curve in Fig. 8A). Binding was detected using a fluorescently labeled anti-human IgG secondary antibody. In fig. 8C shows the binding of P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) to Raji cells ectopically expressing human VISTA at 3125 ng/ml and various pH. pH ₅₀ , the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. In fig. 8D shows the mean fluorescence intensities (MFI) of the isotype-matched nonspecific control antibody (filled and open circles at pH 7.0 and 6.0, respectively), anti-VISTA mAb 2 (control, see Fig. 6C, filled and open squares at pH 7.0 and 6.0, respectively), P1-068761 (filled and open triangles at pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles at pH 7.0 and 6.0, respectively) , binding to human monocytes. Binding was detected using fluorescently labeled anti-human IgG secondary antibodies. In fig. Figure 8E shows comparable blocking of binding of recombinant VISTA multimer to activated human CD4+ T cells at pH 6.0 by P1-061029 (squares), P1-068761 (triangles), and P1-068767 (inverted triangles), whereas a non-VISTA-specific control antibody (circles) did not block VISTA binding. In fig. 8F shows reduced activity of P1-068761 (triangles) and P1-068767 (inverted triangles) in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH. Also shown are P1 -061029 (squares), a VISTA non-specific positive control antibody (circles), and a VISTA non-specific negative control antibody (diamonds). Specific NK cell lysis of target cells as a percentage of total target cells is plotted on the y-axis, and antibody concentrations are plotted on the x-axis. Nonlinear regressions are also shown.

In fig. 9 shows improved pharmacokinetics (PK) of acid pH-selective anti-VISTA antibodies in cynomolgus monkeys. The figure shows the time course of serum antibody concentrations in cynomolgus monkeys treated with anti-VISTA 2 (control, circles, see Fig. 6C), anti-VISTA 3 (acid pH sensitive, squares, see Fig. 6C), or P1- 068767 (triangles).

In fig. 10A and 10B show the effect of the mutations on the binding of acid pH selective antibodies against VISTA '761 and '767. In fig. 10A shows the binding kinetics of the reverse mutants P1-068761 at pH 7.4, pH 6.7 and pH 6.0 and the position of their reverse mutations relative to P1-068761. In fig. 10B shows the binding kinetics of the reverse mutants P1-068767 at pH 7.4, pH 6.7 and pH 6.0 and the position of their reverse mutations relative to P1-068767.

In fig. 11A-C show binning and epitope mapping of various anti-VISTA antibodies. In fig. 11A shows the competition of P1-068761 and P1-068767 for binding to the VISTA epitope compared to P1-061029 and control anti-VISTA antibodies. In fig. 11B and FIG. 11C shows graphical representations of the epitopes of all residues for the hVISTA blocking antibody (Fig. 11B), as listed in table. 14, compared with a non-hVISTA blocking antibody (mAb 1; Fig. 11C). Amino acid residues 66(H) and 162(A) are indicated to indicate the orientation of the molecule. Histidine residues are highlighted in gray and epitope residues are highlighted in black.

In fig. 12A-C show imaged capillary isoelectric focusing (icIEF) data for the following: FIG. 12A: P1-061029, fig. 12B: P1-068761 and fig. 12С: Р1-068767. The isoelectric point of the main peaks (main pI), as well as pI markers, are indicated.

In fig. 13A and B show the alignment of the variable regions for daughter clones '029 and '015. In fig. 13A shows an alignment of the amino acid sequences of the variable regions of '029 and its daughter clones. In fig. 13B shows an alignment of the amino acid sequences of the variable regions of '015 and its daughter clones.

In fig. 14 shows an alignment of the VH sequences of P1-068761, with and without the K16R and T84A substitutions. The double-underlined residues indicate the positions of framework regions 16 and 84, and the shaded regions indicate the CDR regions.

Fig. 15A-O: Wild-type C57BL6 mice were implanted with MC38 tumors and treated with a non-binding, isotype-matched control antibody (black squares), the mouse VISTA blocking antibody VISTA. 10 (upward triangles), a mouse PD-1 blocking antibody (squares) or a combination of anti-VISTA and PD-1 blocking antibodies (downward triangles). All antibodies were of the mouse IgG1-D265A isotype (Fc-inert) (see Fig. 15A-D). These data are representative of three independent experiments. In fig. 15A-D show tumor volumes over time (n=10 per group). TF denotes mice in which tumors fail to engraft. In fig. 15E and F show the frequency of intratumoral CD8+ T cells and CD4+ T cells 7 days after the start of treatment (n=5 per group). One-way ANOVA with Dunnett's multiple comparisons, P=0.0001. In fig. 15G and H show the results for the individual mice shown in FIG. 15A-D. Fig. 15I: VISTA knockout (KO) mice and wild-type (WT) littermates were implanted with MC38 tumors and treated with isotype-matched nonbinding control antibodies (top two curves (0/7 TF and 0/5 TF, marked with circles and triangles pointing down) ), or murine PD-1 blocking antibody (bottom two curves 0/5 TF and 5/8 TF, marked with squares and triangles pointing down). Median tumor growth and number of mice that were tumor free (TF) at the end of the study, versus total mice are shown next to each curve (e.g., 0/7 TF). These data are representative of two independent experiments. Error bars represent interquartile range. Figure 15J-M shows tumor volumes in human VISTA knockin mice (KI ) into which MC38 tumors were implanted and treated with non-binding, isotype-matched control antibodies (Fig. 15J), a mouse PD-1 blocking antibody (Fig. 15K), a combination of a mouse PD-1 blocking antibody and a non-pH selective human VISTA blocking antibody P1 -061029 (Fig. 15L) or a combination of murine PD-1 blocking and acid pH selective human VISTA blocking antibody, P1-068767 (Fig. 15M). All antibodies were of the mouse IgG1-D265A isotype. Tumor volumes over time are shown (n=5-8 per group). These data represent one independent experiment. In fig. 15N shows serum antibody concentrations in human KI VISTA mice and wild-type (WT) littermates following intravenous injection of 5 mg/kg P1-061029 (WT, top-down triangles; KI, squares) or P1-068767 (WT, triangles top up; KI, diamonds).

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The calculated mean serum retention time (MRT) for P1-061029 and P1-068767 in KI mice was 4.1 and 71 hours, respectively. n=4 KI mice and 1-2 WT mice per antibody. These data represent one experiment. In fig. 15O shows antibody concentrations in the serum of cynomolgus monkeys after 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 7.6 hours and 717 hours, respectively (n=1 macaque per antibody). These data represent one experiment. Error bars represent standard error of the mean unless otherwise noted.

In fig. 16A-C show representative histograms of intratumoral CD8+ T cell expression of PD-1 (FIG. 16A), LAG-3 (FIG. 16B), and TIM-3 (FIG. 16C) 7 days after initiation of treatment. Error bars represent standard error of the mean.

In fig. 17A-C show that VISTA binds to PSGL-1 at acidic pH, and that this interaction is blocked by antibodies to VISTA P1-061029, P1-068761, P1-068767 and VISTA.4. In fig. 17A shows BLI binding sensorgrams for the binding of P-Selectin-Fc and VISTA-Fc to immobilized PSGL1 at pH 6.0 and pH 7.4. Fig. 17B is a bar graph showing that antibodies P1-061029, P1068761, P1-068767 and VISTA.4 inhibit the binding of PSGL-1 to hVISTA. In fig. 17C shows the blocking of VISTA-Fc binding to CHO-PSGL-1 cells by VISTA.4 antibody (upward triangles) and anti-PSGL-1 antibody, KPL-1 (circles). These data are representative of two independent experiments. Error bars represent standard error of the mean.

In fig. 18A-E show images of the cocrystal structure of Fab P1-068767 and hVISTA or (in FIG. 18E) the non-blocking antibody VISTA.5 and hVISTA. The IgV VISTA domain has an unusual histidine-rich central β-sheet extension. The IgV VISTA domain was crystallized with the antigen binding fragment P1-068767 (Fab). The crystal structure of the VISTA+P1-068767 complex was determined to 1.6 A resolution. FIG. 18A shows the structure of the IgV VISTA:P1-068767 Fab domain cocrystal. In fig. 18A shows the general structure of the IgV VISTA domain in complex with Fab P1-068767 (heavy chain, dark gray; light chain, light gray). In fig. 18B shows the overlap of the IgV domains of VISTA and PD-L1. VISTA histidine residues are shown as bars. In fig. 18B shows that the IgV VISTA domain has an unusual histidine-rich β-sheet extension. In fig. 18C shows the molecular surface of the P1-068767 epitope (light gray electrostatic surface) represented by the crystal structure of VISTA+P1-068767. In fig. 18C shows that blocking antibodies bind to the histidine-rich β-sheet extension of VISTA. In fig. 18D shows an enlarged view of the contact region between VISTA (gray ribbon image, epitope residues H121, H122 and H123 shown as a rod model) and P1-068767 (shown as an electrostatic surface with residues E100 and D102 as a rod model). In fig. 18D shows that the acidic pH selective P1-068767 binds VISTA histidines with acidic residues. In fig. 18E shows that the non-blocking antibody VISTA.5 binds at a different region of hVISTA than P1-068767.

In fig. 19 shows the VISTA.4 epitope determined by MS-HDX (MS curve).

In fig. 20 shows the position of the VISTA.4 epitope in the hVISTA amino acid sequence based on the data in FIG. 19. Residues 57-68, 86-97 and 148-165, also highlighted in FIG. 19 are shown in lighter gray text and underlining in FIG. 20.

In fig. 21A and 21B show binding of VISTA multimer to activated human CD4+ T cells at pH 6.0 in the presence of VISTA.4 (triangles), VISTA.5 (squares), and non-VISTA binding antibody (control, circles). In fig. 21B shows the blocking efficacy of each antibody on unblocked T cells. One-way ANOVA with Dunnett's multiple comparisons, ***, P < 0.001. These data represent more than four independent experiments. Error bars represent standard error of the mean.

In fig. 22 shows that antibodies that block VISTA binding at acidic pH are functional. Effect of VISTA.4 blocking antibody (squares), non-blocking VISTA.5 antibody (triangles) and non-VISTA binding antibody (control, circles) on proliferation (Fig. 22A) and interferon production (Fig. 22B) of human CD4+ T cells, together cultured with 293T cells modified to express VISTA and a TCR agonist (293T-OKT3-VISTA). One-way ANOVA with Dunnett's multiple comparisons, *, P < 0.05. These data represent more than four independent experiments.

In fig. 23 shows the effect of VISTA.4-induced block on Jurkat T cell activation (by measuring NF-kB inhibition) after co-culture with 293T-OKT3VISTA cells at different pH. These data are representative of three independent experiments.

In fig. 24 shows the effect of pH on VISTA-induced suppression of human CD4+ T cells. Cells were stimulated at the indicated pH using a plate coated with OKT3 and VISTA-Fc in the presence of VISTA.4 (upward triangles), VISTA.5 (upward triangles), or an antibody that does not bind to VISTA (control antibody, squares). Also shown are cells stimulated with OKT3 on the plate and control IgG (VISTA control, black circles) or without OKT3 (no OKT3, gray diamond). These data represent one independent experiment.

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In fig. 25A-E show that the binding specificity of VISTA:PSGL-1 is determined by histidine and sulfothyrosine residues. As shown in FIG. 25A, recombinant human PSGL-1 19-merFc proteins were produced in cells with or without sialyl-Lewis X modification (SLX+ and SLX, respectively). The magnitude of BLI binding at pH 6.0 (white) and 7.4 (black) is shown for VISTA-Fc and P-selectin-Fc as indicated. Data represent one independent experiment. As shown in FIG. 25B, human PSGL-1 19-mer-Fc glycopeptides prepared with sialyl-Lewis X modification were separated into fractions with more than 90% tyrosine sulfation (sY-rich) and less than 1% tyrosine sulfation (sY-poor). The magnitude of BLI binding at pH 6.0 (white) and 7.4 (black) is shown for VISTA-Fc and P-selectin-Fc as indicated. These data represent one independent experiment. As shown in FIG. 25C, 25D, recombinant human VISTA-Fc proteins were produced with histidine residues at positions 153-155, which were left intact (WT VISTA) or replaced with alanine (H2A mutant), aspartic acid (H2D mutant) or arginine (H2R mutant). In fig. 25C shows the magnitude of HI binding for wild-type and mutant VISTA-Fc proteins binding to captured PSGL-1 at pH 6.0 and 7.4. These data represent one experiment. In fig. 25D shows VISTA-Fc binding to CHO-PSGL-1 cells at pH 6.0 for VISTA WT (circles), mutant H2A (squares), mutant H2D (triangles up) and mutant H2R (gray triangles up), and control (diamonds). These data are representative of two independent experiments. In fig. 25E shows a computer model of the 19-mer PSGL-1 glycopeptide (top) complexed with the histidine-rich VISTA ligand interface (gray ribbons, bottom). VISTA residues H98, H100, H153 and H154 are noted. PSGL-1 residues Y46, Y48, E56, T57, and Y58 are also noted.

Detailed description

Definitions.

In the present invention, the use of or means and/or, unless otherwise indicated. In the context of a multiple dependent claim, the use of or refers to more than one preceding independent or dependent claim only in the alternative. The terms comprising, including and having may be used interchangeably herein. According to the present invention, an isolated molecule is a molecule that has been removed from its natural environment. Thus, the term isolated does not necessarily reflect the extent to which the molecule has been purified.

The term polypeptide refers to a polymer of amino acid residues and is not limited to a minimum length. A protein may include one or more polypeptides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. This definition covers both full-length proteins and their fragments. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, as used herein, a polypeptide or protein refers to a polypeptide or protein, respectively, that includes modifications, such as deletions, additions and substitutions (generally conservative in nature), in the native sequence, so long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in protein-producing hosts or errors due to PCR amplification. A protein may contain two or more polypeptides.

VISTA is an abbreviation for English. V-domain immunoglobulin-containing suppressor of T-cell activation protein, which is a member of the B7 family of immune checkpoint regulators. VISTA is also known as PD1 homolog (PD1H), B7-H5, C10orf54, ESC differentiation-1 (Dies-1), platelet receptor precursor Gi24, and death domain 1α (DD1a). 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 presented in SEQ ID NO: 1, while the sequence without the signal peptide is presented in SEQ ID NO: 2 (see sequence table below). The extracellular domain or ECD of VISTA or VISTA-ECD refers to the portion of the VISTA protein that is located in the extracellular space, which, in the case of hVISTA, includes amino acids 1-162 SEQ ID NO: 2 (see also Fig. 1B) Part of hVISTA, IgV domain , includes residues 5-135 SEQ ID NO: 2.

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

The term antibody or Ab is used herein in its broadest sense to cover a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies)

- 6 046477 and antibody fragments, if they demonstrate the required antigen-binding activity. As used herein, the term refers to a molecule comprising at least complementarity determining region (CDR) 1, CDR2 and CDR3 of the heavy chain and at least CDR1, CDR2 and CDR3 of the light chain, where the molecule is capable of binding to an antigen . The term antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single chain Fv (scFv), Fab, Fab' and (Fab') ₂ . The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies from various species such as mouse, cynomolgus, etc.

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

The term heavy chain variable region or VH refers to the region including heavy chain complementarity determining region (CDR) 1, framework region (FR) 2, heavy chain CDR2, FR3 and CDR3. In some embodiments, the heavy chain variable region also includes at least a portion of FR1 and/or at least a portion of FR4. As defined below, in some embodiments, the CDR1 heavy chain includes residues 26-35 of VH SEQ ID NO herein; the CDR2 heavy chain includes residues 50-66 of VH SEQ ID NOs herein and the CDR3 heavy chain includes residues 99-110 of VH SEQ ID NOs herein. In other embodiments, if determined, the CDR1 heavy chain corresponds to Kabat residues 31-35; the heavy chain of CDR2 corresponds to Kabat residues 50-65; and the heavy chain of CDR3 corresponds to Kabat residues 95-102. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NTH, Bethesda, Md.). In some embodiments, the heavy chain CDR regions are as defined herein, for example, in the sequence table below or in table. 2.

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

The term light chain variable region or VL refers to the region including the light chain CDR1, FR2, HVR2, FR3 and HVR3. In some embodiments, the light chain variable region also includes FR1 and/or FR4. As defined below, in some embodiments, the CDR1 light chain includes residues 24-35 of VL SEQ ID NO herein; the CDR2 light chain includes residues 51-57 of VL SEQ ID NOs herein and the CDR3 light chain includes residues 90-98 of VL SEQ ID NOs herein. In other embodiments, if defined, the CDR1 light chain corresponds to Kabat residues 24-34; the light chain of CDR2 corresponds to Kabat residues 50-56; and the light chain of CDR3 corresponds to Kabat residues 89-97. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NTH, Bethesda, Md.). In some embodiments, the light chain CDR regions are as defined herein, for example, in a sequence table.

A chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species and the remainder of the heavy and/or light chain is derived from another source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus, etc.) and at least one constant region from a second species (such as humans, cynomolgus monkeys, etc.). In some embodiments, the chimeric antibody includes at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody includes at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of the chimeric antibody are derived from a first biological species and all of the constant regions of the chimeric antibody are derived from a second biological species.

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

Human antibody as used herein refers to antibodies produced in humans, antibodies produced in non-human animals, including

- 7 046477 human immunoglobulin genes, such as XenoMouse®, as well as antibodies selected using in vitro methods such as phage display, in which the antibody repertoire is based on human immunoglobulin sequences.

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

In some embodiments, an antibody herein may contain one or more conservative substitutions relative to a particular, defined sequence. Conservative amino acid substitutions as used herein refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine , serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains chains (for example, tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted nonessential amino acid residue in an antibody herein is replaced with another amino acid residue from the same side chain family (eg, basic, acidic, beta-branched, aromatic, uncharged polar). Methods for identifying nucleotide and amino acid conservative substitutions that do not interfere with 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)).

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

The KD or dissociation constant for the binding of an antibody to a protein, such as the VISTAECD protein, is a measure of the affinity or specific binding of an antibody to a protein, such as the VISTA-ECD protein. A lower KD indicates improved binding or affinity compared to a higher _KD . KD consists of the ratio of the rate of dissociation, or koff, or kd, and the rate of association, or kon, or k _a, for an antibody and a polypeptide. The dissociation rate and the association rate are the rates at which two binding partners associate and dissociate in a system. Thus, a lower dissociation rate, where the association rate remains approximately constant, results in a higher overall affinity and thus a lower KD. As used herein, koff of a particular value or less indicates that the koff or dissociation rate is as specified or lower than the specified rate.

The terms specific binding or specifically binds or similar terms mean that the K _D for the binding of two polypeptides, such as an antibody and its target polypeptide, is less than that between two random polypeptides existing under the same conditions. In other words, KD is less than the KD value in the case of nonspecific aggregation of polypeptides in the system.

In some embodiments, the antibodies specifically bind to the VISTA-ECD protein at a particular pH or pH range. Acidic pH as used herein generally refers to a pH less than 7.0, basic pH generally refers to a pH above 7.0, and neutral pH generally refers to a pH of approximately 7.0. Physiological pH as used herein refers to the pH under normal (ie, non-cancerous) physiological conditions, for example, 7.35 to 7.45, or 7.3 to 7.4, such as about 7.4. Phrases such as binding under acidic conditions or binding under physiological conditions, etc., used herein in relation to the binding of two molecules, such as VISTA and a VISTA binding partner, or VISTA and a T cell, refer to binding under acidic conditions pH and binding at physiological pH, respectively.

When specifying an antibody that blocks or inhibits binding of a ligand (or receptor) or a competing antibody to a receptor (or ligand), alone or on a cell, binding is blocked if there is an overall decrease that is statistically significant compared to a control, e.g. a reduction of 50% or more, such as a total reduction of 75%, 80%, 85%, 90%, 95% or more. An anti-VISTA blocking antibody, for example, is an antibody that can block the binding of VISTA to PSGL-1 or other VISTA ligand or receptor or heparan sulfate proteoglycans, at least under certain conditions, such as

- 8 046477 slot pH.

A tumor model as used herein refers to a preclinical in vivo study that can be used to study the biological activity of an anti-VISTA-ECD antibody and includes xenograft or native murine tumor assay systems. In some cases, the tumor model may allow the size or growth of the tumor to be monitored after treatment with an antibody and/or the presence of immune cells in the tumor, such as certain types of T cells or NK cells, to be monitored to determine whether the antibody induces or enhances an immune response.

The term immunostimulatory agent as used herein refers to a molecule that stimulates the immune system, either by acting as an agonist of an immunostimulatory molecule, including a co-stimulatory molecule, or by acting as an antagonist of an immunoinhibitory molecule, including a co-inhibitory molecule. The immunostimulatory molecule or immunoinhibitory molecule may be an immune checkpoint regulator such as VISTA or another member of the B7 family, or another molecule as described below. The immunostimulant agent may be a biological substance such as an antibody or antibody fragment, another protein or a vaccine, or may be a small molecule drug. An immunostimulatory molecule includes a receptor or ligand that acts to enhance, stimulate, induce, or otherwise trigger an immune response. Immunostimulatory molecules, as defined herein, include costimulatory molecules. An immunoinhibitory molecule includes a receptor or ligand that acts to reduce, inhibit, suppress, or otherwise turn off the immune response. Immunoinhibitory molecules, as defined herein, include coinhibitory molecules. Such immunostimulatory and immunoinhibitory molecules may be, for example, receptors or ligands present on immune cells, such as T cells, or present on cells involved in innate immunity, such as NK cells.

Percentage (%) amino acid sequence identity and homology with respect to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical in amino acid residues to a defined peptide or polypeptide sequence, after sequence alignment and gapping, if necessary, to obtain maximum percentage of sequence identity, not including any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in various ways that are known in the art, for example, using a publicly available program such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment throughout the sequences being compared.

The terms cause or enhance refer to the initiation or enhancement of any event (such as the binding of a protein ligand) or the initiation or enhancement of any biological activity (such as an immune response) or phenotypic characteristic, or the initiation or enhancement of the frequency, extent or likelihood of such activity or characteristic . To induce or enhance means to initiate or increase activity, function and/or quantity from a reference value. It is not required that initiation or amplification be complete. For example, in some embodiments, enhancement refers to the ability to cause an overall increase of 20% or more. In another embodiment, by amplification is meant the ability to cause an overall increase of 50% or more. In yet another embodiment, by enhancement is meant the ability to cause an overall increase of 75%, 85%, 90%, 95% or more.

The terms inhibition or inhibits more broadly refer to the reduction or cessation of any event (such as the binding of a protein ligand) or the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the frequency, extent or likelihood of such characteristic. To reduce or inhibit means to reduce, reduce, or eliminate activity, function, and/or amount from a reference value. The inhibition or reduction is not required to be complete. For example, in some embodiments, by reducing or inhibiting is meant the ability to cause an overall reduction of 20% or more. In another embodiment, by reducing or inhibiting is meant the ability to cause an overall reduction of 50% or more. In yet another embodiment, by reducing or inhibiting is meant the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

Treatment as used herein covers any administration or use of a therapeutic agent for a disease in a person and includes inhibiting the disease or progression of the disease or one or more symptoms of the disease, inhibiting or delaying

- 9 046477 preventing the disease or its progression, or one or more symptoms of the disease, stopping its development, partial or complete elimination of the disease or one or more symptoms thereof, or preventing the recurrence of one or more symptoms of the disease.

The terms subject and patient are used interchangeably herein to refer to a human being.

The term effective amount or therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a subject, for example, to provide partial or complete relief of one or more symptoms. In some embodiments, an effective amount refers to an amount effective at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

The term malignant tumors is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. The tumor may be benign (also called a benign tumor), precancerous, or malignant. The malignant cells may be solid cancer cells or leukemic cancer cells. The term tumor growth is used herein to refer to the proliferation or growth of a cell or cells that comprise cancer, resulting in a corresponding increase in the size or extent of the cancer.

Examples of forms of cancer applicable to the treatment methods described herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific non-limiting examples of such forms of malignancies include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma salivary gland, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, stomach cancer, melanoma and other types of cancer head and neck (including squamous cell carcinoma of the head and neck).

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

A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material, excipient or carrier commonly used in the art with a therapeutic agent, which together form a pharmaceutical composition for administration to a subject. The pharmaceutically acceptable carrier is non-toxic to recipients at the doses and concentrations used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the composition employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gelatin capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally will not irritate the skin or cause a reaction at the injection site.

A chemotherapy drug is a chemical compound used in the treatment of a malignant tumor. Examples of chemotherapeutic agents that may be used in the methods herein include, but are not limited to, alkylating agents such as thiotepa and Cytoxan® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including a synthetic analogue of topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg, calicheamicin, especially calicheamicin gammaP and calicheamicin omega I1, see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dinemycin, including dinemycin A; bisphosphonates such as clodronate; esperamycin; as well as neocarcinostatin chromophore and related chromophores of enediyne antibiotic chromoproteins), aclacinomycins, actinomycin, outramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carcinophilin, chromomycins, dactinomycin, dauno

- 10 046477 rubicin, detorubicin, 6-diazo-5-oxo-b-norleucine, Adriamycin® doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins , such such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, cinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as antsitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that inhibit the function of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; agents that replenish folic acid deficiency, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; dephosphamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2''trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurine A, riridine A and anhydrine); urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane®, a Cremophor-free, albumin-stabilized nanoparticulate formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, Anthony, France); chlorambucil; Gemzar® gemcitabine; 6thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xelodu; ibandronate; irinotecan (Camptosar, CPT-11) (including a regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; combrestatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (eg, erlotinib (Tarceva®)) and VEGF-A, which reduce cell proliferation, as well as pharmaceutically acceptable salts, acids or derivatives of any of the above.

Other non-limiting examples of chemotherapeutic agents that may be used in the methods herein include antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen ), raloxifene, droloxifene, 4-hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone and Fareston® toremifene; aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen synthesis in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megazu® megestrol acetate, Aromasin® exemestane, formestane, fadrozole, Rivisor® vorozole, Femara® letrozole and Arimidex ® anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (1,3-dioxolane analogue of cytosine nucleoside); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in the proliferation of abnormal cells, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as an inhibitor of VEGF expression (eg Angiozyme® ribozyme) and an inhibitor of HER2 expression; vaccines such as gene therapy vaccines, for example, Allovectin® vaccine, Leuvectin® vaccine and Vaxid® vaccine; Proleukin® rIL-2; topoisomerase-1 inhibitor Lurtotecan®; Abarelix® rmRH; as well as pharmaceutically acceptable salts, acids or derivatives of any of the above.

An antiangiogenic agent or angiogenesis inhibitor refers to a small molecule substance, polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody or conjugates, or fusion proteins thereof, that inhibit angiogenesis, vasculogenesis or unwanted vascular permeability, directly or indirectly. It should be understood that an antiangiogenic agent includes those agents that bind to and block the angiogenic activity of an angiogenic factor or its receptor. For example, an anti-angiogenic agent that may be administered in the methods herein may include an antibody or other antagonist of the angiogenic agent, such as an anti-VEGF-A antibody (e.g., bevacizumab (Avastin®)) or an anti-VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (eg, PTK787/ZK2284, SU6668, Sutent®^III1248 (sunitinib malate),

- 11 046477

AMG706, or compounds described, for example, in international patent application WO 2004/113304). Antiangiogenic agents also include native 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 (eg, Table 3, which lists antiangiogenic therapies for malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (eg, Table 2, which lists known antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (eg, Table 1, which lists antiangiogenic agents used in clinical studies).

A growth inhibitory agent as used herein refers to a compound or composition that inhibits the growth of a cell (such as a cell expressing VEGF), in vitro or in vivo. Thus, a growth inhibitory agent that can be administered in the methods herein may be an agent that significantly reduces the percentage of cells (such as cells expressing VEGF) in S phase. Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (other than S phase), such as agents that induce G1 arrest and M phase arrest. Classic M-phase blockers include the vinca drugs (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that cause G1 arrest also enter S phase, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Additional information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, under the title Cell cycle regulation, oncogenes, and antineoplastic drugs, Murakami et al. (W.B. Saunders, Philadelphia, 1995), for example, page 13. Taxanes (paclitaxel and docetaxel) are antineoplastic agents derived from yew. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from European yew, is a semi-synthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in inhibition of mitosis in cells.

The term antitumor composition refers to a composition used for the treatment of a malignant tumor containing at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiation therapy agents, antiangiogenic agents, antitumor immunotherapy agents, apoptotic agents, antitubulin agents, and other agents for the treatment of malignancy, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonist (eg, tyrosine kinase inhibitor), HER1/EGFR inhibitor (eg, erlotinib (Tarceva®), platelet-derived growth factor inhibitors (eg, Gleevec® (imatinib mesylate) )), COX-2 inhibitor (eg, celecoxib), interferons, CTLA4 inhibitors (eg, anti-CTLA antibody ipilimumab (Yervoy®)), PD-1 or PD-L1 inhibitors (eg, Opdivo®, Keytruda®, Tecentriq®, Bavencio®, Imfinzi®), TIM3 inhibitors (eg, anti-TIM3 antibodies), cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, CTLA4, TIM3 or VEGF receptor(s), TRAIL/Apo2 and other bioactive and organic chemicals etc. Combinations thereof are also included in the present description.

Antibodies that specifically bind to VISTA-ECD at acidic pH Because VISTA contains a large number of histidine residues in the extracellular domain (ECD), its fold and overall structure, as well as the surface available for binding of ligands such as antibodies, may differ at acidic pH in compared to neutral pH, in particular around pH 6.5, which corresponds to the pK _a for histidine. Since the microenvironment of tumors is typically acidic, binding to VISTA in such a microenvironment may require specific binding of the antibody to VISTA at an acidic pH, where at least some of the surface histidine residues are more likely to be protonated.

The sequence table below shows the amino acid sequence of human VISTA (hVISTA) with or without signal peptide (SEQ ID NO: 1 and SEQ ID NO: 2 (mature hVISTA)), respectively. The signal peptide comprises amino acid residues 1-32 of SEQ ID NO:1. The extracellular domain (ECD) consists of amino acid residues 1-162 SEQ ID NO: 2. The IgV domain consists of amino acid residues 37-167 SEQ ID NO: 1 and amino acid residues 5-135 SEQ ID NO: 2. The stalk region includes amino acid residues 172- 194 SEQ ID NO: 1 and amino acid residues 136-162 SEQ ID NO: 2; the transmembrane domain comprises 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 155 of SEQ ID NO: 2 (in bold and underlined) may be D or E, which reflects a polymorphism in hVISTA. This remainder is highlighted in bold and underlined. Thus, SEQ ID NO: 1 and SEQ ID NO: 2 cover both polymorphisms at this residue in humans. Histidine residues in ECD

- 12 046477

VISTA are shaded in gray.

Antibodies (Abs) against VISTA can specifically bind to VISTA-ECD or fragments thereof, for example, comprising the IgV domain of VISTA or the hVISTA region, including, for example, amino acids 20-95, 20-70 35-70, 35-95, 35-127 or 37-125 SEQ ID NO:2, at an acidic pH. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 7.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 6.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 6.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 6.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 6.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH less than pH 5.0.

Some Abs specifically bind to the VISTA-ECD protein at a pH in the pH range of 5.0-7.0. Some Abs specifically bind to the VISTA-ECD protein at a pH in the pH range of 5.0-6.5. Some Abs specifically bind to the VISTA-ECD protein at a pH in the pH range of 5.0-6.0. Some Abs bind specifically to the VISTA-ECD protein at a pH in the pH range of 5.5-7.0. Some Abs specifically bind to the VISTA-ECD protein at a pH in the pH range of 5.5-6.5. Some Abs specifically bind to the VISTA-ECD protein at a pH in the pH range of 6.0-6.5.

Also provided herein are Abs that bind to a VISTA-ECD protein, such as hVISTA-ECD or fragments thereof comprising the IgV domain of VISTA or the hVISTA region including, for example, amino acids 20-95, 20-70, 35-70, 35- 95, 35-127 or 37-125 SEQ ID NO: 2 at a pH of 6.5 or less, with a _{KD of} 10 ^-6 M or less. In some embodiments, the Ab is bound to a KD of 10 ^-7 M or less. In some embodiments, the Ab binds to a KD of 10 ^-8 M or less. In some embodiments, the Ab is bound to a KD of 10 ^-9 M. In some embodiments, the Ab is bound to a KD of 10 ^-10 M or less. For example, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less with a K _{D of} 10 ^-8 M or less.

Also provided herein are Abs that bind to the VISTA-ECD protein in the pH range of 6.0-6.5 with a K _{D of} 10 ^-6 M or less. In some embodiments, the Ab is bound to a KD of 10 ^-7 M or less. In some embodiments, the Ab is bound to a K _{D of} 10 ^-8 M or less. In some embodiments, the Ab is bound to a KD of 10 ^-9 M. In some embodiments, the Ab is bound to a KD of 10 ^-10 M or less. For example, the Ab may bind to the VISTA-ECD protein at a pH of 6.5 or less, eg, in the pH range of 6.0-6.5, with a K _{D of} 10 ^-7 M or less.

In addition, the Ab can bind to the VISTA-ECD protein at a pH of 6.5 or less, for example, in the pH range of 6.0-6.5, with a K _{D of} 10 ^-8 M or less. The Ab may bind to hVISTA-ECD at a pH of 6.5 or less, for example in the pH range of 6.0-6.5, with a K _{D of} 10 ^-9 M or less.

Also provided herein are Abs that specifically bind to a VISTA-ECD protein, such as hVISTA-ECD or fragments thereof comprising the IgV domain of VISTA or the hVISTA region including, for example, amino acids 20-95, 20-70, 35-70, 35- 95, 35-127 or 37-125 SEQ ID NO: 2, for example, at a pH of 6.5 or less, with a koff of 10 ^-5 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 10 ^-4 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 2 10 ^-4 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 5 10 ^-4 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 7 10 ^-4 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 10 ^-3 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 2 10 ^-3 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 5 10 ^-3 with ¹ or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 7 10 ^-3 s ^-1 or less, at 25°C or at 37°C. In some embodiments, the Abs have a koff of 10 ^-2 s ^-1 , at 25°C or at 37°C. In some embodiments, the Abs have a koff of 10 ^-1 s ^-1 , at 25°C or at 37°C. For example, the Ab can specifically bind to the VISTA-ECD protein at pH 6.5 or less with a k _{off of} 10 ^-3 s ^-1 or less, at 25°C or at 37°C. The Ab can specifically bind to hVISTA-ECD at pH 6.5 or less with a koff of 10 ^-3 s ^-1 or less, at 25°C or at 37°C. In addition, the Ab can specifically bind to the VISTA-ECD protein at pH 6.5 or less with a koff of 10 ^-2 s ^-1 or less, at 25°C or at 37°C.

Provided herein are Abs that bind to a VISTA-ECD protein, such as hVISTA-ECD or fragments thereof comprising the IgV domain of VISTA or a region from hVISTA, including, for example, amino acids 20-95, 20-70, 35-70 or 35 -95, 35-95, 35-127 or 37-125 SEQ ID NO: 2, for example, at a pH of 6.5 or less, with: (i) K _D 10 ^-6 M or less, 10 ^-7 M or less , 10 ^-8 M or less, 10 ^-9 M or less or 10 ^-10 M or less and (ii) koff 10 ^-5 s ^-1 or less, 10 ^-4 (or 2, 5 or 7 10 ^-4 ) s - ¹ or less, 10 ^-3 (or 2, 5 or 7 10 ^-4 ) s ^-1 or less, 10 ^-2 s ^-1 or less, or 10 ^-1 s ^-1 or less, when measured, for example, at 25 °C or at 37°C. For example, an Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a K _{D of} 10 ^-7 M or less and koff of 10 ^-3 s ^-1 or less, when measured at, for example, 25°C or

- 13 046477 at 37°C. Ab can bind to the VISTA-ECD protein at pH 6.5 or less with a K _{D of} 10 ^-8 M or less and koff of 10 ^-3 s ^-1 or less, when measured at, for example, 25°C or 37°C. The Ab can bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-8 M or less and koff of 10 ^-2 s ^-1 or less, when measured at, for example, 25°C or 37°C. For example, Ab may bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-7 M or less and koff of 10 ^-3 s ^-1 or less, when measured at, for example, 25°C or 37°C. Ab can bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-9 M or less and koff of 10 ^-3 s ^-1 or less, when measured at, for example, 25°C or 37°C. Ab can bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-9 M or less and koff of 10 ^-2 s ^-1 or less, when measured at, for example, 25°C or 37°C. Ab can bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-8 M or less and koff of 10 ^-4 (or 2.5 or 7 10 ^-4 ) s ^-1 or less, when measured e.g. 25°C or at 37°C. Ab can bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-8 M or less and koff of 10 ^-5 (or 2.5 or 7 10 ^-5 ) s ^-1 or less, when measured, e.g. 25°C or at 37°C. Ab can bind to hVISTAECD at pH 6.5 or less with a KD of 10 ^-9 M or less and koff of 10 ^-4 (or 2, 5 or 7 10 ^-4 ) s ^-1 or less, when measured at, for example, 25° C or at 37°C. Ab can bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-9 M or less and koff of 10 ^-5 (or 2.5 or 7 10 ^-5 ) s ^-1 or less, when measured e.g. 25°C or at 37°C.

Provided herein are Abs that specifically bind to the VISTA-ECD protein, for example, at a pH of 6.5 or less, with a ko _{n of} 104 M ^-1 s ^-1 or higher at 25°C or at 37°C. In some such embodiments, the Ab may bind at kon 10 ⁵ M ^-1 s ^-1 or higher. In some such embodiments, the Ab may bind at kon 10 ⁶ M ^-1 s ^-1 or higher. In some such embodiments, Ab may bind at kon 10 ⁷ M ^-1 s ^-1 or higher. For example, Ab can bind to the VISTA-ECD protein at pH 6.5 or less with kon 10 ⁶ M ^-1 s ^-1 or higher, when measured, for example, at 25°C or at 37°C. For example, Ab can bind to the ECD of hVISTA at pH 6.5 or less with kon 10 ⁶ M ^-1 s ^-1 or higher, when measured, for example, at 25°C or at 37°C.

Provided herein are Abs that bind to the VISTA-ECD protein, for example, at pH 6.5 or less, with: (i) KD 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less , 10 ^-9 M or less or 10 ^-10 M or less and (ii) kon 104 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, when measured, for example, at 25°C or at 37°C. For example, Ab can bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-7 M or less and kon 10 ⁶ M ^-1 s ^-1 or higher, when measured, for example, at 25°C or at 37 °C. For example, an Ab can bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-8 M or less and ko _n 10 ⁶ M ^-1 s ^-1 or higher, when measured, for example, at 25°C or at 37°C. For example, Ab may bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-7 M or less and ko, 10 ⁶ M ^-1 s ^-1 or higher, when measured at, for example, 25°C or 37 °C. For example, Ab may bind to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-8 M or less and kon 10 ⁶ M ^-1 s ^-1 or higher, when measured at, for example, 25°C or 37° C.

In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-7 M or less, as well as a koff of 10 ^-5 s ^-1 or less, 2 10 ^-5 s ^-1 or less , 5 10 ^-5 s ^-1 or less, 7 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less, 2 10 ^-4 s ^-1 or less, 5 10 ^-4 s ^-1 or less, 7 10 ^-4 s ^-1 or less, 10 ^-3 s ^-1 or less, 2 10 ^-3 s ^-1 or less, 5 10 ^-3 s ^-1 or less, 7 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C. In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-9 M or less, as well as a k _{off of} 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less , 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less, or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C. In some such embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-10 M or less, as well as a koff of 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less , 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less, or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C.

In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-7 M or less, as well as kon 10 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, when measured at, for example, 25°C or 37°C. In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-8 M or less, as well as kon 10 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, when measured at, for example, 25°C or 37°C. In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-9 M or less, as well as kon 104 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, when measured, for example, at 25°C or at 37°C. In some such embodiments, the Ab may bind to the VISTAECD protein at pH 6.5 or less with a KD of 10 ^-10 M or less, and ko, 104 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, when measured, for example, at 25°C or at 37°C.

In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-7 M or less, as well as a koff of 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less, 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less, or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C, and ko _n 10 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, when measured at, for example, 25°C or at 37°C. In some embodiments, the Ab may bind to a protein

- 14 046477

VISTA-ECD at pH 6.5 or less with _KD 10 ^-8 M or less, and koff 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less, 10 ^-3 s ^-1 or less , 10 ^-2 s ^-1 or less or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C, and kon 10 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, when measured at, for example, 25°C or 37°C. In some embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-9 M or less, as well as a koff of 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less, 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less or 10 ^{-1 s -1} or less, when measured at, for example, 25°C or 37°C, and kon 10 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, when measured at, for example, 25°C or 37 °C. In some such embodiments, the Ab may bind to the VISTA-ECD protein at pH 6.5 or less with a KD of 10 ^-10 M or less, as well as a koff of 10 ^-5 s ^-1 or less, 10 ^-4 s ^-1 or less , 10 ^-3 s ^-1 or less, 10 ^-2 s ^-1 or less, or 10 ^-1 s ^-1 or less, when measured at, for example, 25°C or 37°C, and ko _n 10 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, when measured at, for example, 25°C or at 37°C.

As also noted above, in some of the above embodiments, the VISTAECD protein is hVISTA-ECD or is part of hVISTA-ECD, such as, for example, an IgV domain. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 20-95 of SEQ ID NO: 2. In some of the above embodiments, the Ab may specifically bind to an epitope comprising amino acids 20-70 of SEQ ID NO: 2. In some of the above embodiments The Ab may specifically bind to an epitope comprising amino acids 35-95 of SEQ ID NO: 2. In some embodiments above, the Ab may specifically bind to an epitope comprising amino acids 35-70 of SEQ ID NO: 2. In some embodiments above, the epitope is a three-dimensional epitope comprising not only one of the above portions of SEQ ID NO: 2 of residues 20-95, 20-70, 35-95 or 35-70, but also another portion of SEQ ID NO: 2 such as residues 95-105 SEQ ID NO: 2. In some embodiments, the Ab binds to an hVISTA epitope to which the Ab described in WO 2015/097536 binds. For example, the Ab may compete or cross-compete for the binding of hVISTA with the Ab disclosed in WO 2015/097536. In some embodiments, the Ab binds to a human VISTA conformational epitope. In some embodiments, the Ab binds to a conformational epitope that includes or is present in residues 103-111 of SEQ ID NO: 2 and 136-146 of SEQ ID NO: 2 of human VISTA. In some embodiments, the Ab binds to a conformational epitope comprising or present at residues 24-36, 54-65, and 100-102 of SEQ ID NO: 2 of human VISTA. In some embodiments, the Ab binds to a conformational epitope comprising amino acid residues in the human FG VISTA loop. In some embodiments, the Ab binds to a polypeptide comprising amino acid residues 35-127 and/or 37-125 of SEQ ID NO: 2. In some embodiments, the Ab binds to a VISTA ECD polypeptide 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 an amino acid substitution, where 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) substitution of one of the following amino acids residues: Y37, T39, R54, F62, Q63, H66, L115, V117, I119, S124 or E125. In some embodiments, the anti-VISTA antibody has the same binding characteristics (or substantially the same binding characteristics) as the antibody described herein, for example, as set forth in the Examples and/or the claims.

Some of the above antibodies may exhibit different binding affinities to VISTA-ECD proteins depending on pH. Some Abs that specifically bind to the VISTAECD protein under acidic conditions, such as pH 6.5 or less, also specifically bind to the VISTA-ECD protein at neutral and/or alkaline pH with similar affinity (i.e., they are universal binders) . For example, some such Abs can bind to the VISTA-ECD protein with a KD of 10 ^-7 M or less at pH 6.5 and at pH 7.0 (at a constant temperature, for example, at 25°C or at 37°C), at In this case, KD at pH 6.5 is 1.5 times greater than KD at pH 7.0. Some such Abs can bind to the VISTA-ECD protein with a KD of 10 ^-8 M or less at pH 6.5 and at pH 7.0 (at constant temperature, e.g., 25°C or 37°C), with a KD at pH 6.5 is 1.5 times higher than KD at pH 7.0. Some such Abs can bind to hVISTA-ECD with a KD of 10 ^-8 M or less at pH 6.5 and at pH 7.0 (at constant temperature, e.g., 25°C or 37°C), with a KD at pH 6.5 is 1.5 times the KD at pH 7.0.

Some Abs that specifically bind to VISTA-ECD protein under acidic conditions, such as pH 6.5 or less, can bind VISTA-ECD protein under neutral, physiological and/or alkaline conditions with lower affinity (pH-sensitive binders or pH-sensitive antibodies). Some Abs that bind specifically to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or less, may exhibit little, e.g., nearly undetectable binding to the VISTA-ECD protein under neutral, physiological, and/or alkaline conditions.

- 15 046477 conditions. For example, in some embodiments, the Ab may bind to the VISTA-ECD protein with a _{KD of} 10 ^-8 M or less at pH 6.5 and with a KD greater than 10 ^-8 M at pH 7.0 and/or pH 7.4. In some such embodiments, the Ab may bind to the VISTA-ECD protein with a KD of 10 ^-8 M or less at pH 6.5 and a KD at pH 7.0 and/or pH 7.4 that is greater than 1.5 times higher than at pH 6.5. In some embodiments, a pH-sensitive Ab is provided that specifically binds to the VISTA-ECD protein with a KD that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300 times, 500 times, 1000 times or 5000 times lower at pH 6.5 than at pH 7.0 (at constant temperature, for example at 25°C or at 37°C). For example, in some cases, the Ab binds to the VISTAECD protein with a KD that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, or 5000 times less at pH 6.0 compared to pH 7.0 and/or pH 7.4 or higher (at constant temperature, e.g. 25°C or 37°C).

In some embodiments, the Ab specifically binds to the VISTA-ECD protein with a koff that is lower under acidic conditions compared to koff under neutral, physiological, or alkaline conditions. In some embodiments, an Ab is provided that binds to the VISTAECD protein under acidic conditions with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000 times lower at pH 6.5 than koff at pH 7.0 and/or pH 7.4, when measured at, for example, 25°C or 37°C. In other words, the dissociation rate at acidic pH is lower than at neutral pH. For example, in some embodiments, the Ab specifically binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6.0 compared to pH 7.0 and/or pH 7.4, when measured at, for example, 25°C or 37°C. In some embodiments, an Ab is provided that binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6.5 than koff at pH 7.4, when measured at, for example, 25°C or 37°C. In some embodiments, the Ab specifically binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6. 0 compared to pH 7.4, when measured at 25°C or 37°C, for example. In some embodiments, an Ab is provided that binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold lower at pH 6.0-6.5 than koff at pH 7.0-7.4, when measured at, for example, 25°C or 37°C.

In some embodiments, an Ab that specifically binds to a VISTA-ECD protein with a ko _n that is higher under acidic conditions compared to neutral physiological or alkaline conditions. In some embodiments, an Ab is provided that binds to the VISTA-ECD protein under acidic conditions with a k _on that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold higher at pH 6 .5 than ko _n at pH 7.0 and/or pH 7.4, when measured at, for example, 25°C or 37°C. For example, in some embodiments, the Ab specifically binds to the VISTA-ECD protein with a k _on that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold higher at pH 6.0. than at pH 7.0 and/or pH 7.4, when measured at, for example, 25°C or 37°C.

In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH at which at least one histidine residue, such as His 98 in SEQ ID NO: 1, is protonated. In some embodiments, the Ab specifically binds to the VISTA-ECD protein at a pH at which most of the histidine residues in the ECD are protonated, and which is expected to be pH 6.5 or less, for example, between pH 6.0 and pH 6. 5.

Also covered herein are Abs that specifically bind to the VISTAECD protein with an affinity that is higher at neutral, physiological, or alkaline pH compared to acidic pH, provided that binding affinity at acidic pH remains high. For example, Abs can bind to a VISTA-ECD protein with a KD of 10 ^-8 M or less at both pH 6.5 and pH 7.0, even if the Abs bind to a KD that is at least 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 300 times, 500 times, 1000 times lower at pH 7.0 than at pH 6.5.

Also covered herein are Abs having one or more of the general properties identified above in this section. The above properties such as specific KD, koff, ko _n , specific epitopes should not be considered separately. Thus, the Ab may bind to an epitope comprising one of the regions of SEQ ID NO: 2 described above, and may also have universal or pH-sensitive or pH-selective binding properties as described above, which is reflected by a change in one or more of its K _D , k _off or k _on at different pH.

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

Determining how well an Ab binds to the VISTA-ECD protein at a given pH can be done using several different methods. For example, using surface plasmon resonance (SPR), such as the BIACORE® analysis. Sample PNR analysis includes capture

- 16 046477 one or more antibodies on a CM4 sensor chip using an immobilized capture reagent (e.g., using the Biacore® Anti-Human Fc Capture Kit, GE Healthcare Part Number #BR-1008-39 or the Biacore® Anti-Mouse Capture Kit, GE Healthcare catalog #BR-1008-39) and passing the VISTA antigen analyte at a series of concentrations to determine binding kinetics and affinity in the passing buffer at the desired pH. In one embodiment, VISTA is administered at two to five concentrations ranging from 0.1 nM to 500 nM (e.g., 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM) at a flow rate of 30 μL/min, time association up to four minutes and dissociation time up to ten minutes. Between bonding cycles, the gripping surface is restored according to the manufacturer's instructions in the appropriate gripping kit. All data are double-checked using a reference flow cell and a blank. Data with simple 1:1 kinetics are fitted to a mass transfer Langmuir binding model using Biacore® T200 data analysis software. The SPR methods described in the examples can also be used.

The affinity of an Ab for a VISTA ECD polypeptide can be determined using cells expressing a VISTA ECD polypeptide, PSGL-1, or heparan sulfate on their surface, where the method involves flow cytometry, and where the binding of the Ab to cell-bound VISTA-ECD is determined at a given pH, e.g. , pH 6.5 or less. An exemplary flow cytometric assay involves the following: 293T cells or other cells ectopically expressing hVISTA ECD are resuspended in a buffer consisting of HBSS+1% BSA adjusted to the desired pH, for example, pH 6.0 using MES or pH 7, 4 using HEPES. Ab (eg, human IgG) against hVISTA is serially diluted from approximately 20 μg/ml and incubated with resuspended cells for 30 min at 4°C. The cells are then washed twice with the same buffers to maintain the desired pH, such as pH 6.0 or 7.4, and incubated with a secondary antibody conjugated to a fluorophore that recognizes the primary antibody (eg, human IgG) and is stable at low temperatures. pH. The cells are then washed as before and immediately recorded, without fixation, on a BD Fortessa or other flow cytometer. The affinity of the Ab for the VISTA ECD polypeptide can be determined as described in the Examples.

In some embodiments, Abs that bind to hVISTA block the binding of hVISTA ECD to its binding partner (eg, the VISTA receptor), for example, on cells. The inhibition or blocking 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 less, and inhibits the binding of VISTA to its binding partner by at least 50%, such as by at least 75%, 80 %, 85%, 90%, 95% or 100%. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 7.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 6.0. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.8. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.5. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.3. In some embodiments, the Ab specifically binds to the VISTA-ECD protein and inhibits the binding of VISTA to its binding partner by at least 50% at a pH less than pH 5.0.

Some Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the pH range of 5.0-7.0. Some Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the pH range of 5.0-6.5. Some Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the pH range of 5.0-6.0. Some Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the pH range of 5.5-7.0. Some Abs specifically bind to the VISTA-ECD protein and inhibit the binding of VISTA to its binding partner by at least 50% at a pH in the pH range of 5.5-6.5. Some Abs specifically bind to the VISTA-ECD protein and inhibit VISTA binding

- 17 046477 with its binding partner by at least 50% at a pH in the pH range of 6.0-6.5. Binding inhibition can be determined as described in the examples.

The binding partner of VISTA may be PSGL-1, such as human PSGL-1. The sequences of human PSGL-1 isoforms are provided in SEQ ID NO: 3-10 herein. VISTA binds to PSGL-1 with or without sialyl-Lewis X. The binding partner may also be heparan sulfate proteoglycans, such as those present on some cells.

Inhibition of binding to a VISTA binding partner can be determined by measuring the inhibition of binding of VISTA (or VISTA ECD or IgV domain of VISTA or VISTA positive cells) to cells to which VISTA binds, e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, activated or not), NK cells or other cells to which VISTA binds in the presence and absence of antibody. An exemplary experiment that can be used to determine whether an antibody inhibits the binding of VISTA to its binding partner or T cells expressing the binding partner is a flow cytometry assay, for example, an assay that includes the following: human peripheral blood mononuclear cells from donor blood, buffy coat or leukopak are resuspended in a buffer consisting of HBSS+1% BSA adjusted to the desired pH, for example pH 6.0 using MES or pH 7.4 using HEPES. Cells were then incubated for 30 min at 4°C with 20 μg/ml recombinant chimeric protein consisting of hVISTA ECD fused to human IgG1 Fc (VISTA-Fc) and various concentrations of candidate VISTA blocking antibodies or control antibodies. The cells are then washed twice in the same buffers, maintaining the desired pH, e.g., pH 6.0 or 7.4, and incubated for an additional 30 minutes at 4°C with a fluorophore-conjugated secondary antibody that recognizes VISTA-Fc but not recognizes candidate blocking antibodies or control antibodies, and is stable at reduced pH. The cells are then washed as before and immediately recorded, without fixation, on a BD Fortessa or other flow cytometer. Binding inhibition can be determined, for example, as described in the examples.

In certain embodiments, the Abs described herein can induce or enhance an immune response, such as an antigen-specific immune response. In some embodiments, Abs stimulate T cell activity, particularly at acidic pH such as is present in the microenvironment of tumors. Stimulation of T cell activity can be measured, for example, in a mixed lymphocyte culture reaction (MLR) or in an in vitro test with an antigen presenting cell (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 determining the secretion of IFN-γ from T cells, where increased secretion of IFN-γ indicates stimulation of T cells. 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-kB levels, is measured. In certain embodiments, the Abs described herein inhibit cell adhesion, which can be measured as described in the Examples.

Anti-VISTA antibody activity can also be demonstrated in monocyte assays, ADCC assays, and ADCP assays, especially at acidic pH such as is present in the tumor microenvironment.

In some embodiments, anti-VISTA antibodies inhibit tumor growth in a tumor model, such as a human VISTA knockin tumor model.

As shown in the Examples herein, recycling anti-VISTA Abs into the endosome, for example, to improve pharmacokinetic properties (PK), i.e. The half-life of the antibody requires that the anti-VISTA antibody bind to VISTA under acidic conditions. Thus, anti-VISTA antibodies that bind at low pH to VISTA, e.g., pH 6.5 or lower, as further described herein, are also expected to have a longer acceptable half-life compared to a VISTA antibody that does not binds to VISTA at acidic pH.

Exemplary Abs that bind hVISTA-ECD.

Provided herein are Abs that selectively bind hVISTA (ECD) at an acidic pH (eg, acidic conditions) relative to physiological pH or neutral pH.

In some embodiments, the anti-hVISTA Ab comprises a heavy chain variable region (VH) including CDR1, CDR2, and/or CDR3 of the VH of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab includes a VH including CDR1, CDR2, and CDR3 of the VH of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH comprising CDR1, CDR2, and/or CDR3 of VH P1-061029 or P1-061015 or their daughter clones, such as P1-061029, P1068757, P1-068759, P1-068761, P1-068763 , P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1- 069071 , P1-069073, P1069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1- 18 046477

068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-0687 61_E30D_E55A, P1-068761_E56N_H100G, P1068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1 -068761_E55A_E100fF, P1068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1068761_E32Y_E55A, P1-0 68761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D 52N_E55A, P1068767_E55A_D102V, P1-068767_D102V , P1-068767_E55A, P1-068767_E30D_D52N, P1068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-068767_E100fF_D102V, P1068767_ E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V10 2D, P1-061029_Y32E or P1061029_Y32E_F100fE. The CDR1, CDR2 and CDR3 VH of each of these antibodies include amino acid positions 26-35 (CDR1 VH), 50-66 (CDR2 VH) and 99-110 (CDR3 VH) of the VH sequences of each of the above antibody types, presented in the sequence table below. The CDR regions are also underlined and in bold on each of the VH sequences of the above antibody types presented in the sequence table below.

In some embodiments, the anti-hVISTA Ab includes a VL including CDR1, CDR2, and CDR3 of the VL of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VL comprising CDR1, CDR2, and CDR3 of the VL of one of P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769 , P1068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1069071, P1-069073, P1-069075, P1-0 69077, P1 -061015, P1-068736, P1-068738, P1-068740, P1-068742, P1068744, P1-068766, P1-068748, P1-068750, P1-068752, P1-068754, P1-068761_E55A, P1 -068761_H100G,

P1-O68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30 D_E56N, P1-068761_E100fF, P1068761_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_E32Y, P1068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1- 068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1068767_E30D_D52N, P1-068767_E30D_D102V, P1 -068767_E30D, P1- 068767_E30D_E55A,P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1 -061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E or P1-061029_Y32E_F100fE. The VL CDR1, CDR2 and CDR3 of each of these antibodies include amino acid positions 24-35 (VL CDR1), 51-57 (VL CDR2) and 90-98 (VL CDR3) of the VL sequences of each of the above antibody types, presented in the sequence table below. The CDR regions are also underlined and in bold on each of these sequences.

In some embodiments, the anti-hVISTA Ab includes a VH including CDR1, CDR2 and/or CDR3, a VH of any of the anti-hVTSTA Abs provided herein, and a VL including CDR1, CDR2 and/or CDR3 of any of the anti-hVISTA Abs provided herein document. In some embodiments, the anti-hVISTA Ab includes a VH including CDR1, CDR2, and CDR3 of a VH of any of the anti-hVISTA Abs provided herein, and a VL including CDR1, CDR2, and CDR3 of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab includes a VH including CDR1, CDR2, and/or CDR3 of VH P1-061029 or P1-061015 or their daughter clones, 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, P1-069 069 , 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, P1, P1 -068750, P1-068752 P1-068754, P1-068761_E55A, P1-O68761_H1OOG, P1-O68761_E56N, P1-068761_E55A_E56N, P1068761_E30D, P1-068761_E30D_E55 A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF,P1068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A,P1068761_E32Y_E56N, P1-0 68761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1-068767_E 55A_D102V,P1068767_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_E5 5A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1061029_V102D , P1- 061029_Y32E or P1-061029_Y32E_F100fE, and VL including CDR1, CDR2 and CDR3 of VL P1-061029 or P1-061015 or their daughter clones 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, P1-068 750, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56N, P1068761_E30D, P1-068761_E30D_E55A, P1-06 8761_E56N_H100G, P1-068761_E30D_H100G, P1- 19 046477

068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1068761_E30D_E100fF, P1-068761_E56N_E100fF, P 1-068761_E32Y, P1-068761_E32Y_E55A, P1068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1068767_ D52N_D102V, P1-068767_D52N, P1- 068767_D52N_E55A, P1-068767_E55A_D102V, P1068767_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_E100f F, P1- 061029_F100fE_V102D, P1-061029_F100fE, P1061029_V102D, P1-061029_Y32E or P1-061029_Y32E_F100fE.

In some embodiments, the anti-hVISTA Ab may include:

(a) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-061029, and VL, including CDR1, CDR2 and CDR3 of VL P1-061029;

(b) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-061015, and VL, including CDR1, CDR2 and CDR3 of VL P1-061015;

(c) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068757, and VL, including CDR1, CDR2 and CDR3 of VL P1-068757;

(d) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068759, and VL, including CDR1, CDR2 and CDR3 of VL P1-068759;

(e) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068761, and VL, including CDR1, CDR2 and CDR3 of VL P1-068761;

(f) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068763, and VL, including CDR1, CDR2 and CDR3 of VL P1-068763;

(g) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068765, and VL, including CDR1, CDR2 and CDR3 of VL P1-068765;

(h) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068767, and VL, including CDR1, CDR2 and CDR3 of VL P1-068767;

(i) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068769, and VL, including CDR1, CDR2 and CDR3 of VL P1-068769;

(j) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068771, and VL, including CDR1, CDR2 and CDR3 of VL P1-068771;

(k) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068773, and VL, including CDR1, CDR2 and CDR3 of VL P1-068773;

(l) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068775, and VL, including CDR1, CDR2 and CDR3 of VL P1-068775;

(m) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069059, and VL, including CDR1, CDR2 and CDR3 of VL P1-069059;

(n) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069061, and VL, including CDR1, CDR2 and CDR3 of VL P1-069061;

(o) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069063, and VL, including CDR1, CDR2 and CDR3 of VL P1-069063;

(p) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069065, and VL, including CDR1, CDR2 and CDR3 of VL P1-069065;

(q) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069067, and VL, including CDR1, CDR2 and CDR3 of VL P1-069067;

(r) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069069, and VL, including CDR1, CDR2 and CDR3 of VL P1-069069;

(s) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069071, and VL, including CDR1, CDR2 and CDR3 of VL P1-069071;

(t) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069073, and VL, including CDR1, CDR2 and CDR3 of VL P1-069073;

(u) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069075, and VL, including CDR1, CDR2 and CDR3 of VL P1-069075;

(v) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-069077, and VL, including CDR1, CDR2 and CDR3 of VL P1-069077;

(w) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068736, and VL, including CDR1, CDR2 and CDR3 of VL P1-068736;

(x) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068738, and VL, including CDR1, CDR2 and CDR3 of VL P1-068738;

(y) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068740, and VL, including CDR1, CDR2 and CDR3 of VL P1-068740;

(z) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068742, and VL, including CDR1, CDR2 and CDR3 of VL P1-068742;

(aa) VH comprising the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068744, and

- 20 046477

VL including CDR1, CDR2 and CDR3 VL P1-068744;

(bb) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068746, and VL, including CDR1, CDR2 and CDR3 of VL P1-068746;

(cc) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068748, and VL, including CDR1, CDR2 and CDR3 of VL P1-068748;

(dd) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068750, and VL, including CDR1, CDR2 and CDR3 of VL P1-068750;

(ee) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068752, and VL, including CDR1, CDR2 and CDR3 of VL P1-068752;

(ff) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-068754, and VL, including CDR1, CDR2 and CDR3 of VL P1-068754;

(gg) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1068761_E55A, and VL, including CDR1, CDR2 and CDR3 of VL P1-068761_E55A;

(hh) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1068761_H100G, and VL, including CDR1, CDR2 and CDR3 of VL P1-068761_H100G;

(ii) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1068761_E56N, and VL, including CDR1, CDR2 and CDR3 of VL P1-068761_E56N;

(jj) VH including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E55A_E56N, and VL including CDR1, CDR2 and CDR3 VL P1-068761_E55A_E56N;

(kk) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1068761_E30D, and VL, including CDR1, CDR2 and CDR3 of VL P1-068761_E30D;

(ll) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761E30DE55A, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E30D_E55A;

(mm) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E56N_H100G, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E56N_H100G;

(nn) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E30D_H100G, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E30D_H100G;

(oo) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E30D_E56N, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E30D_E56N;

(pp) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E100fF;

(qq) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E55A_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E55A_E100fF;

(rr) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_H100G_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_H100G_E100fF;

(ss) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E30D_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E30D_E100fF;

(tt) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E56N_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E56N_E100fF;

(uu) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E32Y, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E32Y;

(vv) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E32Y_E55A, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E32Y_E55A;

(ww) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E32Y_E56N, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E32Y_E56N;

(xx) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E30D_E32Y, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E30D_E32Y;

(yy) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E32Y_H100G, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E32Y_H100G;

(zz) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068761_E32Y_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068761_E32Y_E100fF;

(aaa) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_D52N_D102V, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_D52N_D102V;

(bbb) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_D52N, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_D52N;

(ccc) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_D52N_E55A, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_D52N_E55A;

(ddd) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E55A_D102V, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E55A_D102V;

(eee) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_D102V, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_D102V;

(fff) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1

- 21 046477

068767_E55A, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E55A;

(ggg) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E30D_D52N, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E30D_D52N;

(hhh) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E30D_D102V, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E30D_D102V;

(iii) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1068767_E30D, and VL, including CDR1, CDR2 and CDR3 of VL P1-068767_E30D;

(jjj) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E30D_E55A, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E30D_E55A;

(kkk) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E100fF_D102V, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E100fF_D102V;

(lll) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E55A_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E55A_E100fF;

(mmm) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_D52N_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_D52N_E100fF;

(nnn) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E100fF;

(ooo) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1068767_E30D_E100fF, and VL, including CDR1, CDR2 and CDR3 VL P1-068767_E30D_E100fF;

(ppp) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1061029_F100fE_V102D, and VL, including CDR1, CDR2 and CDR3 VL P1-061029_F100fE_V102D;

(qqq) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1061029_F100fE, and VL, including CDR1, CDR2 and CDR3 VL P1-061029_F100fE;

(pt) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 of VH P1-061029_V102D, and VL, including CDR1, CDR2 and CDR3 of VL P1-061029_V102D;

(sss) VH, including the amino acid sequence of CDR1, CDR2 and CDR3 VH P1061029_Y32E, and VL, including CDR1, CDR2 and CDR3 VL P1-061029_Y32E; or (ttt) VH, including the amino acid sequence CDR1, CDR2 and CDR3 VH P1061029_Y32E_F100fE, and VL, including CDR1, CDR2 and CDR3 VL P1-061029_Y32E_F100fE.

Again, the sequence table below shows the heavy and light chain variable region sequences and the full length heavy and light chain sequences of the antibodies above, with the IgG1.3 heavy chain constant region (provided that no other HC constant region is noted in the table). and the amino acid residue positions of their CDR1, CDR2, and CDR3 VH and CDR1, CDR2, and CDR3 VL are indicated, and the CDR regions in each VH and VL sequence are highlighted in bold and underlined. Thus, for example, CDR1 VH P1-061029 includes amino acids 26-35 of SEQ ID NO: 67, while CDR2 VH includes amino acids 50-66 of SEQ ID NO: 67, and CDR3 VH includes amino acids 99-110 of SEQ ID NO: 67. etc., indicated by the bolded and underlined amino acids in SEQ GO NO: 67 shown in the sequence table.

In some embodiments, the anti-hVISTA Ab comprises a VH comprising the amino acid sequence of the VH of any of the anti-hVISTA Abs provided herein. Individual VH sequences for specific types of antibodies proposed herein are listed in the sequence table. In some embodiments, the anti-hVISTA Ab comprises a VH comprising the amino acid sequence of VH P1-061029 or P1-061015 or daughter clones thereof, 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, P1-069 071, 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, P1068761_E55A, P1-O68761_H1OOG, P1-O68761_E56N, P1-O68761_E55A_E56N, P1-068761_E30D, P1068761_E30D_E55A, P1-068761 _E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E 30D_E100fF , P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068 761_E32Y_E100fF, P1-068767_D52N_D102V, P1068767_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, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E or P1-061029_Y32E_F1 00fE.

In some embodiments, the anti-hVISTA Ab includes the VH of any of P1061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1068773, P1- 068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1069073, P1-069075, P1-069077, P1-061015, P1-0687 36, P1- 068738, P1-068740, P1-068742, P1-068744, P1- 22 046477

068766, P1-068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1068761_E56N, P1-068761_E55A_E56N, P1-068761_E3 0D, P1-068761_E30D_E55A, P1068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1 -068761_E100fF, P1068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068 761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-06876 7_D52N, P1068767_D52N_E55A, P1 -068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A ,P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V10 2D, P1-061029_F100fE, P1 -061029_V102D,P1061029_Y32E or P1-061029_Y32E_F100fE, but with 1, 2, 3, 4 or 5 amino acid substitutions in the framework regions of the VH sequence, for example, 1, 2, 3, 4 or 5 conservative substitutions. In some embodiments, the anti-hVISTA Ab includes the VH of any of the antibodies P1-061029 or its daughter clones, such as 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, P1-068766, P1-068761_E55A, P1-O68761_H1OOG, P1-O68761_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-0687 61_E56N_H100G, P1068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1068761_H100G_E100fF, P1- 068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-06876 1_E32Y_H100G, P1068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1068767_E55A_D102V, P1-068767_D102V , P1-068767_E55A, P1- 068767_E30D_D52N, P1068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-068767_E100fF_D102V, P1068767_E55A_E100fF, P1-0687 67_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E or P 1061029_Y32E_F100fE, but with one or more reversible germline substitutions, for example, one or both of the K16R and/or T84A substitutions, in the VH framework regions of the sequences shown in the sequence table. Exemplary VH sequences with these amino acid substitutions are presented in the sequence table, with residues 16 and 84 in bold and underlined. It should be noted that P1-061015 contains R at position 16 and A at position 84 of its VH framework regions.

In some embodiments, the anti-hVISTA Ab includes CDR1, CDR2, and CDR3 VHs comprising the amino acid sequences of the VH CDR regions of any of the anti-hVISTA Abs provided herein, and includes 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 of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH 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, P1-068 766, P1-068748, P1-068750, P1-068752 P1-068754, P1068761_E55A, P1-O68761_H1OOG, P1-O68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P10687 61_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF, P1 -068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1- 068761_E32Y_E56N, P1068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1068767_D52N, P1-068767_D 52N_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, P1068767_E100fF_D10 2V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D , P1061029_Y32E or P1-061029_Y32E_F100fE. In some embodiments, the antibody VH differs from the VH sequences shown in the sequence table by the presence of 1, 2, 3, 4, or 5 amino acid substitutions in the framework regions of the VH sequence, e.g., 1, 2, 3, 4, or 5 conservative substitutions or, e.g. , one or both of the K16R and/or T84A substitutions in P1-061029 or its daughter clones.

In some embodiments, the anti-hVISTA Ab comprises a VH consisting of the VH amino acid sequence of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab includes a VH that consists of the amino acid sequence of VH P1-061029 or P1-061015 or their daughter clones, such as P1-061029, Pl068757, Pl-068759, P1-068761, P1- 068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-0690 69, P1- 069071, P1-069073, P1069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, PT068748, P1-06875 0, P1-068752 P1 -068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H 100G, P1068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1068761_H100G_E100fF, P1-068761_E30D_E100 fF, P1 -068761_E56N_E100fF, P1-068761_E32Y, P1068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1068761_ E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068 767_E30D_D52N, P1068767_E30D_D102V , P1-068767_E30D, P1-068767_E30D_E55A, P1-068767_E100fF_D102V, P1068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-0 68767_E30D_E100fF, P1061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E or P1061029_Y32E_F100fE, optionally with one or both of the substitutions K16R and/or T84A.

In some embodiments, the anti-hVISTA Ab comprises a VL comprising the amino acid sequence of the VL of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VL comprising the amino acid sequence of VL P1-061029 or P1-061015 or their daughter clones, such as P1-061029, P1068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773, P1068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-06 9073, P1069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, P1068748, P1-068750, P1-068752 P1-0687 54, P1-068761_E55A , P1-068761_H100G, P1-068761_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1068761_E30D_H1 00G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P1068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E10 0fF, P1-068761_E32Y , P1068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1-068761_E32Y_H100G, P1068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1068767_E30D_D102V , P1-068767_E30D, P1 -068767_E30D_E55A, P1-068767_E100fF_D102V, P1068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P 1061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E or P1061029_Y32E_F100fE. In some embodiments, the anti-hVISTA Ab includes CDR1, CDR2, and CDR3 of a VL comprising the amino acid sequences of the VL CDR regions of any of the anti-hVISTA Abs provided herein, and includes 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 of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of VL P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1068765, P1-068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1069065, P1-069 067, P1- 069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1068738, P1-068740, P1-068742, P1-068744, P1-068766, P1-0687 48, P1- 068750, P1-068752 P1-068754, P1068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1068761_E30D_E55 A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF , P1-068761_E30D_E100fF, P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1068761_E30D_E32Y, P1-06 8761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D 102V, P1-068767_E55A , P1-068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1 -068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y3 2E or P1-061029_Y32E_F100fE . In some embodiments, the antibody VL differs from the VL sequences shown in the sequence table by the presence of 1, 2, 3, 4, or 5 amino acid substitutions in the framework regions of the VL sequence, such as 1, 2, 3, 4, or 5 conservative substitutions.

In some embodiments, the anti-hVISTA Ab comprises a VL consisting of the VL amino acid sequence of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a VL consisting of an amino acid

- 24 046477 VL sequence P1-061029 or P1-061015 or their daughter clones, such as P1-061029, Pl068757, Pl-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1 -068771, P1-068773, P1068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1069075, P1-06907 7, P1-061015 , P1-068736, P1-068738, P1-068740, P1-068742, P1-068744, P1-068766, PT068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-068761 _H100G, P1-068761_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761 _E100fF, P1-068761_E55A_E100fF, P1068761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1068761_E32Y_ E55A, P1-068761_E32Y_E56N, P1- 068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-06876 l_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P106876 7_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-068 767_E100fF_D102V , P1068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1-068767_E30D_E100fF, P1061029_F100fE_V102D, P1-061029_F100fE, P 1-061029_V102D, P1-061029_Y32E or P1061029_Y32E_F100fE.

In some embodiments, an anti-hVISTA Ab comprises a VH comprising the VH amino acid sequence of any of the anti-hVISTA Abs provided herein, and includes a VL comprising the VL amino acid sequence of any of the anti-hVISTA Abs provided herein. In some of these embodiments, the anti-hVISTA Ab comprises a VH comprising the amino acid sequence of VH P1-061029 or P1-061015 or their daughter clones, 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, P1-068 750, P1-068752 P1-068754, P1068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1068761_E30D_E55A, P1-068 761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1 -068761_E30D_E100fF, P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1068761_E30D_E32Y, P1-068761 _E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1068767_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, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068 767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E or P 1-061029_Y32E_F100fE; and VL, comprising the amino acid sequence of VL P1-061029 or P1-061015.

In some embodiments, however, the VH of the antibody is VH P1 -061029, P1068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1-068771, P1-068773 , P1068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1069075, P1-069077, P1-061015, P1-0 68736, P1 -068738, P1-068740, P1-068742, P1-068744, P1-068766, P1068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-068761_H100G, P1-0687 61_E56N, P1068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1-068761_E55A_E100fF, P106 8761_H100G_E100fF, P1-068761_E30D_E100fF, P1-068761_E56N_E100fF, P1-068761_E32Y, P1068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761 _E30D_E32Y, P1-068761_E32Y_H100G, P1068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1-068767_D52N_E55A, P1068767_E55A_D102V, P1-068767_D102V, P1-068767_E 55A, P1-068767_E30D_D52N, P1068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1-068767_E100fF_D102V, P1068767_E55A_E100fF, P1 -068767_D52N_E100fF, P1- 068767_E100fF, P1-068767_E30D_E100fF, P1061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1-061029_Y32E or P1061029_Y32E_F10 0fE, but with 1, 2, 3, 4 or 5 amino acid substitutions in the framework regions of the VH sequence, such as 1, 2, 3, 4 or 5 conservative substitutions, and VL is VL P1-061029 or P1061015. In some embodiments, however, the VH of the antibody is VH P1-061029 or its daughter clones, such as P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1068769, P1 -068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-069067, P1069069, P1-069071, P1-069073, P1-069075, P1-0690 77, P1 -068766, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1068761_E56N_H 100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E10 0fF , P1-068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1068761_E32Y_H100G, P1-06 8761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P 1068767_E30D_D52N , P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1- 25 046477

068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102 D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E or P1-061029_Y32E_F100fE, but with one or both of the K16R or T84A substitutions, and VL is VL P1 -061029 or P1-061015.

In some embodiments, the anti-hVISTA Ab comprises VH and VL, comprising the amino acid sequences of VH and VL of P1-061029 or P1-061015 or their daughter clones, 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, P1-069 069, 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, P1068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E5 5A, P1068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, PI-068761_E100fF, P1068761_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, P10 68761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_ D102V, P1 -068767_E55A,P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A,P1068767_E100fF_D102V, P1-068767_E55A _E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D,P 1061029_Y32E or P1-061029_Y32E_F100fE and, optionally, where VH includes one or both of the K16R and/or T84A substitutions.

In some embodiments, the anti-hVISTA Ab includes VH CDR1, CDR2, and CDR3, including the amino acid sequences of the VH CDR regions of any of the anti-hVISTA Abs provided herein, and VL CDR1, CDR2, and CDR3, including the amino acid sequences of the VL CDR regions of any of the anti-hVISTA Abs provided herein, and also includes 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 of the anti-hVISTA Abs proposed in this document. In some embodiments, the VH and VL antibodies differ from the VH and VL sequences shown in the sequence table by the presence of 1, 2, 3, 4, or 5 amino acid substitutions in the framework regions of the sequences, such as 1, 2, 3, 4, or 5 conservative substitutions , or such as one or both of the K16R and/or T84A substitutions in the VH sequence.

In some embodiments, the anti-hVISTA Ab includes a VH and a VL consisting of the amino acid sequence of the VH and VL of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises VH and VL, each consisting of the amino acid sequences VH and VL P1 -061029 or P1-061015 or daughter clones thereof, such as P1-061029, P1-068757, P1-068759, P1- 068761, P1-068763, P1-068765, P1-068767, P1068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, P1-0690 67, P1069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1068742, P1-068744, P1-068766, P1-068748, P1-06 8750, P1-068752 P1-068754, P1-068761_E55A, P1068761_H100G, P1-O68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1068 761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1068761_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, P106876 1_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102 V, P1-068767_E55A ,P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A,P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1- 068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32 E or P1-061029_Y32E_F100fE, and optionally, wherein the VHs include one or both of the K16R and/or T84A substitutions.

Anti-hVISTA antibodies may include:

(a) VH comprising the amino acid sequence of VH P1-061029, and VL comprising the amino acid sequence of VL P1-061029;

(b) VH comprising the amino acid sequence of VH P1-061015, and VL comprising the amino acid sequence of VL P1-061015;

(c) VH comprising the amino acid sequence of VH P1-068757, and VL comprising the amino acid sequence of VL P1-068757;

(d) VH comprising the amino acid sequence of VH P1-068759, and VL comprising the amino acid sequence of VL P1-068759;

(e) VH, including the amino acid sequence of VH P1-068761, and VL, including

- 26 046477 amino acid sequence VL P1-068761;

(f) VH comprising the amino acid sequence of VH P1-068763, and VL comprising the amino acid sequence of VL P1-068763;

(g) VH comprising the amino acid sequence of VH P1-068765, and VL comprising the amino acid sequence of VL P1-068765;

(h) VH comprising the amino acid sequence of VH P1-068767, and VL comprising the amino acid sequence of VL P1-068767;

(i) VH, including the amino acid sequence of VH P1-068769, and VL, including the amino acid sequence of VL P1-068769;

(j) VH comprising the amino acid sequence of VH P1-068771, and VL comprising the amino acid sequence of VL P1-068771;

(k) VH, including the amino acid sequence of VH P1-068773, and VL, including the amino acid sequence of VL - 068773;

(l) VH comprising the amino acid sequence of VH P1-068775, and VL comprising the amino acid sequence of VL P1-068775;

(m) VH comprising the amino acid sequence of VH P1-069059, and VL comprising the amino acid sequence of VL P1-069059;

(n) VH, including the amino acid sequence of VH P1-069061, and VL, including the amino acid sequence of VL P1 -069061;

(o) VH, including the amino acid sequence of VH P1-069063, and VL, including the amino acid sequence of VL P1 -069063;

(p) VH comprising the amino acid sequence of VH P1-069065, and VL comprising the amino acid sequence of VL P1-069065;

(q) VH comprising the amino acid sequence of VH P1-069067, and VL comprising the amino acid sequence of VL P1-069067;

(r) VH comprising the amino acid sequence of VH P1-069069, and VL comprising the amino acid sequence of VL P1-069069;

(s) VH comprising the amino acid sequence of VH P1-069071, and VL comprising the amino acid sequence of VL P1 -069071;

(t) VH, including the amino acid sequence of VH P1-069073, and VL, including the amino acid sequence of VL P1 -069073;

(u) VH comprising the amino acid sequence of VH P1-069075, and VL comprising the amino acid sequence of VL P1-069075;

(v) VH comprising the amino acid sequence of VH P1-069077, and VL comprising the amino acid sequence of VL P1-069077;

(w) VH comprising the amino acid sequence of VH P1-068736, and VL comprising the amino acid sequence of VL P1-068736;

(x) VH comprising the amino acid sequence of VH P1-068738, and VL comprising the amino acid sequence of VL P1-068738;

(y) VH comprising the amino acid sequence of VH P1-068740, and VL comprising the amino acid sequence of VL P1-068740;

(z) VH comprising the amino acid sequence of VH P1-068742, and VL comprising the amino acid sequence of VL P1-068742;

(aa) VH comprising the amino acid sequence of VH P1-068744, and VL comprising the amino acid sequence of VL P1-068744;

(bb) VH comprising the amino acid sequence of VH f P1-068746, and VL comprising the amino acid sequence of VL P1-068746;

(cc) VH comprising the amino acid sequence of VH P1-068748, and VL comprising the amino acid sequence of VL P1-068748;

(dd) VH comprising the amino acid sequence of VH P1-068750, and VL comprising the amino acid sequence of VL P1-068750;

(ee) VH comprising the amino acid sequence of VH P1-068752, and VL comprising the amino acid sequence of VL P1-068752;

(ff) VH comprising the amino acid sequence of VH P1-068754, and VL comprising the amino acid sequence of VL P1-068754;

(gg) VH, including the amino acid sequence of VH P1-068761_E55A, and VL, including the amino acid sequence of VL P1-068761_E55A;

(hh) VH comprising the amino acid sequence VHP 1-068761_H100G, and VL comprising the amino acid sequence VL P1-068761_H100G;

(ii) VH comprising the amino acid sequence of VH P1-068761_E56N, and VL comprising the amino acid sequence of VL P1-068761_E56N;

(jj) VH, including the amino acid sequence VH P1-O68761_E55A_E56N, and VL,

- 27 046477 including the amino acid sequence VL P1-068761_E55A_E56N;

(kk) VH, including the amino acid sequence of VH P1-068761_E30D, and VL, including the amino acid sequence of VL P1-068761_E30D;

(ll) VH comprising the amino acid sequence of VH P1-O68761_E3OD_E55A, and VL comprising the amino acid sequence of VL P1-O68761_E3OD_E55A;

(mm) VH, including the amino acid sequence of VH P1-O68761_E56N_H1OOG, and VL, including the amino acid sequence of VL P1-068761_E56N_H100G;

(nn) VH, including the amino acid sequence of VH P1-O68761_E3OD_H1OOG, and VL, including the amino acid sequence of VL P1-068761_E30D_H100G;

(oo) VH, including the amino acid sequence of VH P1-O68761_E3OD_E56N, and VL, including the amino acid sequence of VL P1-O68761_E3OD_E56N;

(pp) VH, including the amino acid sequence of VH P1-068761_E100fF, and VL, including the amino acid sequence of VLP1-068761_E100fF;

(qq) VH, including the amino acid sequence of VH P1-068761_E55A_E100fF, and VL, including the amino acid sequence of VL P1-068761_E55A_E100fF;

(rr) VH, including the amino acid sequence VH P1-068761_H10()G_E1()()fF. and VL, including the amino acid sequence VL P1-068761_H100G_E100fF;

(ss) VH, including the amino acid sequence of VH P1-068761_E30D_E100fF, and VL, including the amino acid sequence of VL P1-068761_E30D_E100fF;

(tt) VH, including the amino acid sequence of VH P1-068761_E56N_E100fF, and VL, including the amino acid sequence of VL P1-068761_E56N_E100fF;

(uu) VH, including the amino acid sequence of VH P1-068761_E32Y, and VL, including the amino acid sequence of VL P1-068761_E32Y;

(vv) VH comprising the amino acid sequence of VH P1-068761_E32Y_E55A, and VL comprising the amino acid sequence of VL P1-068761_E32Y_E55A;

(ww) VH, including the amino acid sequence of VH P1-068761_E32Y_E56N, and VL, including the amino acid sequence of VL P1-068761_E32Y_E56N;

(xx) VH, including the amino acid sequence of VH P1-068761_E30D_E32Y, and VL, including the amino acid sequence of VL P1-068761_E30D_E32Y;

(UU) VH, including the amino acid sequence of VH P1-068761_E32Y_H100G, and VL, including the amino acid sequence of VL P1-068761_E32Y_H100G;

(zz) VH, including the amino acid sequence of VH P1-068761_E32Y_E100fF, and VL, including the amino acid sequence of VL P1-068761_E32Y_E100fF;

(aaa) VH, including the amino acid sequence of VH P1-068767_D52N_D102V, and VL, including the amino acid sequence of VL P1-068767_D52N_D102V;

(bbb) VH, including the amino acid sequence of VH P1-068767_D52N, and VL, including the amino acid sequence of VL P1-068767_D52N;

(sss) VH, including the amino acid sequence of VH P1-068767_D52N_E55A, and VL, including the amino acid sequence of VL P1-068767_D52N_E55A;

(ddd) VH, including the amino acid sequence of VH P1-068767_E55A_D102V, and VL, including the amino acid sequence of VL P1-068767_E55A_D102V;

(eee) VH, including the amino acid sequence of VH P1-068767_D102V, and VL, including the amino acid sequence of VL P1-068767_D102V;

(iff) VH, including the amino acid sequence of VH P1-068767_E55A, and VL, including the amino acid sequence of VL P1-068767_E55A;

(ggg) VH, including the amino acid sequence of VH P1-068767_E30D_D52N, and VL, including the amino acid sequence of VL P1-068767_E30D_D52N;

(hhh) VH, including the amino acid sequence of VH P1-068767_E30D_D102V, and VL, including the amino acid sequence of VL P1-068767_E30D_D102V;

(iii) VH comprising the amino acid sequence of VH P1-068767_E30D, and VL comprising the amino acid sequence of VL P1-068767_E30D;

(jjj) VH, including the amino acid sequence of VH P1-068767_E30D_E55A, and VL, including the amino acid sequence of VL P1-068767_E30D_E55A;

(kkk) VH, including the amino acid sequence of VH P1-068767_E100fF_D102V, and VL, including the amino acid sequence of VL P1-068767_E100fF_D102V;

(lll) VH, including the amino acid sequence of VH P1-068767_E55A_E100fF, and VL, including the amino acid sequence of VL P1-068767_E55A_E100fF;

(mmm) VH, including the amino acid sequence of VH P1-068767_D52N_E100fF, and VL, including the amino acid sequence of VL P1-068767_D52N_E100fF;

(nnn) VH, including the amino acid sequence of VH P1-068767_E100fF, and VL, including the amino acid sequence of VL P1-068767_E100fF;

(ooo) VH, including the amino acid sequence VH P1-068767_E30D_E100fF, and VL,

- 28 046477 including the amino acid sequence VL P1-068767_E30D_E100fF;

(ppp) VH, including the amino acid sequence of VH P1-061029_F100fE_V102D, and VL, including the amino acid sequence of VL P1-061029_F100fE_V102D;

(qqq) VH, including the amino acid sequence of VH P1-061029_F100fE, and VL, including the amino acid sequence of VL P1-061029_F100fE;

(rrr) VH, including the amino acid sequence of VH P1-061029_V102D, and VL, including the amino acid sequence of VL P1-061029_V102D;

(sss) VH, including the amino acid sequence of VH P1-061029_Y32E, and VL, including the amino acid sequence of VL P1-061029_Y32E; or (ttt) VH, including the amino acid sequence VH P1-061029_Y32E_F100fE, and VL, including the amino acid sequence VL P1-061029_Y32E_F100fE.

Anti-hVTSTA antibodies may include:

(a) VH, including the amino acid sequence of VH P1-061029, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-061029;

(b) VH, including the amino acid sequence of VH P1-068757, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068757;

(c) VH, including the amino acid sequence of VH P1-068759, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068759;

(d) VH, including the amino acid sequence of VH P1-068761, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-068761;

(e) VH, including the amino acid sequence of VH P1-068763, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068763;

(f) VH, including the amino acid sequence of VH P1-068765, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-068765;

(g) VH, including the amino acid sequence of VH P1-068767, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-068767;

(h) VH, including the amino acid sequence of VH P1-068769, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-068769;

(i) VH, including the amino acid sequence of VH P1-068771, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068771;

(j) VH, including the amino acid sequence of VH P1-068773, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068773;

(k) VH, including the amino acid sequence of VH P1-068775, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068775;

(l) VH, including the amino acid sequence of VH P1-069059, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069059;

(m) VH, including the amino acid sequence of VH P1-069061, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069061;

(n) VH, including the amino acid sequence of VH P1-069063, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069063;

(o) VH, including the amino acid sequence of VH P1-069065, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-069065;

(p) VH, including the amino acid sequence of VH P1-069067, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069067;

(q) VH, including the amino acid sequence of VH P1-069069, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069069;

(r) VH, including the amino acid sequence of VH P1-069071, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069071;

(s) VH, including the amino acid sequence of VH P1-069073, modified by K16R and/or T84A substitutions, VL including the amino acid sequence of VL P1-069073;

(t) VH, including the amino acid sequence of VH P1-069075, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-069075;

(u) VH, including the amino acid sequence of VH P1-069077, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence of VL P1-069077;

(v) VH, including the amino acid sequence VH P1-068761_E55A, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence VL P1068761_E55A;

(w) VH, including the amino acid sequence VH P1-068761_H100G, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068761_H100G;

(x) VH, including the amino acid sequence VH P1-068761_E56N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-

- 29 046477

068761_E56N;

(y) VH, including the amino acid sequence VH P1-068761_E55A_E56N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E55A_E56N;

(z) VH, including the amino acid sequence VH P1-068761_E30D, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068761_E30D;

(aa) VH, including the amino acid sequence VH P1-O68761_E3OD_E55A, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E30D_E55A;

(bb) VH, including the amino acid sequence VH P1-O68761_E56N_H1OOG, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E56N_H100G;

(cc) VH, including the amino acid sequence VH P1-O68761_E3OD_H1OOG, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E30D_H100G;

(dd) VH, including the amino acid sequence VH P1-O68761_E3OD_E56N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1 -068761_E30D_E56N;

(ee) VH, including the amino acid sequence VH P1-068761_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068761_E100fF;

(ff) VH, including the amino acid sequence VH P1-068761_E55A_E10()fF. modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence VL P1-068761_E55A_E100fF;

(gg) VH, including the amino acid sequence VH P1-068761_H100G_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_H100G_E100fF;

(hh) VH, including the amino acid sequence of VH P1-068761_E30D_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068761_E30D_E100fF;

(ii) VH, including the amino acid sequence of VH P1-068761_E56N_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1-068761_E56N_E100fF;

(jj) VH, including the amino acid sequence of VH P1-068761_E32Y, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence of VL P1068761_E32Y;

(kk) VH, including the amino acid sequence VH P1-068761_E32Y_E55A, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E32Y_E55A;

(ll) VH, including the amino acid sequence VH P1-068761_E32Y_E56N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1 -068761_E32Y_E56N;

(mm) VH, including the amino acid sequence VH P1-068761_E30D_E32Y, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E30D_E32Y;

(nn) VH, including the amino acid sequence VH P1-068761_E32Y_H100G, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E32Y_H100G;

(oo) VH, including the amino acid sequence VH P1-068761_E32Y_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068761_E32Y_E100fF;

(pp) VH, including the amino acid sequence VH P1-068767_D52N_D102V, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_D52N_D102V;

(qq) VH, including the amino acid sequence VH P1-068767_D52N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068767_D52N;

(rr) VH, including the amino acid sequence VH P1-068767_D52N_E55A, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_D52N_E55A;

(ss) VH, including the amino acid sequence VH P1-068767_E55A_D102V, modified

- 30 046477 cited by substitutions K16R and/or T84A, and VL, including the amino acid sequence VL P1-068767_E55A_D102V;

(tt) VH, including the amino acid sequence VH P1-068767_D102V, modified by K16R and/or T84A substitutions, VL including the amino acid sequence VL P1068767_D102V;

(uu) VH, including the amino acid sequence VH P1-068767_E55A, modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence VL P1068767_E55A;

(vv) VH, including the amino acid sequence VH P1-O68767_E3OD_D52N, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_E30D_D52N;

(ww) VH, including the amino acid sequence VH P1-O68767_E3OD_D1O2V, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_E30D_D102V;

(xx) VH, including the amino acid sequence VH P1-068767_E30D, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068767_E30D;

(yy) VH, including the amino acid sequence VH P1-O68767_E3OD_E55A, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_E30D_E55A;

(zz) VH, including the amino acid sequence VH P1-068767_E100fF_D102V. modified by substitutions K16R and/or T84A, and VL, including the amino acid sequence VL P1-068767_E100fF_D102V;

(aaa) VH, including the amino acid sequence VH P1-068767_E55A_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_E55A_E100fF;

(bbb) VH, including the amino acid sequence VH P1-068767_D52N_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_D52N_E100fF;

(cc) VH, including the amino acid sequence VH P1-068767_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1068767_E100fF;

(ddd) VH, including the amino acid sequence VH P1-068767_E30D_E100fF, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-068767_E30D_E100fF;

(eeee) VH, including the amino acid sequence VH P1-061029_F100fE_V102D, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-061029_F100fE_V102D;

(fff) VH, including the amino acid sequence VH P1-061029_F100fE, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1061029_F100fE;

(ggg) VH, including the amino acid sequence VH P1-061029_V102D, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1061029_V102D;

(hhh) VH, including the amino acid sequence VH P1-061029_Y32E, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1061029_Y32E; or (iii) VH, including the amino acid sequence VH P1-061029_Y32E_F100fE, modified by K16R and/or T84A substitutions, and VL, including the amino acid sequence VL P1-061029_Y32E_F100fE.

Anti-hVTSTA antibodies may include:

(a) VH, including the CDR regions of VH P1-061029, and VL, including the CDR regions of VL P1-061029, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-061029;

(b) VH, including the CDR regions of VH P1-061015, and VL, including the CDR regions of VL P1061015, and the amino acid sequences of VH and VL, which 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 P1-061015;

(c) VH, including the CDR regions of VH of Pl-068757, and VL, including the CDR regions of VL of Pl-068757,

- 31 046477 and amino acid sequences VH and VL, which 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 P1-068757;

(d) VH, including the CDR regions of VH P1-068759, and VL, including the CDR regions of VL P1068759, and the amino acid sequences of 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 VH and VL P1-068759;

(e) VH, including the CDR regions of VH P1-068761, and VL, including the CDR regions of VL P1-068761, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068761;

(f) VH, including the CDR regions of VH P1-068763, and VL, including the CDR regions of VL P1-068763, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068763;

(g) VH, including the CDR regions of VH P1-068765, and VL, including the CDR regions of VL P1068765, and the amino acid sequences of 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 VH and VL P1-068765;

(h) VH, including the CDR regions of VH P1-068767, and VL, including the CDR regions of VL P1068767, and the amino acid sequences of VH and VL, which 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 P1-068767;

(i) VH, including the CDR regions of VH P1-068769, and VL, including the CDR regions of VL P1-068769, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068769;

(j) VH, including the CDR regions of VH P1-068771, and VL, including the CDR regions of VL P1-068771, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068771;

(k) VH, including the CDR regions of VH P1-068773, and VL, including the CDR regions of VL P1068773, and the amino acid sequences of 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 VH and VL P1-068773;

(l) VH, including the CDR regions of VH P1-068775, and VL, including the CDR regions of VL P1-068775, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068775;

(m) VH, including the CDR regions of VH P1-069059, and VL, including the CDR regions of VL P1069059, and the amino acid sequences of VH and VL, which 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 P1-069059;

(n) VH, including the CDR regions of VH P1-069061, and VL, including the CDR regions of VL P1069061, and the amino acid sequences of VH and VL, which 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 P1-069061;

(o) VH, including the CDR regions of VH P1-069063, and VL, including the CDR regions of VL P1069063, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

- 32 046477 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to VH and VL P1-069063;

(p) VH, including the CDR regions of VH P1-069065, and VL, including the CDR regions of VL P1069065, and the amino acid sequences of VH and VL, which 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 P1-069065;

(q) VH, including the CDR regions of VH P1-069067, and VL, including the CDR regions of VL P1069067, and the amino acid sequences of VH and VL, which 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 P1-069067;

(r) VH, including the CDR regions of VH P1-069069, and VL, including the CDR regions of VL P1-069069, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-069069;

(s) VH, including the CDR regions of VH P1-069071, and VL, including the CDR regions of VL P1-069071, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-069071;

(t) VH, including the CDR regions of VH P1-069073, and VL, including the CDR regions of VL P1-069073, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-069073;

(u) VH, including the CDR regions of VH P1-069075, and VL, including the CDR regions of VL P1069075, and the amino acid sequences of VH and VL, which 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 P1-069075;

(v) VH, including the CDR regions of VH P1-069077, and VL, including the CDR regions of VL P1069077, and the amino acid sequences of VH and VL, which 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 P1-069077;

(w) VH, including the CDR regions of VH P1-068736, and VL, including the CDR regions of VL P1068736, and the amino acid sequences of 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 VH and VL P1-068736;

(x) VH, including the CDR regions of VH P1-068738, and VL, including the CDR regions of VL P1068738, and the amino acid sequences of VH and VL, which 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 P1-068738;

(y) VH, including the CDR regions of VH P1-068740, and VL, including the CDR regions of VL P1068740, and the amino acid sequences of VH and VL, which 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 P1-068740;

(z) VH, including the CDR regions of VH P1-068742, and VL, including the CDR regions of VL P1-068742, and the amino acid sequences of VH and VL that are at least 90%, at least 91%, at least 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 P1-068742;

(aa) VH, including the CDR regions of VH P1-068744, and VL, including the CDR regions of VL P1068744, and the amino acid sequences of VH and VL, which 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 P1-068744;

- 33 046477 (bb) VH, including the CDR regions of VH P1-068746, and VL, including the CDR regions of VL Pl068746, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, according to at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% identical to VH and VL P1-068746;

(cc) VH, including the CDR regions of VH P1-068748, and VL, including the CDR regions of VL P1068748, and the amino acid sequences of VH and VL, which 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 P1-068748;

(dd) VH, including the CDR regions of VH P1-068750, and VL, including the CDR regions of VL P1068750, and the amino acid sequences of VH and VL, which 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 P1-068750;

(ee) VH, including the CDR regions of VH P1-068752, and VL, including the CDR regions of VL P1068752, and the amino acid sequences of VH and VL, which 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 P1-068752;

(ff) VH, including the CDR regions of VH P1-068754, and VL, including the CDR regions of VL P1068754, and the amino acid sequences of VH and VL, which 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 P1-068754;

(gg) VH, including the CDR regions of VH P1-068761_E55A, and VL, including the CDR regions of VL P1-068761_E55A, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E55A;

(hh) VH, including the CDR regions of VH P1-068761_H100G, and VL, including the CDR regions of VL P1-068761_H100G, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_H100G;

(ii) VH, including the CDR regions of VH P1-068761_E56N, and VL, including the CDR regions of VL P1068761_E56N, and the amino acid sequences of VH and VL, which 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 P1-068761_E56N;

(jj) VH, including the CDR regions of VH P1-068761_E55A_E56N, and VL, including the CDR regions of VL P1-068761_E55A_E56N, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E55A_E56N;

(kk) VH, including the CDR regions of VH P1-068761_E30D, and VL, including the CDR regions of VL P1-068761_E30D, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E30D;

(ll) VH, including the CDR regions VHP1-068761_E30D_E55A, and VL, including the CDR regions VL P1-068761_E30D_E55A, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least by 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to VH and VL P1-068761_E30D_E55A;

(mm) VH, including the CDR regions of VH P1-068761_E56N_H100G, and VL, including the CDR regions of VL P1-068761_E56N_H100G, and the amino acid sequences of VH and VL, which 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 P1-068761_E56N_H100G;

(nn) VH, including the CDR regions of VH P1-068761_E30D_H100G, and VL, including the CDR regions of VL P1-068761_E30D_H100G, and the amino acid sequences of VH and VL, which are

- 34 046477 by 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 P1-068761_E30D_H100G;

(oo) VH, including the CDR regions VH P1-068761_E30D_E56N, and VL, including the CDR regions VL P1-068761_E30D_E56N, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E30D_E56N;

(pp) VH, including the CDR regions of VH P1-O68761_E1OOFF, and VL, including the CDR regions of VL P1-068761_E100fF, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E100fF;

(qq) VH, including the CDR regions of VH P1-068761_E55A_E100fF, and VL, including the CDR regions of VL P1-068761_E55A_E100fF, and the amino acid sequences of VH and VL, which 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 P1-068761_E55A_E100fF;

(rr) VH, including the CDR regions of VH P1-068761_H100G_E100fF, and VL, including the CDR regions of VL P1-068761_H100G_E100fF, and the amino acid sequences of VH and VL, which 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 P1-068761_H100G_E100fF;

(ss) VH, including the CDR regions of VH P1-068761_E30D_E100fF, and VL, including the CDR regions of VL P1-068761_E30D_E100fF, and the amino acid sequences of VH and VL, which 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 P1-068761_E30D_E100fF;

(tt) VH, including the CDR regions of VH P1-068761_E56N_E100fF, and VL, including the CDR regions of VL P1-068761_E56N_E100fF, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 VH and VL P1-068761_E56N_E100fF;

(uu) VH, including the CDR regions of VH P1-068761_E32Y, and VL, including the CDR regions of VL P1-068761_E32Y, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E32Y;

(vv) VH, including the CDR regions of VH P1-068761_E32Y_E55A, and VL, including the CDR regions of VL P1-068761_E32Y_E55A, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E32Y_E55A;

(ww) VH, including the CDR regions of VH P1-068761_E32Y_E56N, and VL, including the CDR regions of VL P1-068761_E32Y_E56N, and the amino acid sequences of VH and VL, which 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 P1-068761_E32Y_E56N;

(xx) VH, including the CDR regions of VH P1-068761_E30D_E32Y, and VL, including the CDR regions of VL P1-068761_E30D_E32Y, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068761_E30D_E32Y;

(yy) VH, including the CDR regions of VH P1-068761_E32Y_H100G, and VL, including the CDR regions of VL P1-068761_E32Y_H100G, and the amino acid sequences of VH and VL, which 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 P1-068761_E32Y_H100G;

(zz) VH, including the CDR regions of VH P1-068761_E32Y_E100fF, and VL, including the CDR regions of VL P1-068761_E32Y_E100fF, and the amino acid sequences of VH and VL, which 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%, according to

- 35 046477 at least 98% or at least 99% identical to VH and VL P1-068761_E32Y_E100fF;

(aaa) VH, including the CDR regions of VH P1-068767_D52N_D102V, and VL, including the CDR regions of VL P1-068767_D52N_D102V, and the amino acid sequences of VH and VL, which 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! VH and VL P1-068767_D52N_D102V;

(bbb) VH, including the CDR regions of VH P1-068767_D52N, and VL, including the CDR regions of VL P1-068767_D52N, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068767_D52N;

(cc) VH, including the CDR regions of VH P1-068767_D52N_E55A, and VL, including the CDR regions of VL P1-068767_D52N_E55A, and the amino acid sequences of VH and VL, which 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 P1-068767_D52N_E55A;

(ddd) VH, including the CDR regions of VH P1-068767_E55A_D102V, and VL, including the CDR regions of VL P1-068767_E55A_D102V, and the amino acid sequences of VH and VL, which 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! VH and VL P1-068767_E55A_D102V;

(ee) VH, including the CDR regions of VH P1-068767_D102V, and VL, including the CDR regions of VL P1-068767_D102V, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068767_D102V;

(fff) VH, including the CDR regions of VH P1-068767_E55A, and VL, including the CDR regions of VL P1-068767_E55A, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068767_E55A;

(ggg) VH, including the CDR regions of VH P1-068767_E30D_D52N, and VL, including the CDR regions of VL P1-068767_E30D_D52N, and the amino acid sequences of VH and VL, which 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 P1-068767_E30D_D52N;

(hhh) VH, including the CDR regions of VH P1-068767_E30D_D102V, and VL, including the CDR regions of VL P1-068767_E30D_D102V, and the amino acid sequences of VH and VL, which 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! VH and VL P1-068767_E30D_D102V;

(iii) VH, including CDR regions VHP 1-068767E30D, and VL, including CDR regions VL P1068767_E30D, and the amino acid sequences of VH and VL, which 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 P1-068767_E30D;

(jjj) VH, including the CDR regions of VH P1-068767_E30D_E55A, and VL, including the CDR regions of VL P1-068767_E30D_E55A, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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! VH and VL P1-068767_E30D_E55A;

(kkk) VH, including the CDR regions of VH P1-068767_E100fF_D102V, and VL, including the CDR regions of VL P1-068767_E100fF_D102V, and the amino acid sequences of VH and VL, which 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 P1-068767_E100fF_D102V;

(lll) VH, including the CDR regions of VH P1-068767_E55A_E100fF, and VL, including the CDR regions of VL P1-068767_E55A_E100fF, and the amino acid sequences of VH and VL, which 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 P1-068767_E55A_E100fF;

(mmm) VH, including CDR regions VH P1-068767_D52N_E100fF, and VL, including CDR

- 36 046477 VL region 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 P1-068767_D52N_E100fF;

(nnn) VH, including the CDR regions of VH P1-068767_E100fF, and VL, including the CDR regions of VL P1-068767_E100fF, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-068767_E100fF;

(ooo) VH, including the CDR regions of VH P1-068767_E30D_E100fF, and VL, including the CDR regions of VL P1-068767_E30D_E100fF, and the amino acid sequences of VH and VL, which 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 P1-068767_E30D_E100fF;

(ppp) VH, including the CDR regions of VH P1-061029_F100fE_V102D, and VL, including the CDR regions of VL P1-061029_F100fE_V102D, and the amino acid sequences of VH and VL, which 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 P1-061029_F100fE_V102D;

(qqq) VH, including the CDR regions of VH P1-061029_F100fE, and VL, including the CDR regions of VL P1-061029_F100fE, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-061029_F100fE;

(rrr) VH, including the CDR regions of VH P1-061029_V102D, and VL, including the CDR regions of VL P1-061029_V102D, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-061029_V102D;

(sss) VH, including the CDR regions of VH P1-061029_Y32E, and VL, including the CDR regions of VL P1-061029_Y32E, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least 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 P1-061029_Y32E; or (ttt) VH, including the CDR regions of VH P1-061029_Y32E_F100fE, and VL, including the CDR regions of VL P1-061029_Y32E_F100fE, and the amino acid sequences of VH and VL, which are at least 90%, at least 91%, at least by 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to VH and VL P1-061029_Y32E_F100fE;

optionally, wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

In some of the above embodiments, VH and/or VL may differ from each of sequence types (a)-(ttt) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions, such as 1, 2, 3, 4, or 5 conservative substitutions . In some embodiments, the VH may include one or both of the K16R and T84A substitutions.

Anti-hVTSTA antibodies may include:

(a) VH, consisting of the amino acid sequence of VH P1-061029, and VL, consisting of VL P1-061029;

(b) VH consisting of the amino acid sequence of VH P1-061015, and VL consisting of VL P1-061015;

(c) VH, consisting of the amino acid sequence of VH P1-068757, and VL, consisting of VL P1-068757;

(d) VH consisting of the amino acid sequence of VH P1-068759, and VL consisting of VL P1-068759;

(e) VH consisting of the amino acid sequence of VH P1-068761, and VL consisting of VL P1-068761;

(f) VH consisting of the amino acid sequence of VH P1-068763, and VL consisting of VL P1-068763;

(g) VH consisting of the amino acid sequence of VH P1-068765, and VL consisting of VL P1-068765;

(h) VH, consisting of the amino acid sequence of VH P1-068767, and VL, consisting of VL P1-068767;

(i) VH, consisting of the amino acid sequence VH P1-068769, and VL, consisting of VL

- 37 046477

P1-068769;

(j) VH consisting of the amino acid sequence of VH P1-068771, and VL consisting of VL P1-068771;

(k) VH consisting of the amino acid sequence of VH P1-068773, and VL consisting of VL P1-068773;

(l) VH, consisting of the amino acid sequence of VH P1-068775, and VL, consisting of VL P1-068775;

(m) VH, consisting of the amino acid sequence of VH P1-069059, and VL, consisting of VL P1-069059;

(n) VH consisting of the amino acid sequence of VH P1-069061, and VL consisting of VL P1-069061;

(o) VH consisting of the amino acid sequence of VH P1-069063, and VL consisting of VL P1-069063;

(p) VH, consisting of the amino acid sequence of VH P1-069065, and VL, consisting of VL P1-069065;

(q) VH consisting of the amino acid sequence of VH P1-069067, and VL consisting of VL P1-069067;

(r) VH, consisting of the amino acid sequence of VH P1-069069, and VL, consisting of VL P1-069069;

(s) VH, consisting of the amino acid sequence of VH P1-069071, and VL, consisting of VL P1-069071;

(t) VH, consisting of the amino acid sequence of VH P1-069073, and VL, consisting of VL P1-069073;

(u) VH consisting of the amino acid sequence of VH P1-069075, and VL consisting of VL P1-069075;

(v) VH, consisting of the amino acid sequence of VH P1-069077, and VL, consisting of VL P1-069077;

(w) VH, consisting of the amino acid sequence of VH P1-068736, and VL, consisting of VL P1-068736;

(x) VH, consisting of the amino acid sequence of VH P1-06873 8, and VL, consisting of VL P1-068738;

(y) VH, consisting of the amino acid sequence of VH P1-068740, and VL, consisting of VL P1-068740;

(z) VH consisting of the amino acid sequence of VH P1-068742, and VL consisting of VL P1-068742;

(aa) VH consisting of the amino acid sequence of VH P1-068744, and VL consisting of VL P1-068744;

(bb) VH consisting of the amino acid sequence of VH P1-068746, and VL consisting of VL P1-068746;

(cc) VH, consisting of the amino acid sequence of VH P1-068748, and VL, consisting of VL P1-068748;

(dd) VH, consisting of the amino acid sequence of VH P1-068750, and VL, consisting of VL P1-068750;

(ee) VH, consisting of the amino acid sequence of VH P1-068752, and VL, consisting of VL P1-068752;

(ff) VH, consisting of the amino acid sequence of VH P1-068754, and VL, consisting of VL P1-068754;

(gg) VH consisting of the amino acid sequence VH P1-068761_E55A, and VL consisting of the amino acid sequence VL ofP1-068761_E55A;

(hh) VH consisting of the amino acid sequence VH P1-068761_H100G, and VL consisting of the amino acid sequence VL P1-068761_H100G;

(ii) VH consisting of the amino acid sequence VH P1-068761_E56N, and VL consisting of the amino acid sequence VL P1-068761_E56N;

(jj) VH consisting of the amino acid sequence VH P1-O68761_E55A_E56N, and VL consisting of the amino acid sequence VL P1-O68761_E55A_E56N;

(kk) VH consisting of the amino acid sequence VH P1-068761_E30D, and VL consisting of the amino acid sequence VL P1-068761_E30D;

(ll) VH consisting of the amino acid sequence VH P1-O68761_E3OD_E55A, and VL consisting of the amino acid sequence VL P1-O68761_E3OD_E55A;

(mm) VH, consisting of the amino acid sequence VH P1-O68761_E56N_H1OOG, and VL, consisting of the amino acid sequence VL P1-O68761_E56N_H1OOG;

(nn) VH, consisting of the amino acid sequence VH P1-O68761_E3OD_H1OOG, and VL,

- 38 046477 consisting of the amino acid sequence VL P1-068761_E30D_H100G;

(oo) VH. consisting of the amino acid sequence VH P1-O68761_E3OD_E56N, and VL consisting of the amino acid sequence VL P1-O68761_E3OD_E56N;

(pp) VH, consisting of the amino acid sequence VH P1-068761_E100fF, and VL, consisting of the amino acid sequence VL P1-068761_E100fF;

(qq) VH, consisting of the amino acid sequence VH P1-068761_E55A_E10()fF. and VL, consisting of the amino acid sequence VL P1-068761_E55A_E10()fF;

(rr) VH, consisting of the amino acid sequence VH P1-068761_H100G_E100fF, and VL, consisting of the amino acid sequence VL P1-068761_H100G_E100fF;

(ss) VH, consisting of the amino acid sequence VH P1-068761_E30D_E100fF, and VL, consisting of the amino acid sequence VL P1-068761_E30D_E100fF;

(tt)VH. consisting of the amino acid sequence VH P1-068761_E56N_E10()fF. and VL, consisting of the amino acid sequence VL P1-068761_E56N_E100fF;

(uu)VH. consisting of the amino acid sequence VH P1-068761_E32Y. and VL. consisting of the amino acid sequence VL P1-068761_E32Y;

(vv)VH. consisting of the amino acid sequence VH P1-068761_E32Y_E55A. and VL. consisting of the amino acid sequence VL P1-068761_E32Y_E55A;

(ww)VH. consisting of the amino acid sequence VH P1-068761_E32Y_E56N. and VL. consisting of the amino acid sequence VL P1-068761_E32Y_E56N;

(xx) VH. consisting of the amino acid sequence VH P1-068761_E30D_E32Y. and VL. consisting of the amino acid sequence VL P1-068761_E30D_E32Y;

(yay) VH. consisting of the amino acid sequence VH P1-068761_E32Y_H100G. and VL. consisting of the amino acid sequence VL P1-068761_E32Y_H100G;

(zz)VH. consisting of the amino acid sequence VH P1-068761_E32Y_E100fF. and VL. consisting of the amino acid sequence VL P1-068761_E32Y_E100fF;

(aaah) VH. consisting of the amino acid sequence VH P1-068767_D52N_D102V, and VL. consisting of the amino acid sequence VL P1-068767_D52N_D102V;

(bbb) VH. consisting of the amino acid sequence VH P1-068767_D52N, and VL. consisting of the amino acid sequence VL P1-068767_D52N;

(sss) VH. consisting of the amino acid sequence VH P1-068767_D52N_E55A, and VL. consisting of the amino acid sequence VL P1-068767_D52N_E55A;

(ddd)VH. consisting of the amino acid sequence VH P1-068767_E55A_D102V, and VL. consisting of the amino acid sequence VL P1-068767_E55A_D102V;

(eee) VH. consisting of the amino acid sequence VH P1-068767_D102V, and VL. consisting of the amino acid sequence VL P1-068767_D102V;

(iff)VH. consisting of the amino acid sequence VH P1-068767_E55A, and VL. consisting of the amino acid sequence VL P1-068767_E55A;

(ggg)VH. consisting of the amino acid sequence VH P1-068767_E30D_D52N, and VL. consisting of the amino acid sequence VL P1-068767_E30D_D52N;

(hhh) VH. consisting of the amino acid sequence VH P1-068767_E30D_D102V, and VL. consisting of the amino acid sequence VL P1-068767_E30D_D102V;

(iii) VH. consisting of the amino acid sequence VH P1-068767_E30D, and VL. consisting of the amino acid sequence VL P1-068767_E30D;

(jjj) VH. consisting of the amino acid sequence VH P1-068767_E30D_E55A, and VL. consisting of the amino acid sequence VL P1-068767_E30D_E55A;

(kkk) VH. consisting of the amino acid sequence VH P1-068767_E100fF_D102V, and VL. consisting of the amino acid sequence VL P1-068767_E100fF_D102V;

(lll)VH. consisting of the amino acid sequence VH P1-068767_E55A_E100fF, and VL. consisting of the amino acid sequence VL P1-068767_E55A_E100fF;

(mmm) VH. consisting of the amino acid sequence VH P1-068767_D52N_E100fF, and VL. consisting of the amino acid sequence VL P1-068767_D52N_E100fF;

(nnn)VH. consisting of the amino acid sequence VH P1-068767_E100fF, and VL. consisting of the amino acid sequence VL P1-068767_E100fF;

(oooh) VH. consisting of the amino acid sequence VH P1-068767_E30D_E100fF, and VL. consisting of the amino acid sequence VL P1-068767_E30D_E100fF;

(rrr) VH. consisting of the amino acid sequence VH P1-061029_F100fE_V102D. and VL. consisting of the amino acid sequence VL P1-061029_F100fE_V102D;

(qqq) VH. consisting of the amino acid sequence VH P1-061029_F100fE. and VL. consisting of the amino acid sequence VL P1-061029_F100fE;

(rrrr)VH. consisting of the amino acid sequence VH P1-061029_V102D. and VL. consisting of the amino acid sequence VL P1-061029_V102D;

(sss)VH. consisting of the amino acid sequence VH P1-061029_Y32E. and VL. state

- 39 046477 from the amino acid sequence VL P1-061029_Y32E; or (ttt) VH, consisting of the amino acid sequence VH P1-061029_Y32E_F100fE, and VL, consisting of the amino acid sequence VL P1-061029_Y32E_F100fE;

optionally, wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

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

(1) one or more VH CDR1, CDR2 and CDR3;

(2) CDR1, CDR2 and CDR3 VH: (3) VH;

(4) one or more VH CDR1, CDR2 and CDR3 and one or more VL CDR1, CDR2 and CDR3;

(5) CDR1, CDR2 and CDR3 VH and CDR1, CDR2 and CDR3 VL;

(6) VH and VL; or (7) VL and VH, excluding one or both of the K16R and T84A substitutions in VH (in the case of P1-061029 or its daughter clones) from:

P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1068771, P1-068773, P1-068775, P1-069059, P1-06 9061, P1-069063, P1-069065, P1-069067, P1-069069, P1069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-06 8742, P1068744, P1-068766, P1-068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-O68761_H1OOG, P1068761_E56N, P1-068761_E55A_E56N, P1 -O68761_E3OD, P1-068761_E30D_E55A, Р1068761_E56N_H100G, P1-068761_E30D_H100G, P1 -068761_E30D_E56N, P1-068761_E100fF, P1068761_E55A_E100fF, 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_E32Y, P1068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767 _D52N_D102V, P1-068767_D52N , P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E 30D, P1-068767_E30D_E55A, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100 fF, P1-061029_F100fE_V102D, P1 -061029_F100fE, P1-061029_V102D,P1061029_Y32E or P1-061029_Y32E_F100fE; and The anti-VISTA Ab is also an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody or a modified form thereof, as described in the section below. In some embodiments, the constant region has an effector function, and in some embodiments, the constant region is not an effector. In some embodiments, the constant region is an IgG1.3 constant region.

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

(1) one or more VH CDR1, CDR2 and CDR3;

(2) CDR1, CDR2 and CDR3 VH: (3) VH;

(4) one or more VH CDR1, CDR2 and CDR3 and one or more VL CDR1, CDR2 and CDR3;

(5) CDR1, CDR2 and CDR3 VH and CDR1, CDR2 and CDR3 VL;

(6) VH and VL; or (7) VL and VH, excluding one or both of the K16R and T84A substitutions in VH (in the case of P1-061029 and its daughter clones) from:

P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1-068767, P1-068769, P1068771, P1-068773, P1-068775, P1-069059, P1-06 9061, P1-069063, P1-069065, P1-069067, P1-069069, P1069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-06 8742, P1068744, P1-068766, P1-068748, P1-068750, P1-068752 P1-068754, P1-068761_E55A, P1-O68761_H1OOG, P1068761_E56N, P1-068761_E55A_E56N, P1 -O68761_E3OD, P1-068761_E30D_E55A, Р1068761_E56N_H100G, P1-068761_E30D_H100G, P1 -068761_E30D_E56N, P1-068761_E100fF, P1068761_E55A_E100fF, 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_E32Y, P1068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767 _D52N_D102V, P1-068767_D52N , P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E 30D, P1-068767_E30D_E55A, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100 fF, P1-061029_F100fE_V102D, P1 -061029_F100fE, P1-061029_V102D, P1061029_Y32E or P1-061029_Y32E_F100fE, and additionally includes one or more of the following characteristics:

binds specifically to hVISTA, for example, the histidine-rich region of the ECD, or a polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2, at an acidic pH, for example, pH 6.0 or pH 6.5;

does not demonstrate significant binding to hVISTA, such as the histidine-rich ECD region, or the polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2, under physiological conditions

- 40 046477 pH or neutral pH, for example pH 7.4 or pH 7.0;

binds specifically to cynomolgus VISTA, eg the histidine-rich region of the ECD, at acidic pH, eg pH 6.0 or pH 6.5;

does not show significant binding to cynomolgus VISTA, such as the histidine-rich region of the ECD, at physiological pH or neutral pH, such as pH 7.4 or pH 7.0;

demonstrates reduced binding to hVISTA-ECD having one or more of the following amino acid substitutions: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127 compared to hVISTA ECD having SEQ ID NO: 2;

cross-competes for hVISTA binding with P1-061029, P1-068761, P1-068767 and/or P1061015;

inhibits the binding of hVISTA to human T cells expressing VISTA (eg, naïve or activated T cells) at an acidic pH, such as pH 6.0 or pH 6.5;

inhibits the binding of hVISTA to PSGL-1 at an acidic pH, e.g., pH 6.0 or pH 6.5 (e.g., inhibits the interaction between H153 and H154 of hVISTA having SEQ ID NO: 1, and PSGL-1 tyrosines Y46 and Y48), wherein PSGL-1 has or does not have sialyl Lewis X, and wherein the tyrosines are preferably sulfothyrosines;

a mean retention time (MRT) of at least 100, 200, 300, 350, 400, 450, 500, 600, or 700 hours (eg, at least 350 hours) in cynomolgus monkeys, measured, for example, as described in the examples;

stimulates T cell activation, for example, by increasing T cell proliferation; increasing IFN-γ production from T cells; and/or stimulates T cell receptor-mediated NF-kB signaling;

inhibits VISTA-mediated cell:cell adhesion;

binds specifically to hVISTA in human tumor cell samples or inflamed human tissue samples that express VISTA;

contacts hVISTA through one or more (e.g., at least 1-3, 1-5, 1-10, 5-10, 5-15, or all) energetically important contact residues Y37, T39, R54, F62, H66, V117 , I119 or S124, as determined, for example, by yeast surface display and NGS analysis described in Example 15; and where the numbering corresponds to the numbering of the mature hVISTA;

contacts

Region 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) hVISTA having SEQ ID NO: 1, and optionally where binding is strongest in Region 2 as determined by MS-HDX as described in Example 21;

binds to the histidine-rich β-sheet extension of hVISTA, as determined, for example, by crystallography described, for example, in the examples;

contacts H121, H122 and/or H123 of mature hVISTA (distance of 4.0 angstroms (A) or less), for example, through hydrogen bonds, as determined, for example, by crystallography described, for example, in the examples;

contacts hVISTA through at least one or more glutamic acid, aspartic acid or histidine residues located in CDR1, CDR2 or CDR3 of VH; and any additional characteristic presented in the claims and/or examples.

In some embodiments, the anti-hVISTA Ab comprises a heavy chain (HC) comprising the heavy chain amino acid sequence of any of the anti-hVISTA Abs provided herein. In some embodiments, the anti-hVISTA Ab comprises a heavy chain comprising the heavy chain amino acid sequence of P1-061029 or P1-061015 or daughter clones thereof, as shown below in the table of sequences comprising the heavy chain constant region of IgG1.3, such as P1-061029 .IgG1.3 (SEQ ID NO: 69), P1-068757.IgG1.3, P1068759.IgG1.3,

068769.IgG1.3,

069061.IgG1.3,

069071.IgG1.3,

068736.IgG1.3,

068766.IgG1.3,

P1-068761.IgG1.3,

P1-068771.IgG1.3,

P1-069063.IgG1.3,

P1-069073.IgG1.3,

P1-068738.IgG1.3,

P1-068748.IgG1.3,

P1-068763.IgG1.3

P1-068773.IgG1.3

P1-069065.IgG1.3

P1-069075.IgG1.3

P1-068740.IgG1.3

P1-068750.IgG1.3

P1-068765.IgG1.3,

P1-068775.IgG1.3,

P1-069067.IgG1.3,

P1-069077.IgG1.3,

P1-068742.IgG1.3,

P1-068752.IgG1.3,

P1-068767.IgG1.3, P1P1-069059.IgG1.3, P1P1-069069.IgG1.3, P1P1-061015.IgG1.3, P1P1-068744.IgG1.3, P1P1-068754.IgG1.3, P1

068761_E55A.IgG1.3, P1-068761_H100G.IgG1.3, P1-068761_E56N.IgG1.3, P1-068761_E55A_E56N.IgG1.3,

P1-068761_E30D.IgG1.3, P1-068761_E30D_E55A.IgG1.3, P1-068761_E56N_H100G.IgG1.3, P1068761_E30D_H100G.IgG1.3, P1-068761_E30D_E56N. IgG1.3, P1-068761_E100fF.IgG1.3, P1068761_E55A_E100fF.IgG1. 3, P1-068761_H100G_E100fF.IgG1.3, P1-068761_E30D_E100fF.IgG1.3, P1068761_E56N_E100fF.IgG1.3, P1-068761_E32Y.IgG1.3, P1-068761_E3 2Y_E55A.IgG1.3,P1068761_E32Y_E56N.IgG1.3, P1-068761_E30D_E32Y. Igg1.3, p1-068761_e32y_h100g.igg1.3, p1068761_e32y_e100FF.igg1.3, p1-068767_d52n_d102v.igg1.3, p1-068767_d52n.iggg1.3, p1- 41 0464777

068767_D52N_E55A.IgG1.3, P1-068767_E55A_D102V.IgG1.3, P1-068767_D102V.IgG1.3, P1068767_E55A.IgG1.3, P1-068767_E30D_D52N.IgG1.3, P1-068767_E30D_D102V.IgG1.3,P1068767_E30D.IgG1.3, P1-068767_E30D_E55A.IgG1.3, P1-068767_E100fF_D102V.IgG1.3, P1068767_E55A_E100fF.IgG1.3, P1-068767_D52N_E100fF.IgG1.3, P1-068767_ E100fF.IgG1.3, P1068767_E30D_E100fF.IgG1.3, P1-061029_F100fE_V102D.IgG1. 3, P1-061029_F100fE.IgG1.3, P1061029_V102D.IgG1.3, P1-061029_Y32E.IgG1.3 or P1-061029_Y32E_F100fE.IgG1.3, optional, where

VH include one or both of the K16R and T84A substitutions.

In some embodiments, the anti-hVISTA Ab comprises a heavy chain including the heavy chain amino acid sequence of any of the anti-hVISTA Abs provided herein, which includes the heavy chain constant region of IgG1.3, and the light chain amino acid sequence of any of the anti-hVISTA Abs provided herein. this document. In some embodiments, the anti-hVISTA Ab comprises a heavy chain comprising the VH amino acid sequence of P1-061029 or P1-061015 or daughter clones thereof comprising the IgG1.3 HC constant region, such as P1-061029.IgG1.3 (SEQ ID NO : 69), P1-068757.^1.3, P1068759.IgG1.3,

068769.IgG1.3,

069061.IgG1.3,

069071.IgG1.3,

068736.IgG1.3,

068766.IgG1.3,

P1-068761.IgG1.3,

P1-068771.IgG1.3,

P1-069063.IgG1.3,

P1-069073.IgG1.3,

P1-068738.IgG1.3,

P1-068748.IgG1.3,

P1-068763.IgG1.3

P1-068773.IgG1.3

P1-069065.IgG1.3

P1-069075.IgG1.3

P1-068740.IgG1.3

P1-068750.IgG1.3

P1-068765.IgG1.3,

P1-068775.IgG1.3,

P1-069067.IgG1.3,

P1-069077.IgG1.3,

P1-068742.IgG1.3,

P1-068752.IgG1.3,

P1-068767.IgG1.3,P1P1-069059.IgG1.3,P1P1-069069.IgG1.3,P1P1-061015.IgG1.3,P1P1-068744.IgG1.3,P1P1-068754.IgG1.3,P1

068761_E55A.IgG1.3, P1-068761_H100G.IgG1.3, P1-068761_E56N.IgG1.3, P1-068761_E55A_E56N.IgG1.3,

P1-068761_E30D.IgG1.3, P1-068761_E30D_E55A.IgG1.3, P1-068761_E56N_H100G.IgG1.3, P1068761_E30D_H100G.IgG1.3, P1-068761_E30D_E56N. IgG1.3, P1-068761_E100fF.IgG1.3, P1068761_E55A_E100fF.IgG1. 3, P1-068761_H100G_E100fF.IgG1.3, P1-068761_E30D_E100fF.IgG1.3, P1068761_E56N_E100fF.IgG1.3, P1-068761_E32Y.IgG1.3, P1-068761_E3 2Y_E55A.IgG1.3,P1068761_E32Y_E56N.IgG1.3, P1-068761_E30D_E32Y. IgG1.3, P1-068761_E32Y_H100G.IgG1.3, P1068761_E32Y_E100fF.IgG1.3, P1-068767_D52N_D102V.IgG1.3, P1-068767_D52N.IgG1.3, P1068767_D52N _E55A.IgG1.3, P1-068767_E55A_D102V.IgG1.3, P1- 068767_D102V.IgG1.3,P1068767_E55A.IgG1.3, P1-068767_E30D_D52N.IgG1.3, P1-068767_E30D_D102V.IgG1.3,P1068767_E30D.IgG1.3, P1-068 767_E30D_E55A.IgG1.3, P1-068767_E100fF_D102V.IgG1.3, P1068767_E55A_E100fF.IgG1.3, P1-068767_D52N_E100fF.IgG1.3, P1-068767_E100fF.IgG1.3, P1068767_E30D_E100fF.IgG1.3, P1-061029_F100f E_V102D.IgG1.3, P1-061029_F100fE.IgG1.3, P1061029_V102D.IgG1.3, P1-061029_Y32E.IgG1.3 or P1-061029_Y32E_F100fE.IgG1.3, optional, where

VH includes one or both of the K16R and T84A substitutions; and a light chain comprising the amino acid sequence of the light chain P1-061029 or P1-061015.

Anti-hVTSTA antibodies may include:

(a) a heavy chain comprising the amino acid sequence of the heavy chain P1061029.IgG1.3 (SEQ ID NO: 69), and a light chain comprising the amino acid sequence of the light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain comprising the amino acid sequence of the heavy chain P1061015.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1061015;

(c) a heavy chain comprising the amino acid sequence of the heavy chain P1068757.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068757;

(d) a heavy chain comprising the amino acid sequence of the heavy chain P1068759.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068759;

(e) a heavy chain comprising the amino acid sequence of the heavy chain P1068761.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068761;

(f) a heavy chain comprising the amino acid sequence of the heavy chain P1068763.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068763;

(g) a heavy chain comprising the amino acid sequence of the heavy chain P1068765.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068765;

(h) a heavy chain comprising the amino acid sequence of the heavy chain P1068767.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068767;

(i) a heavy chain comprising the amino acid sequence of the heavy chain P1068769.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1-

- 42 046477

068769;

(j) a heavy chain including the amino acid sequence of the heavy chain P1068771.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068771;

(k) a heavy chain comprising the amino acid sequence of the heavy chain P1068773.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068773;

(l) a heavy chain comprising the amino acid sequence of the heavy chain P1068775.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068775;

(m) a heavy chain comprising the amino acid sequence of the heavy chain P1069059.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069059;

(n) a heavy chain comprising the amino acid sequence of the heavy chain P1069061.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069061;

(o) a heavy chain comprising the amino acid sequence of the heavy chain P1069063.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069063;

(p) a heavy chain comprising the amino acid sequence of the heavy chain P1069065.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069065;

(q) a heavy chain comprising the amino acid sequence of the heavy chain P1069067.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069067;

(r) a heavy chain comprising the amino acid sequence of the heavy chain P1069069.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069069;

(s) a heavy chain comprising the amino acid sequence of the heavy chain P1069071.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069071;

(t) a heavy chain comprising the amino acid sequence of the heavy chain P1069073.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069073;

(u) a heavy chain comprising the amino acid sequence of the heavy chain P1069075.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069075;

(v) a heavy chain comprising the amino acid sequence of the heavy chain P1069077.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1069077;

(w) a heavy chain comprising the amino acid sequence of the heavy chain P1068736.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068736;

(x) a heavy chain comprising the amino acid sequence of the heavy chain P1068738.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068738;

(y) a heavy chain comprising the amino acid sequence of the heavy chain P1068740.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068740;

(z) a heavy chain comprising the amino acid sequence of the heavy chain P1068742.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068742;

(aa) a heavy chain comprising the amino acid sequence of the heavy chain P1068744.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068744;

(bb) a heavy chain including the amino acid sequence of the heavy chain P1068746.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068746;

(cc) a heavy chain comprising the amino acid sequence of the heavy chain P1068748.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068748;

(dd) a heavy chain comprising the amino acid sequence of the P1 heavy chain

- 43 046477

068750.IgG1.3, and a light chain comprising the amino acid sequence of the light chain Pl068750;

(ee) a heavy chain comprising the amino acid sequence of the heavy chain P1068752.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068752;

(ff) a heavy chain including the amino acid sequence of the heavy chain P1068754.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068754;

(gg) a heavy chain including the amino acid sequence of the heavy chain P1068761_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068761_E55A;

(hh) a heavy chain including the amino acid sequence of the heavy chain P1068761_H100G.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_H100G;

(ii) a heavy chain comprising the amino acid sequence of the heavy chain P1068761_E56N.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068761_E56N;

(jj) a heavy chain including the amino acid sequence of the heavy chain P1068761_E55A_E56N.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E55A_E56N;

(kk) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068761_E30D;

(ll) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E30D_E55A;

(mm) a heavy chain including the amino acid sequence of the heavy chain P1068761_E56N_H100G.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D_H100G.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E30D_H100G;

(oo) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D_E56N.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E30D_E56N;

(pp) a heavy chain including the amino acid sequence of the heavy chain P1068761_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-O68761_E1OOSH;

(qq) a heavy chain including the amino acid sequence of the heavy chain P1068761_E55A_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E55A_E100fF;

(rr) a heavy chain including the amino acid sequence of the heavy chain P1068761_H100G_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain including the amino acid sequence of the heavy chain P1068761_E56N_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_Ε56Ν_Ε10ΟNo;

(uu) a heavy chain including the amino acid sequence of the heavy chain P1068761_E32Y.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068761_E32Y;

(vv) a heavy chain including the amino acid sequence of the heavy chain P1068761_E32Y_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E32Y_E55A;

(ww) a heavy chain including the amino acid sequence of the heavy chain P1068761_E32Y_E56N.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-O68761_E32Y_E56N;

(xx) a heavy chain including the amino acid sequence of the heavy chain P1068761_E30D_E32Y.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761E30DE32Y;

- 44 046477 (yy) a heavy chain, including the amino acid sequence of the heavy chain P1068761_E32Y_H100G.IgG1.3, and a light chain, including the amino acid sequence of the light chain P1-O68761_E32Y_H1OOG;

(zz) a heavy chain including the amino acid sequence of the heavy chain P1068761_E32Y_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068761_E32Y_E100fF;

(aaa) a heavy chain including the amino acid sequence of the heavy chain P1068767_D52N_D102V.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_D52N_D102V;

(bbb) a heavy chain including the amino acid sequence of the heavy chain P1068767_D52N.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_D52N;

(cc) a heavy chain including the amino acid sequence of the heavy chain P1068767_D52N_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain including the amino acid sequence of the heavy chain P1068767_E55A_D102V.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E55A_D102V;

(eeee) a heavy chain including the amino acid sequence of the heavy chain P1068767_D102V.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_D102V;

(fff) a heavy chain including the amino acid sequence of the heavy chain P1068767_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1068767_E55A;

(ggg) a heavy chain including the amino acid sequence of the heavy chain P1068767_E30D_D52N.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E30D_D52N;

(hhh) a heavy chain including the amino acid sequence of the heavy chain P1068767_E30D_D102V.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E30D_D102V;

(iii) a heavy chain comprising the amino acid sequence of the heavy chain P1068767_E30D.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1068767_E30D;

(jjj) a heavy chain including the amino acid sequence of the heavy chain P1068767_E30D_E55A.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E30D_E55A;

(kkk) a heavy chain including the amino acid sequence of the heavy chain P1068767_E100fF_D102V.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E100fF_D102V;

(lll) a heavy chain including the amino acid sequence of the heavy chain P1068767_E55A_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E55A_E100fF;

(mmm) a heavy chain including the amino acid sequence of the heavy chain P1068767_D52N_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain including the amino acid sequence of the heavy chain P1068767_E100fF.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-068767_E100fF; or (ooo) a heavy chain comprising the heavy chain amino acid sequence P1068767_E30D_E100fF.IgG1.3, and a light chain comprising the light chain amino acid sequence P1-068767_E30D_E100fF.

(ppp) a heavy chain comprising the amino acid sequence of the heavy chain P1061029_F100fE_V102D and a light chain comprising the amino acid sequence of the light chain P1061029_F100fE_V102D;

(qqq) a heavy chain including the amino acid sequence of the heavy chain P1061029_F100fE.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-061029_F100fE;

(rrr) a heavy chain including the amino acid sequence of the heavy chain P1061029_V102D.IgG1.3, and a light chain including the amino acid sequence of the light chain P1-061029_V102D;

(sss) a heavy chain comprising the amino acid sequence of the heavy chain P1061029_Y32E.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1-

- 45 046477

061029_Y32E; or (ttt) a heavy chain comprising the amino acid sequence of the heavy chain P1061029_Y32E_F100fE.IgG1.3, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E_F100fE;

optionally, wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

Anti-hVTSTA antibodies may include:

(a) a heavy chain (HC) including the HC CDR regions of P1-061029, and a light chain (LC) including the LC CDR regions of P1-061029, and the amino acid sequences of the HC and LC, which 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 at least 98% or at least 99% identical to the HC and LC of P1-061029.IgG1.3, respectively;

(b) HC, including the CDR regions of HC P1-061015, and LC, including the CDR regions of LC P1-061015, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-061015.IgG1.3, respectively;

(c) HC, including the CDR regions of HC P1-068757, and LC, including the CDR regions of LC P1-068757, and the amino acid sequences of the HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068757.IgG1.3, respectively;

(d) HC, including the CDR regions of HC P1-068759, and LC, including the CDR regions of LC P1-068759, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068759.IgG1.3, respectively;

(e) HC, including the CDR regions of HC P1-068761, and LC, including the CDR regions of LC P1-068761, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068761.IgG1.3, respectively;

(f) HC, including the CDR regions of HC P1-068763, and LC, including the CDR regions of LC P1-068763, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068763.IgG1.3, respectively;

(g) HC, including the CDR regions of HC P1-068765, and LC, including the CDR regions of LC P1-068765, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068765.IgG1.3, respectively;

(h) HC, including the CDR regions of HC P1-068767, and LC, including the CDR regions of LC P1-068767, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068767.IgG1.3, respectively;

(i) HC, including the CDR regions of HC P1-068769, and LC, including the CDR regions of LC P1-068769, and the amino acid sequences of the HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068769.IgG1.3, respectively;

(j) HC, including the CDR regions of HC P1-068771, and LC, including the CDR regions of LC P1-068771, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068771.IgG1.3, respectively;

(k) HC, including the CDR regions of HC P1-068773, and LC, including the CDR regions of LC P1-068773, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068773.IgG1.3, respectively;

(l) HC, including the CDR regions of HC P1-068775, and LC, including the CDR regions of LC P1-068775,

- 46 046477 and amino acid sequences HC and LC, which 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 NS and LC P1-068775.IgG1.3, respectively;

(m) HC, including the CDR regions of HC P1-069059, and LC, including the CDR regions of LC P1069059, and the amino acid sequences of HC and LC, which 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 NS and LC P1-069059.IgG1.3, respectively;

(n) HC, including the CDR regions of HC P1-069061, and LC, including the CDR regions of LC P1-069061, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069061.IgG1.3, respectively;

(o) HC, including the CDR regions of HC P1-069063, and LC, including the CDR regions of LC P1-069063, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069063.IgG1.3, respectively;

(p) HC, including the CDR regions of HC P1-069065, and LC, including the CDR regions of LC P1-069065, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069065.IgG1.3, respectively;

(q) HC, including the CDR regions of HC P1-069067, and LC, including the CDR regions of LC P1-069067, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069067.IgG1.3, respectively;

(r) HC, including the CDR regions of HC P1-069069, and LC, including the CDR regions of LC P1-069069, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069069.IgG1.3, respectively;

(s) HC, including the CDR regions of HC P1-069071, and LC, including the CDR regions of LC P1-069071, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069071.IgG1.3, respectively;

(t) HC, including the CDR regions of HC P1-069073, and LC, including the CDR regions of LC P1-069073, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069073.IgG1.3, respectively;

(u) HC, including the CDR regions of HC P1-069075, and LC, including the CDR regions of LC P1-069075, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069075.IgG1.3, respectively;

(v) HC, including the CDR regions of HC P1-069077, and LC, including the CDR regions of LC P1-069077, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-069077.IgG1.3, respectively;

(w) HC, including the CDR regions of HC P1-068736, and LC, including the CDR regions of LC P1068736, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068736.IgG1.3, respectively;

(x) HC, including the CDR regions of HC P1-068738, and LC, including the CDR regions of LC P1-06873 8, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, according to at least 92%, at least 93%, at least 94%, at least

- 47 046477 are 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to NS and LC P1-068738.IgG1.3, respectively;

(y) HC, including the CDR regions of HC P1-068740, and LC, including the CDR regions of LC P1-068740, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068740.IgG1.3, respectively;

(z) HC, including the CDR regions of HC P1-068742, and LC, including the CDR regions of LC P1-068742, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068742.IgG1.3, respectively;

(aa) HC, including the CDR regions of HC P1-068744, and LC, including the CDR regions of LC P1068744, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068744.IgG1.3, respectively;

(bb) HC, including the CDR regions of HC P1-068746, and LC, including the CDR regions of LC P1068746, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068746.IgG1.3, respectively;

(cc) HC, including the CDR regions of HC P1-068748, and LC, including the CDR regions of LC P1068748, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068748.IgG1.3, respectively;

(dd) HC, including the CDR regions of HC P1-068750, and LC, including the CDR regions of LC P1068750, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068750.IgG1.3, respectively;

(ee) HC, including the CDR regions of HC P1-068752, and LC, including the CDR regions of LC P1068752, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068752.IgG1.3, respectively;

(ff) HC, including the CDR regions of HC P1-068754, and LC, including the CDR regions of LC P1-068754, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, at least 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 NS and LC P1-068754.IgG1.3, respectively;

(gg) HC, including the CDR regions of HC P1-068761_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068761_E55A, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068761_E55A.IgG1.3, respectively;

(hh) HC, including the CDR regions of HC P1-068761_H100G.IgG1.3, and LC, including the CDR regions of LC P1-068761_H100G, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_H100G.IgG1.3, respectively;

(ii) HC, including the CDR regions of HC P1-068761_E56N.IgG1.3, and LC, including the CDR regions of LC P1-068761_E56N, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068761_E56N.IgG1.3, respectively;

(jj) HC, including the CDR regions of HC P1-068761_E55A_E56N.IgG1.3, and LC, including the CDR regions of LC P1-068761_E55A_E56N, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E55A_E56N.IgG1.3,

- 48 046477 respectively;

(kk) HC, including the CDR regions of HC P1-068761_E30D.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068761_E30D.IgG1.3, respectively;

(ll) HC, including the CDR regions of HC P1-068761_E30D_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D_E55A, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E30D_E55A.IgG1.3, respectively;

(mm) HC, including the CDR regions of HC P1-068761_E56N_H100G.IgG1.3, and LC, including the CDR regions of LC P1-068761_E56N_H100G, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068761_E56N_H100G.IgG1.3, respectively;

(nn) HC, including the CDR regions of HC P1-068761_E30D_H100G.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D_H100G, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E30D_H100G.IgG1.3, respectively;

(oo) HC, including the CDR regions of HC P1-068761_E30D_E56N.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D_E56N, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E30D_E56N.IgG1.3, respectively;

(pp) HC, including the CDR regions of HC P1-068761_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E100fF.IgG1.3, respectively;

(qq) HC, including the CDR regions of HC P1-068761_E55A_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_E55A_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E55A_E100fF.IgG1.3, respectively;

(rr) HC, including the CDR regions of HC P1-068761_H100G_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_H100G_E100fF, and the amino acid sequences of HC and LC, which 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 NS and LC P1068761_H100G_E100fF.IgG1.3, respectively;

(ss) HC, including the CDR regions of HC P1-068761_E30D_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E30D_E100fF.IgG1.3, respectively;

(tt) HC, including the CDR regions of HC P1-068761_E56N_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_E56N_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E56N_E100fF.IgG1.3, respectively;

(uu) HC, including the CDR regions of HC P1-068761_E32Y.IgG1.3, and LC, including the CDR regions of LC P1-068761_E32Y, and the amino acid sequences of HC and LC, which are at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%,

- 49 046477 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to NS and LC P1-068761_E32Y.IgG1.3, respectively ;

(vv) HC, including the CDR regions of HC P1-068761_E32Y_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068761_E32Y_E55A, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E32Y_E55A.IgG1.3, respectively;

(ww) HC, including the CDR regions of HC P1-068761_E32Y_E56N.IgG1.3, and LC, including the CDR regions of LC P1-068761_E32Y_E56N, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E32Y_E56N.IgG1.3, respectively;

(xx) HC, including the CDR regions of HC P1-068761_E30D_E32Y.IgG1.3, and LC, including the CDR regions of LC P1-068761_E30D_E32Y, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E30D_E32Y.IgG1.3, respectively;

(yy) HC, including the CDR regions of HC P1-068761_E32Y_H100G.IgG1.3, and LC, including the CDR regions of LC P1-O68761_E32Y_H1OOG, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E32Y_H100G.IgG1.3, respectively;

(zz) HC, including the CDR regions of HC P1-068761_E32Y_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068761_E32Y_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068761_E32Y_E100fF.IgG1.3, respectively;

(aaa) HC, including the CDR regions of HC P1-068767_D52N_D102V.IgG1.3, and LC, including the CDR regions of LC P1-068767_D52N_D102V, and the amino acid sequences of HC and LC, which 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 NS and LC P1068767_D52N_D102V.IgG1.3, respectively;

(bbb) HC, including the CDR regions of HC P1-068767_D52N.IgG1.3, and LC, including the CDR regions of LC P1-068767_D52N, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_D52N.IgG1.3, respectively;

(cc) HC, including the CDR regions of HC P1-068767_D52N_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068767_D52N_E55A, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_D52N_E55A.IgG1.3, respectively;

(ddd) HC, including the CDR regions of HC P1-068767_E55A_D102V.IgG1.3, and LC, including the CDR regions of LC P1-068767_E55A_D102V, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068767_E55A_D102V.IgG1.3, respectively;

(eeee) HC, including the CDR regions of HC P1-068767_D102V.IgG1.3, and LC, including the CDR regions of LC P1-068767_D102V, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_D102V.IgG1.3, respectively;

(fff) HC, including the CDR regions of HC P1-068767_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068767_E55A, and the amino acid sequences of HC and LC, which are at least 90%,

- 50 046477 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 NS and LC P1-068767_E55A.IgG1.3, respectively;

(ggg) HC, including the CDR regions of HC P1-068767_E30D_D52N.IgG1.3, and LC, including the CDR regions of LC P1-068767_E30D_D52N, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_E30D_D52N.IgG1.3, respectively;

(hhh) HC, including the CDR regions of HC P1-068767_E30D_D102V.IgG1.3, and LC, including the CDR regions of LC P1-068767_E30D_D102V, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068767_E30D_D102V.IgG1.3, respectively;

(iii) HC, including the CDR regions of HC P1-068767_E30D.IgG1.3, and LC, including the CDR regions of LC P1-068767_E30D, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068767_E30D.IgG1.3, respectively;

(jjj) HC, including the CDR regions of HC P1-068767_E30D_E55A.IgG1.3, and LC, including the CDR regions of LC P1-068767_E30D_E55A, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_E30D_E55A.IgG1.3, respectively;

(kkk) HC, including the CDR regions of HC P1-068767_E100fF_D102V.IgG1.3, and LC, including the CDR regions of LC P1-068767_E100fF_D102V, and the amino acid sequences of HC and LC, which 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 NS and LC P1068767_E 100fF_D 102V.IgG 1.3, respectively;

(lll) HC, including the CDR regions of HC P1-068767_E55A_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068767_E55A_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_E55A_E100fF.IgG1.3, respectively;

(mmm) HC, including the CDR regions of HC P1-068767_D52N_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068767_D52N_E100fF, and the amino acid sequences of HC and LC, which 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 NS and LC P1068767_D52N_E100fF.IgG1.3, respectively;

(nnn) HC, including the CDR regions of HC P1-068767_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068767_E100fF, and the amino acid sequences of HC and LC, which are at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by at least 99% identical to NS and LC P1-068767_E100fF.IgG1.3, respectively;

(ooo) HC, including the CDR regions of HC P1-068767_E30D_E100fF.IgG1.3, and LC, including the CDR regions of LC P1-068767_E30D_E100fF, and the amino acid sequences of HC and LC, which 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 NS and LC P1-068767_E30D_E100fF.IgG1.3, respectively;

(ppp) HC, including the CDR regions of HC P1-061029_F100fE_V102D.IgG1.3, and LC, including the CDR regions of LC P1-061029_F100fE_V102D, and the amino acid sequences of HC and LC, which 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 NS and LC P1061029_F100fE_V102D.IgG1.3, respectively;

(qqq) NS, including the CDR regions of NS P1-061029_F100fE.IgG1.3, and LC, including the CDR

- 51 046477 LC region P1-061029_F100fE, and amino acid sequences HC and LC, which 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 NS and LC P1-061029_F100fE.IgG1.3, respectively;

(rrr) HC, including the CDR regions of HC P1-061029_V102D.IgG1.3, and LC, including the CDR regions of LC P1-061029_V102D, and the amino acid sequences of HC and LC, which 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 NS and LC P1-061029_V102D.IgG1.3, respectively;

(sss) HC, including the CDR regions of HC P1-061029_Y32E.IgG1.3, and LC, including the CDR regions of LC P1-061029_Y32E, and the amino acid sequences of HC and LC, which 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 NS and LC P1-061029_Y32E.IgG1.3, respectively; or (ttt) HC, including the CDR regions of HC P1-061029_Y32E_F100fE.IgG1.3, and LC, including the CDR regions of LC P1-061029_Y32E_F100fE, and the amino acid sequences of HC and LC, which 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 NS and LC P1-061029_Y32E_F100fE.IgG1.3, respectively.

In some of the above embodiments, the HC and/or LC may differ from each of sequence types (a)-(ttt) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions, such as 1, 2, 3, 4, or 5 conservative substitutions . In some embodiments, for example, the HC of P1-061029 or one of its daughter clones may include one or both of the K16R and T84A substitutions in the VH region of the HC (P1-061015 and its daughter clones already have R and A at these positions, respectively) .

In some embodiments, the anti-hVISTA Ab may include:

(a) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1061029;

(b) a heavy chain consisting of the amino acid sequence of the heavy chain P1061015.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1061015;

(c) a heavy chain consisting of the amino acid sequence of the heavy chain P1068757.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068757;

(d) a heavy chain consisting of the amino acid sequence of the heavy chain P1068759.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068759;

(e) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068761;

(f) a heavy chain consisting of the amino acid sequence of the heavy chain P1068763.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068763;

(g) a heavy chain consisting of the amino acid sequence of the heavy chain P1068765.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068765;

(h) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068767;

(i) a heavy chain consisting of the amino acid sequence of the heavy chain P1068769.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068769;

(j) a heavy chain consisting of the amino acid sequence of the heavy chain P1068771.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068771;

(k) a heavy chain consisting of the amino acid sequence of the heavy chain P1068773.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068773;

(l) a heavy chain consisting of the amino acid sequence of the heavy chain P1068775.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068775;

- 52 046477 (m) a heavy chain consisting of the amino acid sequence of the heavy chain P1069059.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069059;

(n) a heavy chain consisting of the amino acid sequence of the heavy chain P1069061.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069061;

(o) a heavy chain consisting of the amino acid sequence of the heavy chain P1069063.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069063;

(p) a heavy chain consisting of the amino acid sequence of the heavy chain P1069065.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069065;

(q) a heavy chain consisting of the amino acid sequence of the heavy chain P1069067.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069067;

(r) a heavy chain consisting of the amino acid sequence of the heavy chain P1069069.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069069;

(s) a heavy chain consisting of the amino acid sequence of the heavy chain P1069071.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069071;

(t) a heavy chain consisting of the amino acid sequence of the heavy chain P1069073.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069073;

(u) a heavy chain consisting of the amino acid sequence of the heavy chain P1069075.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069075;

(v) a heavy chain consisting of the amino acid sequence of the heavy chain P1069077.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1069077;

(w) a heavy chain consisting of the amino acid sequence of the heavy chain P1-068736, and a light chain consisting of the amino acid sequence of the light chain P1-068736;

(x) a heavy chain consisting of the amino acid sequence of the heavy chain P1068738.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068738;

(y) a heavy chain consisting of the amino acid sequence of the heavy chain P1068740.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068740;

(z) a heavy chain consisting of the amino acid sequence of the heavy chain P1068742.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068742;

(aa) a heavy chain consisting of the amino acid sequence of the heavy chain P1068744.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068744;

(bb) a heavy chain consisting of the amino acid sequence of the heavy chain P1068746.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068746;

(cc) a heavy chain consisting of the amino acid sequence of the heavy chain P1068748.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068748;

(dd) a heavy chain consisting of the amino acid sequence of the heavy chain P1068750.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068750;

(ee) a heavy chain consisting of the amino acid sequence of the heavy chain P1068752.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068752;

(ff) a heavy chain consisting of the amino acid sequence of the heavy chain P1-068754, and a light chain consisting of the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068761_E55A;

(hh) a heavy chain consisting of the amino acid sequence of the P1 heavy chain

- 53 046477

068761_H100G.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E56N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068761_E56N;

(jj) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E55A_E56N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A_E56N;

(kk) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068761E30D;

(ll) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E55A;

(mm) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E56N_H100G.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D_H100G.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_H100G;

(oo) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D_E56N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E56N;

(pp) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E1OO£F;

(qq) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E55A_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A_E100fF;

(rr) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_H100G_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E56N_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E56N_E1OO!F;

(uu) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E32Y.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068761_E32Y;

(vv) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E32Y_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y_E55A;

(ww) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E32Y_E56N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y_E56N;

(xx) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E30D_E32Y.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E32Y;

(yy) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E32Y_H100G.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y_H1OOG;

(zz) a heavy chain consisting of the amino acid sequence of the heavy chain P1068761_E32Y_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y_E1OO!F;

(aaa) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_D52N_D102V.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_D102V;

(bbb) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_D52N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N;

- 54 046477 (cc) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_D52N_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E55A_D102V.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E55A_D102V;

(eeee) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_D102V.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D102V;

(fff) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068767_E55A;

(ggg) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E30D_D52N.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D_D52N;

(hhh) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E30D_D102V.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D_D102V;

(iii) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E30D.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1068767E30D;

(jjj) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E30D_E55A.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D_E55A;

(kkk) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E100fF_D102V.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E100fF_D102V;

(lll) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E55A_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E55A_E100fF;

(mmm) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_D52N_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E100fF;

(ooo) a heavy chain consisting of the amino acid sequence of the heavy chain P1068767_E30D_E100fF.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D_E100fF;

(ppp) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029_F100fE_V102D.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-061029_F100fE_V102D;

(qqq) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029_F100fE.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-O61O29_F1OOIE;

(rrr) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029_V102D.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-061029_V102D;

(sss) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029_Y32E.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1061029_Y32E; or (ttt) a heavy chain consisting of the amino acid sequence of the heavy chain P1061029_Y32E_F100fE.IgG1.3, and a light chain consisting of the amino acid sequence of the light chain P1-061029_Y32E_F100fE, where optionally the VH in any of (a)-(ttt) includes one or both of the K16R and T84A replacements.

In some embodiments, anti-VISTA mAbs are provided including:

a heavy chain consisting of the heavy chain amino acid sequences (a)-(ttt) above followed by a Lys residue; and a light chain consisting of the light chain amino acid sequences (i-i)-(ttt) above;

wherein the amino acid sequences of the heavy chain and light chain are selected from the same types of an

- 55 046477 title from (a)-(ttt) above.

In some embodiments, the anti-hVISTA Ab may comprise a heavy chain amino acid sequence including the amino acid sequence of the VH types of antibodies herein, but rather than the IgG1.3 heavy chain constant region presented in the HC sequences in the sequence table herein (and see SEQ ID NO: 163), the antibody may include another heavy chain constant region sequence, such as a wild-type human IgG1 constant region, such as human IgG1 allotype f (IgG1f) (SEQ ID NO: 182), or a modified human IgG1 constant region, such as IgG1.If (SEQ ID NO: 183), or a modified human IgG1 constant region such as IgG1.P238K (SEQ ID NO: 184). Thus, embodiments of the present invention include anti-VISTA Abs comprising:

(a) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain comprising: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-061015;

(c) a heavy chain comprising: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068757;

(d) a heavy chain comprising: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068759;

(e) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761;

(f) a heavy chain comprising: (i) the amino acid sequence of VH P1-068763 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068763;

(g) a heavy chain comprising: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068765;

(h) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767;

(i) a heavy chain comprising: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068769;

(j) a heavy chain comprising: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068771;

(k) a heavy chain comprising: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068773;

(l) a heavy chain comprising: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068775;

(m) a heavy chain comprising: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069059;

(n) a heavy chain comprising: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069061;

(o) a heavy chain comprising: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069063;

(p) a heavy chain comprising: (i) the amino acid sequence of VH P1-069065 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069065;

(q) a heavy chain comprising: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069067;

- 56 046477 (r) a heavy chain comprising: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069069;

(s) a heavy chain comprising: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069071;

(t) a heavy chain comprising: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069073;

(u) a heavy chain comprising: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069075;

(v) a heavy chain comprising: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-069077;

(w) a heavy chain comprising: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068736;

(x) a heavy chain comprising: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068738;

(y) a heavy chain comprising: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068740;

(z) a heavy chain comprising: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068742;

(aa) a heavy chain comprising: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain comprising the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain comprising: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain comprising: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068750;

(its) a heavy chain comprising: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068752;

(ff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E55A;

(hh) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_H1OOG and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E55A_E56N;

(kk) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E3OD and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E30D;

(ll) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain including

- 57 046477 amino acid sequence of the light chain P1-068761_E30D_E55A;

(mm) a heavy chain comprising: (i) the amino acid sequence of VH P106876l_E56N_H100G and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_H100G and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-O68761_E3OD_H1OOG;

(oo) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-O68761_E3OD_E56N;

(pp) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E1()()fF;

(qq) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E55A_E100fF;

(rr) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E56N_E100fF;

(uu) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E32Y;

(vv) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E32Y_E55A;

(ww) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E32Y_E56N;

(xx) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068761_E30D_E32Y;

(yy) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068761_E32Y_H100G;

(zz) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068761_E32Y_E100fF;

(aaa) a heavy chain comprising: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N;

(cc) a heavy chain comprising: (i) the amino acid sequence VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068767_D52N_E55A;

(ddd) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_D102V;

(iff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain comprising: (i) the amino acid sequence VH P1

- 58 046477

068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the light chain amino acid sequence P1-O68767_E3OD_D52N;

(hhh) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68767_E3OD_D1O2V;

(iii) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68767_E3OD and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D;

(jjj) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-O68767_E3OD_E55A;

(kkk) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E100fF_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068767_E 100fF_D102V;

(lll) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-()68767_E55A_E1O()GG;

(mmm) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68767_E1O()GE and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-068767_E100fF;

(ooo) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-068767_E30D_E100fF;

(ppp) a heavy chain comprising: (i) the amino acid sequence VH P1061029_F100fE_V102D and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-061029_F100fE_V102D;

(qqq) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029_F100fE and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-061029_F100fE;

(rrr) a heavy chain comprising: (i) the amino acid sequence VH P1-O61O29_V1O2D and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-061029_V102D;

(sss) a heavy chain comprising: (i) the amino acid sequence of VH P1-O61O29_Y32E and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain comprising: (i) the amino acid sequence VH P1061029_Y32E_F100fE and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain comprising the amino acid sequence light chain P1-O61O29_Y32E_F1OOIE, optionally, wherein VH is in any of (a )-(ttt) includes one or both of the K16R and T84A substitutions.

In some embodiments, the LC may be as defined in (a)-(III) above, and the HC may differ from the sequence of each of the antibody types (a)-(ttt) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions , such as 1, 2, 3, 4 or 5 conservative substitutions. In some embodiments, for example, the HC P1-061029 or one of its daughter clones may include one or both of the K16R and T84A substitutions in the VH region of the HC (P1-061015 and its daughter clones already have an R at position 16 of the VH and an A at position 84 VH).

Some embodiments of the present invention include anti-VISTA Abs including:

(a) a heavy chain consisting of: (i) the amino acid sequence VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain consisting of: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-061015;

(c) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068757;

(d) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068759;

- 59 046477 (e) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761;

(f) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068763 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068763;

(g) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068765;

(h) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767;

(i) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068769;

(j) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068771;

(k) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068773;

(l) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068775;

(m) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069059;

(n) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of light chain P1 -069061;

(o) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069063;

(p) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069065 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069065;

(q) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069067;

(r) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069069;

(s) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069071;

(t) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069073;

(u) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069075;

(v) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-069077;

(w) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068736;

(x) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068738;

(y) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid after

- 60 046477 light chain reactivity P1-068740;

(z) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068742;

(aa) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain consisting of the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068750;

(its) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068752;

(ff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain consisting of: (i) the amino acid sequence VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068761E55A;

(hh) a heavy chain consisting of: (i) the amino acid sequence VH P1-O68761H1OOG and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068761_H100G;

(ii) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E55A_E56N;

(kk) a heavy chain consisting of: (i) the amino acid sequence VH P1-O68761_E3OD and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068761_E30D;

(ll) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the light chain amino acid sequence P1-O68761_E3OD_E55A;

(mm) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E56N_H100G and (ii) the amino acid sequence SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain consisting of: (i) the amino acid sequence VH P1068761E30DH100G and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the light chain amino acid sequence P1-O68761_E3OD_H1OOG;

(oo) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E3OD_E56N;

(pp) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E10()fF;

(qq) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A_E1()()fF;

(rr) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain consisting of: (i) the amino acid sequence VH P1

- 61 046477

068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the light chain amino acid sequence P1-068761_E56N_E10()fF;

(uu) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y;

(vv) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y_E55A;

(ww) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E32Y_E56N;

(xx) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E3OD_E32Y;

(yy) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y_H100G;

(zz) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain Ρ1-Ο68761_Ε32Υ_Ε1()()ίΈ;

(aaa) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N;

(cc) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_D102V;

(fff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D_D52N;

(hhh) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D_D102V;

(iii) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_E30D and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767E30D;

(ϋί) a heavy chain consisting of: (i) the amino acid sequence of VH P1068767_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D_E55A;

(kkk) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E100fF_D102V and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_E100fF_D102V;

(lll) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-068767_E55A_E100fF;

(mmm) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767_E100fF and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E100fF;

- 62 046477 (ooo) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_E100fF and (ii) the amino acid sequence SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E3OD_E1OOIF;

(ppp) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_F100fE_V102D and (ii) the amino acid sequence SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-O61O29_F1OOIE_V1O2D;

(qqq) a heavy chain consisting of: (i) the amino acid sequence of VH P1-061029_F100fE and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-061029F100fE;

(rrr) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_V1O2D and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-061029_V102D;

(sss) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_Y32E and (ii) the amino acid sequence of SEQ ID NO: 182, and a light chain consisting of the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_Y32E_F100fE and (ii) the amino acid sequence SEQ ID NO: 182, and a light chain consisting of the amino acid sequence light chain P1-061029_Y32E_F100fE, where the C-terminal amino acid is VH and N-terminal amino acid SEQ ID NO: 182 form a peptide bond; and optionally wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

In some embodiments, an anti-VISTA mAb is provided, including:

a heavy chain consisting of the amino acid sequences: (i) VH (a)-(ttt) above, (ii) SEQ ID NO: 182, and (iii) a Lys residue, where the C-terminal amino acid is VH and the N-terminal amino acid SEQ ID NO: 182 form a peptide bond, and wherein the C-terminal amino acid of SEQ ID NO: 182 is connected to the N-terminal Lys; and a light chain consisting of the amino acid sequence of light chain (a)-(III) above;

wherein the VH and light chain amino acid sequences are selected from the same antibody types from (a)-(ttt) above.

Some embodiments of the present invention include anti-VISTA Abs including:

(a) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain comprising: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061015;

(c) a heavy chain comprising: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068757;

(d) a heavy chain comprising: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068759;

(e) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068761;

(f) a heavy chain comprising: (i) the amino acid sequence of VH P1-068763 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068763;

(g) a heavy chain comprising: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068765;

(h) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767;

(i) a heavy chain comprising: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068769;

(j) a heavy chain comprising: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068771;

- 63 046477 (k) a heavy chain comprising: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068773;

(l) a heavy chain comprising: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068775;

(m) a heavy chain comprising: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069059;

(n) a heavy chain comprising: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069061;

(o) a heavy chain comprising: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069063;

(p) a heavy chain comprising: (i) the amino acid sequence of VH P1-069065 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069065;

(q) a heavy chain comprising: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069067;

(r) a heavy chain comprising: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069069;

(s) a heavy chain comprising: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069071;

(t) a heavy chain comprising: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069073;

(u) a heavy chain comprising: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069075;

(v) a heavy chain comprising: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-069077;

(w) a heavy chain comprising: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068736;

(x) a heavy chain comprising: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068738;

(y) a heavy chain comprising: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068740;

(z) a heavy chain comprising: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068742;

(aa) a heavy chain comprising: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain comprising the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain comprising: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain comprising: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068750;

(its) a heavy chain comprising: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid

- 64 046477 light chain sequence P1-068752;

(ff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068761_E55A;

(hh) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761H1OOG and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E55A_E56N;

(kk) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E3OD and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068761_E30D;

(ll) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_E55A;

(mm) a heavy chain comprising: (i) the amino acid sequence of VH P1 068761_E56N_H100G and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_H100G and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_H1OOG;

(oo) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-O68761_E3OD_E56N;

(pp) a heavy chain comprising: (i) the amino acid sequence VH P1-068761_E100!B and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-()68761_E1()()GE ;

(qq) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E55A_E1OO!B;

(rr) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain Ρ1-Ο68761_Ε56Ν_Ε1ΟΟ1Έ;

(uu) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y;

(vv) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y_E55A;

(ww) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y_E56N;

(xx) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_E32Y;

(yy) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-O68761_E32Y_H1OOG;

(zz) a heavy chain comprising: (i) the amino acid sequence VH P1

- 65 046477

068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the light chain amino acid sequence P1-O68761_E32y_E1OOiF;

(aaa) a heavy chain comprising: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N;

(cc) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_D102V;

(fff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D_D52N;

(hhh) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D_D102V;

(iii) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E30D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D;

(jjj) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D_E55A;

(kkk) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E100fF_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E100fF_D102V;

(111) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E55A_E100fF;

(mmm) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E100fF;

(ooo) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D_E100fF;

(ppp) a heavy chain comprising: (i) the amino acid sequence VH P1061029_F100fE_V102D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence light chain P1-061029_F100fE_V102D;

(qqq) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029_F100fE and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061029_F100fE;

(rrr) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029_V102D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061029_V102D;

(sss) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029_Y32E and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain comprising: (i) the amino acid sequence of VH P1061029_Y32E_F100fE and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E_F100fE;

- 66 046477 optional, where VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

Some embodiments of the present invention include anti-VISTA Abs including:

(a) a heavy chain consisting of: (i) the amino acid sequence VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain consisting of: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-061015;

(c) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068757;

(d) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068759;

(e) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761;

(f) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068763 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068763;

(g) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068765;

(h) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068767;

(i) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068769;

(j) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068771;

(k) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068773;

(l) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068775;

(m) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069059;

(n) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069061;

(o) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069063;

(p) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069065 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069065;

(q) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069067;

(r) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069069;

(s) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069071;

(t) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence

- 67 046477 light chain reactivity P1-069073;

(u) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069075;

(v) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-069077;

(w) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068736;

(x) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068738;

(y) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068740;

(z) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068742;

(aa) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain consisting of the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068750;

(ee) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068752;

(ff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A;

(hh) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_H1OOG and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E55A_E56N;

(kk) a heavy chain consisting of: (i) the amino acid sequence VH P1-O68761_E3OD and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068761_E30D;

(ll) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E3OD_E55A;

(mm) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E56N_H100G and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_H100G and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E3OD_H1OOG;

(oo) a heavy chain consisting of: (i) the amino acid sequence VH P1

- 68 046477

068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the light chain amino acid sequence P1-O68761_E3OD_E56N;

(pp) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E10()fF;

(qq) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A_E10()fF;

(rr) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E56N_E100fF;

(uu) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y;

(vv) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the light chain amino acid sequence P1-068761_E32Y_E55A;

(ww) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068761_E32Y_E56N;

(xx) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068761_E30D_E32Y;

(yy) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y_H100G;

(zz) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y_E100fF;

(aaa) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N;

(cc) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_D102V;

(iff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D_D52N;

(hhh) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D_D102V;

(iii) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767_E30D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E30D;

- 69 046477 (jjj) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_E55A and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E3OD_E55A;

(kkk) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E100fF_D102V and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-O68767_E1OOIF_D1O2V;

(lll) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-()68767_E55A_E1O()GG;

(mmm) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain consisting of: (i) the amino acid sequence VH P1-O68767_E1O()GE and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_E100fF;

(ooo) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_E100fF and (ii) the amino acid sequence SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D_E100fF;

(ppp) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_F100fE_V102D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-O61O29_F1OOIE_V1O2D;

(qqq) a heavy chain consisting of: (i) the amino acid sequence of VH P1-061029_F100fE and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-061029_F100fE;

(rrr) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_V1O2D and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-061029_V102D;

(sss) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_Y32E and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_Y32E_F100fE and (ii) the amino acid sequence of SEQ ID NO: 183, and a light chain consisting of the amino acid sequence light chain P1-061029_Y32E_F100fE.

wherein the C-terminal amino acid VH and the N-terminal amino acid of SEQ ID NO: 183 form a peptide bond; and optionally wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

In some embodiments, anti-VISTA mAbs are provided including:

heavy chain consisting of the amino acid sequences: (i) VH (a)-(ttt) listed above, (ii) SEQ ID NO: 183, and (iii) a Lys residue, where the C-terminal amino acid is VH and the N-terminal amino acid SEQ ID NO: 183 form a peptide bond, and wherein the C-terminal amino acid of SEQ ID NO: 183 is connected to the N-terminal Lys; and a light chain consisting of the light chain amino acid sequence of (a) to (III) listed above;

wherein the VH and light chain amino acid sequences are selected from the same antibody types from (a)(ttt) listed above.

Other embodiments of the present invention include anti-VISTA Abs including:

(a) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain comprising: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061015;

(c) a heavy chain comprising: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068757;

(d) a heavy chain comprising: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068759;

(e) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068761;

(f) a heavy chain comprising: (i) the amino acid sequence of VH P1-068763 and (ii) ami

- 70 046477 amino acid sequence SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068763;

(g) a heavy chain comprising: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068765;

(h) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767;

(i) a heavy chain comprising: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068769;

(j) a heavy chain comprising: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068771;

(k) a heavy chain comprising: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068773;

(l) a heavy chain comprising: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068775;

(m) a heavy chain comprising: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069059;

(n) a heavy chain comprising: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069061;

(o) a heavy chain comprising: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069063;

(p) a heavy chain comprising: (i) the amino acid sequence of VH P1-069065 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069065;

(q) a heavy chain comprising: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069067;

(r) a heavy chain comprising: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069069;

(s) a heavy chain comprising: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069071;

(t) a heavy chain comprising: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069073;

(u) a heavy chain comprising: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069075;

(v) a heavy chain comprising: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-069077;

(w) a heavy chain comprising: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068736;

(x) a heavy chain comprising: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068738;

(y) a heavy chain comprising: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068740;

(z) a heavy chain comprising: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068742;

- 71 046477 (aa) a heavy chain comprising: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain comprising the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain comprising: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain comprising: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068750;

(its) a heavy chain comprising: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068752;

(ff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain comprising: (i) the amino acid sequence of VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068761_E55A;

(hh) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_H1OOG and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E55A_E56N;

(kk) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E3OD and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068761_E30D;

(ll) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_E55A;

(mm) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E56N_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N_H1OOG;

(nn) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_H1OOG;

(oo) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-O68761_E3OD_E56N;

(pp) a heavy chain comprising: (i) the amino acid sequence of VH E1-068761_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E1O(^;

(qq) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E55A_E1OOIF;

(rr) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E3OD_E1OOIF;

(tt) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E56N_E1OOIF;

(uu) a heavy chain comprising: (i) the amino acid sequence of VH P1-O68761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising amino acids

- 72 046477 light chain sequence P1-068761_E32Y;

(vv) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y_E55A;

(ww) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y_E56N;

(xx) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-O68761_E3OD_E32Y;

(yy) a heavy chain comprising: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-O68761_E32Y_H1OOG;

(zz) a heavy chain comprising: (i) the amino acid sequence of VH P1068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-O68761_E32Y_E1O()GG;

(aaa) a heavy chain comprising: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N;

(cc) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_D102V;

(fff) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_E30D_D52N;

(hhh) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_E30D_D102V;

(iii) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E30D and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D;

(ϋί) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_E30D_E55A;

(kkk) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E100fF_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_E100fF_D102V;

(lll) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_E55A_E100fF;

(mmm) a heavy chain comprising: (i) the amino acid sequence of VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_D52N_E100fF;

(nnn) a heavy chain comprising: (i) the amino acid sequence of VH P1-068767_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-068767_E100fF;

(ooo) a heavy chain comprising: (i) the amino acid sequence VH P1068767_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-068767_E30D_E100fF;

(ppp) a heavy chain comprising: (i) the amino acid sequence VH P1

- 73 046477

061029_F100fE_V102D and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061029_F100fE_V102D;

(qqq) a heavy chain comprising: (i) the amino acid sequence of VH P1-061029_F100fE and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061029_F100fE;

(rrr) a heavy chain comprising: (i) the amino acid sequence VH P1-O61O29_V1O2D and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence light chain P1-061029_V102D;

(sss) a heavy chain comprising: (i) the amino acid sequence of VH P1-O61O29_Y32E and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain comprising: (i) the amino acid sequence of VH P1061029_Y32E_F100fE and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of the light chain P1-061029_Y32E_F100fE; and optionally wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

Other embodiments of the present invention include anti-VISTA Abs including:

(a) a heavy chain consisting of: (i) the amino acid sequence VH P1-061029 (SEQ ID NO: 67) and (ii) the amino acid sequence SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-061029 (SEQ ID NO: 70);

(b) a heavy chain consisting of: (i) the amino acid sequence of VH P1-061015 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-061015;

(c) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068757 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068757;

(d) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068759 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068759;

(e) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761;

(f) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068763 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068763;

(g) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068765 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068765;

(h) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068767;

(i) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068769 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068769;

(j) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068771 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068771;

(k) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068773 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068773;

(l) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068775 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068775;

(m) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069059 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069059;

(n) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069061 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069061;

(o) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069063 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069063;

(p) a heavy chain consisting of: (i) the amino acid sequence VH P1-069065 and (ii) ami

- 74 046477 amino acid sequence SEQ ID NO: 184, and a light chain consisting of the light chain amino acid sequence P1-069065;

(q) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069067 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069067;

(r) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069069 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069069;

(s) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069071 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069071;

(t) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069073 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of light chain P1 -069073;

(u) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069075 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069075;

(v) a heavy chain consisting of: (i) the amino acid sequence of VH P1-069077 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-069077;

(w) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068736 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068736;

(x) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068738 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068738;

(y) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068740 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068740;

(z) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068742 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068742;

(aa) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068744 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068744;

(bb) a heavy chain consisting of the amino acid sequence of VH P1-068746 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068746;

(cc) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068748 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068748;

(dd) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068750 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068750;

(ee) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068752 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068752;

(ff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068754 and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068754;

(gg) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A;

(hh) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_H1OOG and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_H1OOG;

(ii) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68761_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E56N;

(jj) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E55A_E56N;

- 75 046477 (kk) a heavy chain consisting of: (i) the amino acid sequence VH P1-068761_E30D and (ii) the amino acid sequence SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D;

(ll) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the light chain amino acid sequence P1-O68761_E3OD_E55A;

(mm) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E56N_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E56N_H100G;

(nn) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E30D_H100G;

(oo) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68761_E3OD_E56N;

(pp) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E100fF;

(qq) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E55A_E10()fF;

(rr) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_H100G_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_H100G_E100fF;

(ss) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E30D_E100fF;

(tt) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E56N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E56N_E10()fF;

(uu) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068761_E32Y and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y;

(vv) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E32Y_E55A;

(ww) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E56N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E32Y_E56N;

(xx) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E30D_E32Y and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E30D_E32Y;

(yy) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_H100G and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068761_E32Y_H100G;

(zz) a heavy chain consisting of: (i) the amino acid sequence VH P1068761_E32Y_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068761_E32Y_E100fF;

(aaa) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N_D102V;

(bbb) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D52N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068767_D52N;

(cc) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068767_D52N_E55A;

(ddd) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068767_E55A_D102V;

(eeee) a heavy chain consisting of: (i) the amino acid sequence VH P1-068767_D102V and (ii) the amino acid sequence SEQ ID NO: 184, and a light chain consisting of the amino acid

- 76 046477 light chain sequence P1-068767_D102V;

(fff) a heavy chain consisting of: (i) the amino acid sequence of VH P1-068767_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E55A;

(ggg) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D52N and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E3OD_D52N;

(hhh) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E3OD_D1O2V;

(iii) a heavy chain consisting of: (i) the amino acid sequence VH P1-O68767_E3OD and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-068767_E30D;

(jjj) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_E55A and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E3OD_E55A;

(kkk) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E100fF_D102V and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_E1OOIF_D1O2V;

(lll) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E55A_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-()68767_E55A_E1O()GG;

(mmm) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_D52N_E100fF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-O68767_D52N_E1OOIF;

(nnn) a heavy chain consisting of: (i) the amino acid sequence of VH P1-O68767_E1OOIF and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-068767_E100fF;

(ooo) a heavy chain consisting of: (i) the amino acid sequence VH P1068767_E30D_E100fF and (ii) the amino acid sequence SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-O68767_E3OD_E1OOIF;

(ppp) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_F100fE_V102D and (ii) the amino acid sequence SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-O61O29_F1OOIE_V1O2D;

(qqq) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_F1OOIE and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1 -061029_F100fE;

(rrr) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_V1O2D and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-061029_V102D;

(sss) a heavy chain consisting of: (i) the amino acid sequence VH P1-O61O29_Y32E and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence of the light chain P1-061029_Y32E; or (ttt) a heavy chain consisting of: (i) the amino acid sequence VH P1061029_Y32E_F100fE and (ii) the amino acid sequence of SEQ ID NO: 184, and a light chain consisting of the amino acid sequence light chain P1-061029_Y32E_F100fE.

wherein the C-terminal amino acid VH and the N-terminal amino acid of SEQ ID NO: 184 form a peptide bond; and optionally wherein VH in any of (a)-(ttt) includes one or both of the K16R and T84A substitutions.

In some embodiments, anti-VISTA mAbs are provided including:

heavy chain consisting of the amino acid sequences: (i) VH (a)-(ttt) listed above, (ii) SEQ ID NO: 184, and (iii) a Lys residue, where the C-terminal amino acid is VH and the N-terminal amino acid SEQ ID NO: 184 form a peptide bond, and wherein the C-terminal amino acid of SEQ ID NO: 184 is connected to the N-terminal Lys; and a light chain consisting of the light chain amino acid sequence of (a) to (III) listed above;

wherein the VH and light chain amino acid sequences are selected from the same antibody types from (a)(ttt) listed above.

In some embodiments, the anti-hVISTA Ab may comprise a VH amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH P1-061029, where the antibody includes CDR1, CDR2 and/or CDR3 of VH Pl061029, in which at least one residue is replaced by D, E, or H. In some embodiments, CDR1, CDR2, and CDR3 of VH P1-061029 each contains one, two, or three residues replaced by D, E, or H. In some embodiments, the anti-hVISTA Ab may comprise a VH amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH P1-061029, where the antibody includes CDR1 VH, including one or two D or E residues at amino acid positions 4, 5 or 7 of CDR1, and/or includes CDR2 VH with one, two or three D, E or H residues at positions 3,5,6 or 7 of CDR2, and/or CDR3 VH with one, two or three D, E or H residues at positions 6, 12 or 14 of CDR 3 (see table 5 below for examples of antibodies included within these embodiments). In such cases, the light chain variable region may include CDR1, CDR2 and/or CDR3 of P1-061029 or P1-061015 or their daughter clones, 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, P1-068 750, P1-068752 P1-068754, P1-068761_E55A, P1-O68761_H1OOG, P1-O68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1068 761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1068761_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, P10 68761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_ D102V, P1 -068767_E55A,P1068767_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A,P1068767_E100fF_D102V, P1-068767_E55A _E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D,P 1061029_Y32E, or P1- 061029_Y32E_F100fE, and/or the light chain variable region may be 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 light chain variable region of P1061029 or P1-061015 or their daughter clones, 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, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1, P1. -069063, P1-069065, P1-069067, P1-069069, P1-069071, P1-069073, P1-069075, P1-069077, P1-061015, P1-068736, P1-068738, P1-068740, P1-068 742 , P1-068744, P1-068766, P1-068748, P1-068750, P1-068752 P1068754, P1-068761_E55A, P1-068761_H100G, P1-068761_E56N, P1-068761_E55A_E56 N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF, P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761 _E32Y_E100fF, P1-068767_D52N_D102V, P1068767_D52N, P1-068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P1-068767_E30D_D52N, P1-068767_E30D_D10 2V, P1-068767_E30D, P1-068767_E30D_E55A, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P106 8767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E or P1-061029_Y32E_F100fE.

In some embodiments, the anti-hVISTA Ab may comprise a VH amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH P1-061015, where the antibody includes CDR1, CDR2 and/or CDR3 VH P1061015, in which at least one residue is replaced by D, E, or H. In some embodiments, CDR1, CDR2, and CDR3 of VH P1-061015 each contain one, two, or three residues that are replaced by D, E, or H. In some In embodiments, the anti-hVISTA Ab may comprise a VH amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least is 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH P1-061015, where the antibody includes CDR1 VH, including one or two residues D, E or H at amino acid positions 6, 7, 8 and 9 of CDR1, and/or includes CDR2 VH with one, two or three D, E or H residues at positions 1, 2, 4 or 8-11 of CDR2, and/ or CDR3 VH with one, two or three D, E or H residues at positions 2, 3, 6, 7 or 12 of CDR 3 (see table 6 below for examples of antibodies included within these embodiments). In such cases, the light chain variable region may include CDR1, CDR2 and/or CDR3 of P1-061029 or P1-061015 or their daughter clones, such as P1-061029, P1-068757, P1-068759, P1-068761, P1-068763, P1-068765, P1- 78 046477

068767, P1-068769, P1-068771, P1-068773, P1-068775, P1-069059, P1-069061, P1-069063, P1-069065, Pl069067, Pl-069069, P1-069071, P1-0690 73, P1- P1-068752 P1-06875 4, P1-068761_E55A , P1-O68761_H1OOG, P1-068761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-068761_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H 100G, P1-068761_E30D_E56N, P1-068761_E100fF, P1068761_E55A_E100fF, P1-06876 l_H100G_E100fF, P1-068761_E30D_E100fF, P1- 06876l_E56N_E100fF, P1068761_E32Y, P1-068761_E32Y_E55A, P1-068761_E32Y_E56N, P1-068761_E30D_E32Y, P1068761_E32Y_H100G, P1-068761_ E32Y_E100fF, P1-068767_D52N_D102V, P1-068767_D52N, P1068767_D52N_E55A, P1-068767_E55A_D102V, P1-068767_D102V, P1-068767_E55A, P10687 67_E30D_D52N, P1-068767_E30D_D102V, P1-068767_E30D, P1-068767_E30D_E55A, P1068767_E100fF_D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P10687 67_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F100fE, P1-061029_V102D, P1061029_Y32E, or P1-061029_Y32E_F100fE, and/or light chain variable region may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% is at least 97%, at least 98%, or at least 99% identical to the light chain variable region of P1061029 or P1-061015 or their daughter clones, 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, P1-068 766, P1-068748, P1-068750, P1-068752 P1068754, P1-068761_E55A, P1-068761_H100G, P1-O68761_E56N, P1-068761_E55A_E56N, P1-068761_E30D, P1-06 8761_E30D_E55A, P1-068761_E56N_H100G, P1-068761_E30D_H100G, P1-068761_E30D_E56N, P1068761_E100fF , P1-068761_E55A_E100fF, P1-068761_H100G_E100fF, P1-068761_E30D_E100fF, P1068761_E56N_E100fF, P1-068761_E32Y, P1-068761_E32Y_E55A , P1-068761_E32Y_E56N, P1068761_E30D_E32Y, P1-068761_E32Y_H100G, P1-068761_E32Y_E100fF, P1-068767_D52N_D102V, P1068767_D52N, P1-0687 67_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, P1068767_E100fF _D102V, P1-068767_E55A_E100fF, P1-068767_D52N_E100fF, P1-068767_E100fF, P1068767_E30D_E100fF, P1-061029_F100fE_V102D, P1-061029_F10 0fE,P1 -061029_V102D, P1061029_Y32E or P1-061029_Y32E_F100fE.

In some embodiments, such modified anti-hVISTA daughter clones P1-061029 or P1-061015 have one or more of the following characteristics:

specifically bind to hVISTA, for example, the histidine-rich region of the ECD or the polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2, at an acidic pH, for example, pH 6.0 or pH 6.5;

do not demonstrate significant binding to hVISTA, for example, the histidine-rich region of the ECD or the polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2, at physiological pH or neutral pH, for example, pH 7.4 or pH 7.0;

bind specifically to cynomolgus VISTA, eg the histidine-rich region of the ECD, at acidic pH, eg pH 6.0 or pH 6.5;

do not show significant binding to cynomolgus VISTA, such as the histidine-rich region of the ECD, at physiological pH or neutral pH, such as pH 7.4 or pH 7.0;

demonstrate reduced binding to hVISTA-ECD having one or more of the following amino acid substitutions: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127 compared to hVISTA ECD having SEQ ID NO: 2;

cross-compete for hVISTA binding with P1-061029, P1-068761, P1-068767 and/or P1061015;

inhibit the binding of hVISTA to human T cells expressing VISTA (eg, naïve or activated T cells) at an acidic pH, eg, pH 6.0 or pH 6.5;

inhibit the binding of hVISTA to PSGL-1 at an acidic pH, e.g., pH 6.0 or pH 6.5 (e.g., inhibit the interaction between H153 and H154 of hVISTA having SEQ ID NO: 1, and PSGL-1 tyrosines Y46 and Y48), wherein PSGL-1 has or does not have sialyl Lewis X, and wherein the tyrosines are preferably sulfothyrosines;

a mean retention time (MRT) of at least 100, 200, 300, 350, 400, 450, 500, 600, or 700 hours (eg, at least 350 hours) in cynomolgus monkeys, measured, for example, as described in the Examples;

stimulate T cell activation, for example, by increasing T cell proliferation; increasing IFN-γ production from T cells; and/or stimulate T cell receptor-mediated NF-kB signaling;

inhibit VISTA-mediated cell:cell adhesion;

bind specifically to hVISTA in human tumor cell samples or inflamed human tissue samples that express VISTA;

contact hVISTA through one or more (for example, at least 1-3, 1-5, 1-10, 5-10, 5-15

- 79 046477 or all) energetically important contact residues Y37, T39, R54, F62, H66, V117, I119 or S124, as determined, for example, by yeast surface display and NGS analysis described in example 15; and where the numbering corresponds to the numbering of the mature hVISTA;

contact

Region 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) hVISTA having SEQ ID NO: 1, and optionally where binding is strongest in Region 2 as determined by MS-HDX as described in Example 21;

bind to the histidine-rich β-sheet extension of hVISTA, as determined, for example, by crystallography described, for example, in the examples;

contacts H121, H122 and/or H123 of mature hVISTA (distance of 4.0 angstroms (A) or less), for example, through hydrogen bonds, as determined, for example, by crystallography described, for example, in the Examples;

contact hVISTA through at least one or more glutamic acid, aspartic acid or histidine residues located in CDR1, CDR2 or CDR3 of VH; and and any additional characteristic presented in the claims and/or in the Examples.

Exemplary antibody constant regions.

In some embodiments, an antibody described herein includes one or more human constant regions. In some embodiments, the human heavy chain constant region has an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region has an isotype selected from k and λ. In some embodiments, an antibody described herein includes a human IgG constant region, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, an antibody described herein includes a human IgG4 heavy chain constant region. In some such embodiments, the antibody described herein includes the S241P mutation in the human IgG4 constant region. In some embodiments, an antibody described herein includes a human IgG4 constant region and a human k light chain.

The choice of heavy chain constant region can determine whether the antibody will have effector function in vivo. Such effector function in some embodiments includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) and can result in the killing of the cell to which the antibody binds. In some treatments, including treatments for certain forms of cancer, it may be desirable to kill cells, for example, when an antibody binds to a cell that is supporting or growing a tumor. Examples of cells that may mediate tumor maintenance or growth include, but are not limited to, the tumor cells themselves, cells that promote vasculature recruitment to the tumor, and cells that provide ligands, growth factors, or counterreceptors that promote or promote tumor growth or tumor survival. In some embodiments, when effector function is required, an antibody comprising a human IgG1 heavy chain or a human IgG3 heavy chain is selected.

In some embodiments, an antibody provided herein is modified to increase or decrease the extent to which the antibody is glycosylated. The addition or removal of glycosylation sites in an antibody can be conveniently achieved by changing the amino acid sequence such that one or more glycosylation sites are created or removed.

When an antibody includes an Fc region, the carbohydrate attached to it may be changed. Native antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide that is typically N-linked to Asn297 CH2 of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary structure of the oligosaccharide. In some embodiments, modifications to the oligosaccharide may be made to the antibody of the invention to create antibodies with certain improved properties. For example, in some embodiments, the antibody may be afucosylated, for example, by mutation of residues such as Asn297 that are typically glycosylated by fucose-containing glycosylation, or other means. In some embodiments, the antibodies herein may include an afucosylated human IgG1 constant region.

Antibodies are further provided with bisected oligosaccharides, for example, in which the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US Patent 6,602,684 (Umana

- 80 046477 et al.); and US 2005/0123546 (Umana et al.). Also provided are antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies may have improved CDC function. Such antibodies are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

Antibodies are also provided with N-terminal leader extensions. For example, one or more amino acid residues of an N-terminal leader sequence are present at the N-terminus of any one or more heavy or light chains of an antibody. An exemplary leader extension at the N-terminus includes or consists of three amino acid residues, VHS, present on one or both light chains of the antibody.

The in vivo or serum half-life of high-binding affinity human FcRn polypeptides can be examined, for example, in transgenic mice, human or non-human primates administered with variant Fc region polypeptides. See also, for example, Petkova et al. International Immunology 18(12): 1759–1769 (2006).

In some embodiments, the afucosylated antibody mediates ADCC in the presence of human effector cells more effectively than the parent antibody that includes fucose. Typically, ADCC activity can be determined using an in vitro ADCC assay as disclosed herein, but other assays or methods for determining ADCC activity are also provided, such as in an animal model, etc.

In some embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330 and/or 331 may be replaced by another amino acid residue, resulting in the antibody having an altered affinity for the effector ligand, but will retain the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may, for example, be an Fc receptor or a complement component C1. This approach is described in more detail in US patents 5,624,821 and 5,648,260 (Winter et al.).

In some examples, one or more amino acids selected from amino acid residues 329, 331 and 322 may be replaced with another amino acid residue, resulting in the antibody having altered C1q binding and/or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described in more detail in US Pat. No. 6,194,551 to Idusogie et al.

In some examples, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is also described in PCT publication WO 94/29351 by Bodmer et al. In some examples, the Fc region can be modified to reduce antibody-dependent cellular cytotoxicity (ADCC) and/or reduce affinity for the FcY receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243 , 244, 245, 247, 248, 249, 252, 254, 255,

256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293,294,

295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331,332,

333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435,436,

437, 438, or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Example options include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T. Other Fc modifications that can be made to the Fc are modifications to reduce or eliminate binding to FcyR and/or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330 and/or 331 (eg, 330 and 331), where the numbering corresponds to the EU index. Exemplary replacements include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), where the numbering corresponds to the EU index. Option Fc may include 236R/328R. Other modifications to reduce FcyR-complement interactions include substitutions at 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutational or enzymatic means, or when produced in organisms such as bacteria that do not glycosylate proteins. These and other modifications are discussed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. For example, the human IgG1.3 Fc constant region contains the substitutions L234A, L235E and G237A. IgG1fa.P238K (or IgG1.P238K) contains the P238K substitution. IgG1.1f includes substitutions L234A, L235E, G237A, A330S and P331S.

Fc variants that increase affinity for the inhibitory receptor FcYRIIb can also be used. Such variants may produce an Fc fusion protein with immunomodulatory activity associated with FcYRIIb cells, including, for example, B cells and monocytes. In one embodiment, Fc variants provide selectively increased affinity for FcYRIIb compared to one or more activating receptors. Modifications to change binding to

- 81 046477

FcYRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331 and 332 according to the EU index. Examples of substitutions to increase the affinity of FcYRIIb include, but are not limited to, 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E7, 266M, 267E, 268D , 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 331S and 332E. Examples of replacements include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W and 328Y. Other Fc variants to enhance binding to FcYRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.

Other modifications to strengthen the interactions of FCYR and complement include, without limiting the listed, replacement 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292R, 300L, 396L, 305I and 396L. These and other modifications are discussed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. Fc modifications that enhance binding to the Fcy receptor include amino acid modifications at any one or more amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337 , 338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 Fc region, where the numbering of residues in the Fc region corresponds to EU -index as specified in patent publication WO 00/42072.

It is not necessary to include the remainder of the non-living amino acid in additional and/or alternative provisions known to the specialist in this field (see, for example, US patents 5.624.821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; patent publications of the WO 00/420 72; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040 217, WO 05/092925 and WO 06/020114).

The affinity and binding properties of the Fc region for its ligand can be determined by various in vitro assays (biochemical or immunoassays) known in the art, including, but not limited to, equilibrium techniques (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) ) or kinetic (eg, BIACORE assay), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (eg, gel filtration). These and other methods may use a label on one or more components of interest and/or various detection methods including, but not limited to, chromogenic, fluorescent, luminescent or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

In some embodiments, the antibody is modified to increase its biological half-life. Different approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, one or more of the following residues may be mutated: 252, 254, 256, 433, 435, 436, as described in US Pat. US 6,277,375. Specific exemplary replacements include one or more of the following: T252L, T254S, and/or T256F. Alternatively, to increase the biological half-life, the antibody can be modified in the CH1 or CL region to contain a rescue receptor binding epitope taken from the two CH2 loops of the IgG Fc region domain, as described in US Patents 5,869,046 and 6,121,022 (Presta et al.) . Other exemplary variants that increase FcRn binding and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 43411, 434F, 434Y, and 434X1. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem. 279(8):62136216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9) :65916604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 433Q, 434H , 434F, 434Y, 252Y/254T/ 256E, 433K/434F/436H, 308T/309P/311S (DallAcqua et al. Journal of Immunology, 2002, 169:5171-5180, DallAcqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182: 7663-7671.

In some embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, an IgG1/IgG3 hybrid variant can be constructed by replacing the IgG1 positions in the CH2 and/or CH3 region with amino acids from IgG3 at positions where the two isotypes differ. Thus, it is possible to construct a hybrid variant of an IgG antibody that includes one or more substitutions, for example, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R and 436F. In some embodiments described herein, an IgG1/IgG2 hybrid variant can be constructed by replacing the IgG2 positions in the CH2 and/or CH3 region with amino acids from IgG1 at positions at which the two isotypes differ. Thus, it is possible to construct a hybrid version of an IgG antibody that includes one or more

- 82 046477 to substitutions, for example one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236) and 327A.

Moreover, binding sites for FcyRI, FcyRII, FcyRIII and FcRn have been mapped on human IgG1, and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604) . Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcyRIII. In addition, the following combination mutants have been shown to improve FcyRIIIa binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A, which have been shown to exhibit increased FcYRIIIa binding and ADCC activity (Shields et al. , 2001). Other IgG1 variants with highly increased binding to FcYRIIIa have been identified, including variants with the S239D/I332E and S239D/I332E/A330L mutations, which showed the greatest increase in affinity for FcYRIIIa, decreased FcYRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20-specific) and cetuximab (EGFR-specific) led to a significant increase in ADCC activity in vitro, with the S239D/ variant I332E showed increased ability to eliminate B cells in monkeys (Lazar et al., 2006). In addition, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations were identified that exhibited increased binding to FcYRIIIa and concomitant increased ADCC activity in transgenic mice expressing human FcYRIIIa in models of B-cell malignancies and breast cancer. (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L and

M428L/N434S.

In some embodiments, an Fc with reduced binding to FcyR is selected. An exemplary Fc, eg IgG1 Fc, with reduced FcyR binding includes the following three amino acid substitutions: L234A, L235E and G237A.

In some embodiments, an Fc with reduced complement fixation is selected. An exemplary Fc, eg IgG1 Fc, with reduced complement fixation has the following two amino acid substitutions: A330S and P331S.

In some embodiments, an Fc is selected that has substantially no effector function, i.e. it has decreased binding to FcyR and decreased complement fixation. An exemplary Fc, such as the IgG1 Fc, which is not an effector, includes the following five mutations: L234A, L235E, G237A, A330S, and P331S.

When using the IgG4 constant domain, it may include the S228P substitution, which mimics the hinge sequence in IgG1 and thus stabilizes IgG4 molecules.

Fc modifications described in WO 2017/087678 or WO 2016081746 can also be used.

In some embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (ie, an antibody without glycosylation) can be produced. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be made, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more glycosylation sites in the framework regions of the variable region, resulting in elimination of glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in more detail in US patents 5,714,350 and 6,350,861 (Co et al.).

Glycosylation of the constant region at N297 can be prevented by mutating residue N297 to another residue, eg N297A, and/or mutating an adjacent amino acid, eg 298, thereby reducing glycosylation at N297.

In addition or alternatively, an antibody may be produced that has an altered glycosylation pattern, such as a hypofucosylated antibody containing a reduced amount of fucose residues, or an antibody containing an increased amount of GlcNac branching structures. These altered glycosylation profiles have been shown to enhance the anti-ADCC potency of antibodies. Such modifications of carbohydrates can be made, for example, by expressing antibodies in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which the recombinant antibodies described herein can be expressed, resulting in an altered glycosylation antibody. For example, EP 1176195 (Hanai et al.) describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, resulting in antibodies expressed in such a cell line exhibiting hypofucosylation. PCT publication WO 03/035835 (Presta) describes a variant of the CHO cell line, Lec 3 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed by such host cells (see also Shields, R.L. et al.

- 83 046477 (2002) J. Biol. Chem. 277:26733-26740). PCT publication WO 99/54342 (Umana et al.) describes cell lines modified to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), resulting in antibodies expressed by the modified lines cells exhibit an increased number of GlcNac branching structures, which leads to increased ADCC activity of antibodies (see also Umana et al. (1999) Nat. Biotech., 17:176-180).

Another option for modifying antibodies according to the present invention is pegylation. The antibody may be pegylated, for example, to increase the biological half-life (eg, in serum) of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. In some embodiments, the pegylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol covers any form of PEG that is used to derivatize other proteins, such as mono(C1-C10)alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0 154 316 (Nishimura et al.) and EP 0 401 384 (Ishikawa et al.).

In various embodiments, antibody binding to the VISTA described herein is modified to selectively block antigen binding in tissues and environments where antigen binding may be undesirable, but allows antigen binding where it may be beneficial (activated antibody). In one embodiment, a blocking peptide mask is created that specifically binds to the antigen-binding surface of the antibody and prevents antigen binding, the mask being connected to each of the binding arms of the antibody by a peptidase-cleavable linker. See, for example, US Patent 8,518,404 (CytomX). Such constructs can be used to treat forms of malignant tumors in which the levels of proteases in the tumor microenvironment are significantly increased compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment causes dissociation of the masking/blocking peptide, allowing selective antigen binding in the tumor rather than in peripheral tissues where antigen binding may cause unwanted side effects. Examples of blocking peptides attached to antibodies are presented in WO 2018/08555.

Alternatively, such an embodiment provides a bivalent binding compound (masking ligand) comprising two antigen-binding domains that binds to both antigen-binding surfaces of the (bivalent) antibody and prevents binding of an antigen in which the two mask binding domains are linked to each other (but not to antibody) cleavable linker, for example, cleavable peptidase. See, for example, pub. international application for patent. WO 2010/077643 (Tegopharm Corp.). Masking ligands may include or may be derived from the antigen to which the antibody is intended to bind, or may be created independently. Such masking ligands can be used to treat malignant tumors in which protease levels are significantly elevated in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment causes the two binding domains to dissociate from each other, reducing avidity toward the antigen-binding surfaces of the antibody. As a result, dissociation of the masking ligand from the antibody allows for selective antigen binding in the tumor rather than in peripheral tissues where antigen binding may cause unwanted side effects.

Nucleic acids and host cells.

Nucleic acids encoding an antibody or a heavy or light chain thereof, or a part thereof, are also provided. Exemplary nucleic acids are presented in the sequence table. Any nucleic acid that constitutes at least 80%, 85%, 90%, 95%, 97%, 98% or 99% of the nucleic acid in the sequence table is included in the present description. Compositions comprising nucleic acids encoding an antibody provided herein are also included, as are cells comprising them, as well as methods for producing antibodies comprising culturing a cell transformed with a nucleic acid encoding an anti-VISTA antibody and isolating the antibody from the medium or cell. .

Methods of treatment using VISTA-ECD binding antibodies and corresponding pharmaceutical compositions.

In some embodiments, an anti-VISTA antibody that binds to VISTA at low pH and, for example, does not exhibit significant binding at neutral or physiological pH, may be an antagonistic anti-VISTA antibody, i.e. an antibody that inhibits the action of VISTA, resulting in stimulation of the immune response. Such antibodies can be used for le

- 84 046477 treatment of diseases that require stimulation of the immune system or immune response, such as proliferative diseases (benign or malignant), malignant tumors and infectious diseases (for example, viral infections).

In some embodiments, an anti-VISTA antibody that binds to VISTA at low pH and, for example, does not exhibit significant binding at neutral or physiological pH, may be an agonistic anti-VISTA antibody, i.e. an antibody that enhances the action of VISTA, resulting in inhibition of the immune response. Such antibodies may be used to treat diseases that require inhibition of the immune system or immune response, such as autoimmune diseases and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, celiac disease, Sjögren's syndrome, Graves' disease, inflammatory bowel disease, psoriasis, ankylosing spondylitis, graft-versus-host disease, allergies and asthma.

The antibodies described herein can be used, for example, to treat cancer. In some embodiments, methods of treating cancer are provided, comprising administering to a patient an effective amount of an antibody described herein. In some embodiments, Abs can induce or enhance an immune response in a patient, such as an antigen-specific immune response. In some embodiments, Abs can stimulate T cell activity. In some embodiments, the Abs can inhibit the growth of at least one tumor in a patient.

Provided herein are methods of treating a cancer patient, comprising administering to the subject a therapeutically effective amount of an anti-VISTA antibody described herein, thereby treating the subject. The anti-VISTA antibody can be used alone. Alternatively, the anti-VISTA antibody may be used in combination with another agent, as further described below.

Examples of cancers that can be treated with an Ab that specifically binds to the VISTA-ECD protein under acidic conditions as described herein include, but are not limited to, cancer, lymphoma, blastoma, sarcoma, and leukemia. Cancers that can be treated with the Abs described herein also include cancers that are generally responsive to immunotherapy and cancers that are not typically responsive to immunotherapy. Cancers that can be treated also include VISTA-positive cancers, for example, cancers containing VISTA-positive tumor-infiltrating cells, such as lymphocytes, myeloid or monocytic cells. Oncological diseases can be malignant solid tumors or hemoblastoses (tumors of liquid tissues).

Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer (eg, clear cell carcinoma), ovarian cancer, cancer liver, colorectal cancer, endometrial cancer, kidney cancer (eg, renal cell carcinoma (RCC)), prostate cancer (eg, hormone-resistant prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), stomach cancer, germ cell tumor, childhood sarcoma, sinonasal NK cell lymphoma, melanoma (eg , metastatic malignant melanoma such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small bowel cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, cancers caused by environmental factors, including those caused by asbestos, virus-related malignancies or malignancies of viral origin (eg, tumors associated with or caused by human papillomavirus (HPV)), and hemoblastic malignancies originating from one of the two major blood cell lines, that is, the myeloid cell lineage (from which granulocytes, red blood cells, platelets, macrophages and mast cells) or lymphoid cell lines (which give rise to B, T, NK and plasma cells), e.g. all types of leukemias, lymphomas and myelomas, e.g. acute, chronic, lymphocytic and/or myelogenous leukemias such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (UM), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia

- 85 046477 (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, such as B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosal lymphoid tissue (MALT) lymphoma , anaplastic (eg, Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio-immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, Tlymphoblastic lymphoma from progenitor cells, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/TOLL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell lymphoma, cutaneous T-cell lymphoma (CTLC) ( also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytoid lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), isolated plasmacytoma and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid origin, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including, but not limited to, T-cell disorders , such as T-prolymphocytic leukemia (T-PLL), including small cell and medullary type; T-cell type large granular lymphocyte leukemia (LGL); a/d T-NHL hepatosplenic lymphoma; peripheral/postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, colorectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these forms of cancer. The methods described in the present invention can also be used to treat metastatic cancers, inoperable, refractory cancers (eg, cancers resistant to previous immunotherapy, such as using a blocking antibody against CTLA-4 or PD-1) and/or recurrent malignant tumors.

In some embodiments, methods of treating cancer are provided, wherein the methods include administering an isolated antibody that specifically binds to huVISTA under acidic conditions, as described herein, to a subject with cancer. In some embodiments, the use of an antibody described herein is provided for the treatment of cancer.

In some embodiments, an antibody described herein is administered to patients having cancer that has demonstrated an insufficient response or has progressed with previous treatment, such as previous treatment with an immuno-oncology or immunotherapy agent. In some embodiments, the cancer is refractory or resistant to previous treatment, or is initially refractory or resistant (eg, refractory to a PD-1 pathway antagonist), or the resistant or refractory condition is acquired. For example, an antibody described herein may be administered to subjects who do not respond or insufficiently respond to a first therapy, or who experience disease progression after treatment, such as treatment with an anti-PD-1 pathway antagonist, alone or in combination with other therapy ( for example, with PD-1 pathway antagonist therapy). In other embodiments, an antibody described herein is administered to patients who have not previously received (ie, been treated with) an immuno-oncology agent, such as a PD-1 pathway antagonist.

In some embodiments, a method of treating cancer in a subject includes first determining tumor mutational burden (TMB) in the subject and administering an anti-VISTA antibody based on the results, for example, to subjects who are determined to have high TMB.

Combinations with immunostimulating agents

In some embodiments, an antibody described herein, for example, an anti-VISTA antagonist antibody described herein, is administered in combination with at least one immunostimulatory agent. For example, the therapeutic agents may be administered together by infusion or administered by injection at approximately the same time. In some embodiments, the antibody and at least one immunostimulant agent are administered sequentially. For example, in some embodiments, the antibody is administered sequentially, before or after at least one immunostimulatory agent, wherein the two therapeutic

- 86 046477 agents are administered at intervals of 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days or two weeks.

In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of the antibody are administered prior to administration of the at least one immunostimulant agent. In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of at least one immunostimulant agent are administered before administering the antibody. In some embodiments, the last dose of the immunostimulant agent is administered at least one, two, three, five, or ten days, or one, two, three, five, twelve, or twenty-four weeks before the first dose of the antibody. In some embodiments, the last dose of the antibody is administered at least one, two, three, five or ten days, or one, two, three, five, twelve or twenty-four weeks before the first dose of the at least one immunostimulant agent. In some embodiments, the subject has received or is receiving therapy with at least one immunostimulant agent, and a VISTA-ECD binding antibody is added to the therapeutic regimen.

In some embodiments, the at least one immunostimulant agent includes a T cell activation inhibitor antagonist, while in some embodiments, the at least one immunostimulatory agent includes a T cell activation stimulator agonist. In some embodiments, the at least one immunostimulatory agent includes an antagonist of CTLA4, LAG-3, PD-1, PD-L1, Galectin 1, Galectin 9, SEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFe, VEGF, KIR, LAG-3, adenosine receptor A2A, PI3K delta or IDO. In some embodiments, the at least one immunostimulatory agent includes a B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40 agonist , CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, the at least one immunostimulatory agent includes an agent that binds to another member of the B7 family of membrane-bound proteins, such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, and B7-H6. In some embodiments, the at least one immunostimulatory agent includes an agent that binds to a member of the TNF receptor family, or a co-stimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR , XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, LTpR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2, FAS FASL, RELT, DR6, TROY or NGFe. In some embodiments, the at least one immunostimulatory agent includes an agent that antagonizes or inhibits a cytokine that inhibits T cell activation, such as IL-6, IL-10, TGFe, VEGF. In some embodiments, the at least one immunostimulatory agent includes a cytokine agonist that stimulates T cell activation, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, the at least one immunostimulatory agent includes a chemokine antagonist such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, the at least one immunostimulating agent includes an antibody. In some embodiments, the at least one immunostimulant agent may include a vaccine, such as a mesothelin vaccine or an attenuated Listeria cancer vaccine, such as CRS-207.

For example, the anti-VISTA antibody described herein may be administered with one or more of the following agents.

(1) Antagonist (inhibitor or blocking agent) of a protein that inhibits T cell activation (eg, immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2 and LAG3, Galectin 9, SEACAM -1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, B7-H3, B7-H-4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-3 and TIM-4; and/or (2) A T cell activation protein agonist such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Examples of agents that may be combined with the anti-VISTA antibodies described herein for the treatment of cancer include: YERVOY® (ipilimumab) or Tremelimumab (anti-CTLA4), galiximab (anti-B7.1), BMS-936558 (anti-PD-1 ), MK-3475 (to PD-1), atezolizumab (TECENTRIQ®), Avelumab, Durvalumab, PDR001 (Novartis), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1) , MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (to CD27), anti-OX40 antibody (Providence Health Services), huMAbOX40L (anti-OX40L), Atacicept (anti-TACI), CP-870893 (anti-CD40), Lucatumumab (anti-CD40), Dacetuzumab (anti-CD40),

- 87 046477

CD40), MypoMOHa6-CD3 (to CD3); antibodies against GITR MK4166, TRX518, Medi1873, INBRX-110, LK2145, GWN-323, GITRL-Fc, or any combination thereof.

Other molecules that can be combined with anti-VISTA antibodies to treat cancer include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells, such as KIR antagonists (eg, lirilumab).

T cell activation can also be regulated by soluble cytokines. In some embodiments, anti-VISTA antibodies may be administered in combination with cytokine antagonists that are designed to inhibit T cell activation or cytokine agonists that promote T cell activation. For example, anti-VISTA antibodies can be used in combination with: (i) antagonists (or inhibitors or blocking agents) of IgSF family or B7 family or TNF family proteins that inhibit T cell activation, or antagonists of cytokines that inhibit T cell activation (eg , IL-6, IL-10, TGF-β, VEGF; immunosuppressive cytokines) and/or (ii) stimulatory receptor agonists of the IgSF family, B7 family or TNF family, or cytokines that stimulate T-cell activation.

Other agents for combination therapy include agents that inhibit or eliminate macrophages or monocytes, including, but not limited to, CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO 11/70024, WO 11/107553, WO 11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO 13169264; WO 14/036357).

Antibodies against VISTA can also be administered with agents that inhibit TGF-β signaling.

Additional agents that can be combined with the anti-VISTA antibody include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF-secreting cell vaccines, CpG oligonucleotides and imiquimod, or therapeutic agents that enhance the immunogenicity of tumor cells (eg anthracyclines).

Other therapeutic agents that can be combined with an anti-VISTA antibody include therapeutic agents that eliminate or block Treg cells, for example, an agent that specifically binds to CD25.

Another therapy that can be combined with an anti-VISTA antibody is a therapy that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthetase.

Another class of agents that can be used with an anti-VISTA antibody include agents that inhibit adenosine formation, such as CD73 inhibitors, or inhibit the A2A adenosine receptor.

Other therapies that can be combined with anti-VISTA antibody to treat cancer include therapies that reverse/prevent T cell anergy or exhaustion, and therapies that induce innate immune activation and/or inflammation at the tumor site.

Other therapies that can be combined with anti-VISTA antibody to treat cancer include therapies that block IL-8, such as HuMax®-IL8.

An anti-VISTA antibody may be combined with more than one immuno-oncology agent and, for example, may be combined with a combinatorial approach that is designed to target multiple elements of the immune pathway, such as one or more of the following: a therapy that enhances tumor antigen presentation (eg, a vaccine based on dendritic cells, GM-CSF secreting cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibiting the CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immunosuppressive cells; therapy that stimulates positive immune regulation, for example, using agonists that stimulate the CD-137, OX-40 and/or CD40 or GITR pathway and/or stimulate T cell effector function; therapies that systemically increase the frequency of antitumor T cells; therapy that depletes or inhibits Tregs, such as Tregs in a tumor, for example, using a CD25 antagonist (eg, daclizumab) or by ex vivo elimination with anti-CD25 antibody beads; therapies that affect the function of myeloid suppressor cells in the tumor; therapy that increases the immunogenicity of tumor cells (for example, anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, such as chimeric antigen receptor modified cells (CAR-T therapy); therapy that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; therapies that reverse/prevent anergy or T cell exhaustion; therapy that causes activation of innate immunity and/or inflammation in the area of tumor localization; administration of immunostimulating cytokines; or blocking immunorepressive cytokines.

The anti-VISTA antibodies described herein can be used in conjunction with one

- 88 046477 or more agonistic agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome various immunosuppressive pathways in the microenvironment tumors (eg, block inhibitory receptor interaction (eg, PD-L1/PD-1 interactions), eliminate or inhibit Tregs (eg, using anti-CD25 monoclonal antibodies (eg, daclizumab) or by ex vivo elimination with anti-CD25 beads ), inhibit metabolic enzymes such as IDO, or reverse/prevent anergy or T cell exhaustion), and agents that cause innate immune activation and/or inflammation in tumor sites.

In some embodiments, an anti-VISTA antibody is administered to a subject along with a BRAF inhibitor if the subject is positive for the BRAF V600 mutation.

Suitable PD-1 antagonists for use in the combination therapy described herein include, but are not limited to, ligands, antibodies (eg, monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, for example, an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

An example of an anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody that contains the CDRs or variable regions of one of the antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In some embodiments, the anti-PD-1 antibody is MK3475 (lambrolizumab), described in WO 2012/145493; AMP-514, described in WO 2012/145493; or PDR001. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, US patents 7,635,757 and 8,217,149 and US Patent Publication 2009/0317368. Any of the anti-PD-1 antibodies disclosed in WO 2013/173223 may also be used. An anti-PD-1 antibody that competes for binding and/or binds to the same epitope on PD-1 as one of these antibodies can also be used in combination therapy.

In some embodiments, the anti-PD-L1 antibody used for combination therapy is BMS-936559 (referred to as 12A4 in WO 2007/005874 and US Pat. No. 7,943,743) or an antibody that includes the CDR regions or variable regions 3G10, 12A4, 10A5 , 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT publication WO 07/005874 and US patent 7,943,743. In a particular embodiment, the anti-PD-L1 antibody is MEDI4736 (also known as durvalumab and anti-B7-H1), MPDL3280A (also known as atezolizumab and RG7446), MSB0010718C (also known as avelumab; WO 2013/79174) or rHigM12B7. Any of the anti-PD-L1 antibodies disclosed in WO 2013/173223, WO 2011/066389, WO 2012/145493, US Patents 7,635,757 and 8,217,149 and US Publication 2009/145493 may also be used. Antibodies against PD-L1 that compete and/or bind to the same epitope as either of these antibodies can also be used in combination therapy.

In some embodiments, an anti-VISTA antibody of the invention may be used with a CTLA-4 antagonist, such as an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody selected from the group consisting of: YERVOY® (ipilimumab or antibody 10D1 described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206), a monoclonal antibody, or anti-CTLA-4 antibody described in any of the following publications: WO 98/42752; WO 00/37504; US Patent 6,207,156; Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301–5304. Any of the anti-CTLA-4 antibodies disclosed in WO 2013/173223 may also be used.

In some embodiments, an anti-VISTA antibody of the invention is used in combination with a LAG3 antagonist. Examples of anti-LAG3 antibodies include antibodies comprising the CDR regions or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which are described in US patent publication US 2011/0150892, WO 10/19570 and WO 2014/008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other known anti-LAG3 antibodies that may be used include IMP731 and IMP 321, described in US 2011/007023, WO 08/132601 and WO 09/44273. Antibodies against LAG-3 that compete and/or bind to the same epitope as either of these antibodies can also be used in combination treatments.

In some embodiments, an anti-VISTA antibody of the invention may be administered in combination with a CD137 agonist (4-1BB), such as an anti-CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab or PF-05082566 (WO 12/32433).

In some embodiments, an anti-VISTA antibody may be administered in combination with an OX40 agonist, such as an anti-OX40 agonist antibody. Suitable anti-OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO 06/029879).

In one embodiment, an anti-VISTA antibody is administered in combination with a CD40 agonist,

- 89 046477 such as an agonistic antibody to CD40. In some embodiments, the immuno-oncology agent is a CD40 antagonist, such as an anti-CD40 antagonist antibody. Suitable anti-CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4.

In one embodiment, the anti-VISTA antibody is administered in combination with a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab (CDX-1127).

In some embodiments, the anti-VISTA antibody is administered together with an anti-GITR antibody, for example, an antibody having 6C8 CDR sequences, for example, a humanized antibody having 6C8 CDR regions, as described, for example, in WO 2006/105021; an antibody comprising the CDR regions of the anti-GITR antibody described in WO 2011/028683; an antibody comprising the CDR regions of an anti-GITR antibody described in JP 2008278814, an antibody comprising the CDR regions of an anti-GITR antibody described in WO 2015/031667, WO 2015/187835, WO 2015/184099, WO 2016/054638, WO 2016/0 57841 or WO 2016/057846, or other anti-GITR antibody described or referenced herein.

In some embodiments, an anti-VISTA antibody is administered in combination with MGA271 (anti-B7H3) (WO 11/109400).

In some embodiments, an anti-VISTA antibody is administered in combination with a KIR antagonist, such as lirilumab.

In some embodiments, an anti-VISTA antibody is administered in combination with an IDO antagonist. Suitable IDO antagonists include, for example, INCB 024360 (WO 2006/122150, WO 07/75598, WO 08/36653, WO 08/36642), indoximod, NLG-919 (WO 09/73620, WO 09/1156652, WO 11/ 56652, WO 12/142237) or F001287.

In some embodiments, the anti-VISTA antibody is administered in combination with a Toll-like receptor agonist, for example, a TLR2/4 agonist (eg, Bacillus Calmette-Guerin); a TLR7 agonist (eg Hiltonol or Imiquimod); TLR7/8 agonist (eg, Resiquimod); or a TLR9 agonist (eg, CpG7909).

In one embodiment, an anti-VISTA antibody is administered in combination with a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

In some embodiments, an anti-VISTA agent, such as an antibody, is administered with an anti-PSGL-1 antibody.

Additional combination therapy.

Abs herein may also be presented before, substantially simultaneously with, or after other treatments, such as surgery, chemotherapy, radiation therapy, or administration of a biologic, such as another therapeutic antibody. In some embodiments, the cancer has recurred or progressed after therapy selected from surgery, chemotherapy and radiation therapy, or a combination thereof. For example, an anti-VISTA antibody as described herein may be administered as adjuvant therapy if there is a risk that micrometastases may be present and/or to reduce the risk of relapse.

For the treatment of cancer, the combinations may be administered in combination with one or more additional antitumor agents, such as a chemotherapeutic agent, a growth inhibitory agent, an anticancer vaccine such as a gene therapy vaccine, an antiangiogenic agent, and/or an antitumor composition. Non-limiting examples of a chemotherapeutic agent, a growth inhibitory agent, an anticancer vaccine, an antiangiogenic agent, and an antitumor composition that can be used in combination with antibodies of the present invention are provided herein in the Definitions section.

In some embodiments, an anti-inflammatory agent, such as a steroidal or nonsteroidal anti-inflammatory drug (NSAID), may be administered with the combination. In cases where aberrantly proliferating cells need to be quiescent in combination with or prior to treatment with the anti-VISTA antibodies described herein, the patient may also be administered hormones and steroids (including synthetic analogues) such as 17a-ethinyl estradiol, diethylstilbestrol, testosterone , prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megesterol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX®. When using the methods or compositions described herein, other agents used to modulate the growth or metastasis of tumors in clinical settings, such as antimimetics, may also be administered, if necessary.

The antibodies described herein can also be combined with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and cells transfected with a gene encoding immunostimulatory cytokines (He et al., ( 2004) J Immunol 173:4919-28). Non-limiting examples against

- 90 046477 tumor vaccines that can be used include peptides of melanoma antigens, such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF (further discussed below).

In humans, some tumors have been shown to be immunogenic, such as melanomas. By lowering the threshold for T cell activation by inhibiting VISTA, antitumor responses in the recipient can be activated, providing the opportunity to treat non-immunogenic tumors or tumors with limited immunogenicity.

The anti-VISTA antibody described herein may also be combined with a vaccination protocol. Many experimental tumor vaccination strategies have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023 -3043, in DeVita et al (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, a vaccine is produced using autologous or allogeneic tumor cells. Such cell-based vaccines have been shown to be most effective when tumor cells are transformed to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large-scale gene expression profiles in various tumors has led to the identification of so-called tumor-specific antigens (Rosenberg, S.A. (1999) Immunity 10:281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cell from which the tumor developed, for example, melanocyte antigens gp100, MAGE and Trp-2 antigens. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells present in the host. VISTA inhibition can be used in combination with a collection of recombinant proteins and/or peptides expressed in the tumor to generate an immune response to these proteins. These proteins are usually considered by the immune system as its own antigens, and therefore it is tolerant to them. The tumor antigen may include the protein telomerase, which is required for chromosome telomere synthesis and which is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266:2011-2013). A tumor antigen may also be a neoantigen, expressed in cancer cells as a result of somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (ie, bcr-abl on the Philadelphia chromosome), or an idiotype from B-cell tumors.

Other cancer vaccines may include proteins from viruses implicated in the pathogenesis of human cancers, such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpesvirus (KHSV). Another form of tumor-specific antigen that can be used in combination with VISTA inhibition is purified heat shock proteins (HSPs) isolated directly from tumor tissue. Such heat shock proteins contain protein fragments from tumor cells, and such HSPs are highly effective in delivery to antigen presenting cells to induce antitumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117 -120).

Oncolytic viruses can also be used in combination with anti-VISTA antibodies.

Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be generated ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express such tumor antigens. DCs have also been directly fused to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with VISTA inhibition to activate more potent antitumor responses.

Treatment of infectious diseases.

The methods described herein may also be used to treat patients who have been exposed to certain toxins or pathogens. Thus, the present invention also provides methods for treating an infectious disease in a subject, comprising administering to the subject an antibody as described herein, for example, an anti-VISTA antagonist antibody, thereby treating the subject for the infectious disease. Similar to the use against tumors as discussed above, antibody-mediated inhibition of VISTA can be used alone or as an adjuvant in combination with vaccines to stimulate an immune response to pathogens, toxins and autoantigens. Examples of pathogens against which such a therapeutic modality may be particularly useful include pathogens for which no effective vaccine currently exists or pathogens for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (A, B and C),

- 91 046477 influenza, herpes, lamblia, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. VISTA inhibition can be used against established infections caused by agents such as HIV that present altered antigens during infections.

Some examples of pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis (A, B, or C), herpes virus (eg, VZV, HSV-1, HAV-6, HSV-II, and CMV , Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections that can be treated by the methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Serrationa, Pseudomonas, Legionella, Diphtheria Bacillus, Salmonella , bacilli, Vibrio cholerae, tetanus bacillus, botulism, anthrax bacillus, plague bacillus, leptospirosis and bacteria that cause Lyme disease.

Some examples of pathogenic fungi that cause infections that can be treated by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections that can be treated by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodiumvivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis.

In all of the methods described above, VISTA inhibition can be combined with other forms of immunotherapy, such as those described herein, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which can provide for improved presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

Routes of administration and carriers.

In various embodiments, antibodies can be administered in vivo by various routes, including, without limitation, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or otherwise manner, by implantation or inhalation. The subject compositions may be formulated as preparations in solid, semi-solid, liquid or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, rinses, injectables, inhalants and aerosols. The nucleic acid molecule encoding the antibody can be coated on gold microparticles and delivered intradermally using a particle bombardment device or gene gun, as described in the literature (see, for example, Tang et al., Nature 356:152-154 (1992) ). A suitable composition and route of administration can be selected according to the intended use.

In various embodiments, antibody-containing compositions may be provided in formulations with various pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al. al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers are available, which include carriers, adjuvants and diluents. In addition, various pharmaceutically acceptable excipients such as pH adjusters and buffers, tonicity agents, stabilizers, wetting agents and the like are also available. Non-limiting examples of carriers include sodium chloride solution, buffered sodium chloride solution, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, the antibody-containing compositions can be formulated for injection, including subcutaneous administration, by dissolving, suspending or emulsifying them in an aqueous or non-aqueous vehicle, such as vegetable or other oils, synthetic aliphatic acid glycerides, higher aliphatic acid esters, or propylene glycol; and, if necessary, with standard additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In various embodiments, the compositions may be formulated for inhalation, for example, using suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. In various embodiments, the compositions may also be formulated as microcapsules with

- 92 046477 slow release, for example with biodegradable or non-biodegradable polymers. A non-limiting example of a biodegradable composition includes a polylactic acid-glycolic acid polymer. A non-limiting example of a biodegradable composition includes a glycerol fatty acid ester polymer. Some methods for preparing such compositions are described, for example, in EP 1 125 584 A1.

Also provided are pharmaceutical packages and kits comprising one or more containers, each of which contains one or more doses of an antibody or combination of antibodies. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising an antibody or combination of antibodies, with or without one or more additional agents. In some embodiments, such unit dose is provided in a disposable prefilled injection syringe. In various embodiments, the composition contained in the unit dose may include sodium chloride solution, sucrose or the like; a buffer such as phosphate or the like; and/or can be manufactured in a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid, such as sterile water. In some embodiments, the composition contains one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the composition of the invention contains heparin and/or proteoglycan.

The pharmaceutical compositions are administered in an amount effective to treat or prevent a particular indication. The therapeutically effective amount will generally depend on the weight of the subject being treated, his or her physical or health condition, the extent of the condition being treated, or the age of the subject being treated. Typically, antibodies may be administered in amounts ranging from about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibodies may be administered in an amount ranging from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, the antibodies may be administered in an amount ranging from about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies may be administered in an amount ranging from about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies may be administered in an amount ranging from about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.

Antibody compositions can be administered to subjects as needed.

Determination of the frequency of administration can be made by those skilled in the art, such as a physician, based on consideration of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In some embodiments, an effective dose of the antibody is administered to a subject one or more times. In various embodiments, an effective dose of the antibody is administered to a subject once per month, less than once per month, such as once every two months or once every three months. In other embodiments, an effective dose of the antibody is administered more than once per month, such as once every three weeks, once every two weeks, or once a week. In some embodiments, an effective dose of the antibody is administered once every 1, 2, 3, 4, or 5 weeks. In some embodiments, an effective dose of the antibody is administered two or three times per week. An effective dose of the antibody is administered to the subject at least once. In some embodiments, an effective dose of the antibody may be administered multiple times, including over periods of at least a month, at least six months, or at least a year.

In some embodiments, the combination of an anti-VISTA antibody and a second agent discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier or simultaneously, as separate compositions, with an anti-VISTA antibody and a second agent in a pharmaceutically acceptable carrier. In one embodiment, the combination of an anti-VISTA antibody and a second agent may be administered sequentially. Administration of the two agents may be started at points in time, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days or one or more weeks, or administration of the second agent may begin, for example, after 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days or one or more weeks after administration of the first remedy. Methods for Identifying Antibodies that Bind hVISTA-ECD at Low pH Also provided herein are methods for identifying antibodies that specifically bind to VISTA-ECD protein under acidic (or low pH) conditions. In some embodiments, a method for identifying an Ab that specifically binds to a VISTA-ECD protein at a pH of 6.5 or less comprises contacting the test Ab or a plurality of test Abs with the VISTA-ECD protein at a pH of 6.5 or less and selecting the test Ab. if it binds to the ECD of the VISTA protein with a KD of 10 ^-7 M, 10 ^-8 M, 10 ^-9 M or less. In some embodiments, the method is carried out at pH 6.5, while in other cases it is carried out at pH 6.0,

- 93 046477 either at pH 5.5 or at pH 5.0. In some embodiments, the VISTA-ECD protein is an hVISTA-ECD protein, or includes the IgV domain of hVISTA, or is a polypeptide comprising amino acids 20-95 of SEQ ID NO: 2 or amino acids 20-70, 35-95, or 35-70 of SEQ ID NO: 2 : 2. In some embodiments, the polypeptide also includes amino acids 95-105 of SEQ ID NO: 2. In some embodiments, the polypeptide also includes amino acids 35-127 or 37-125 of SEQ ID NO: 2.

In some embodiments, the method further includes testing the binding of a test Ab or multiple test Abs at a neutral, physiological, or alkaline pH, such as pH 7.0 or pH 7.4. In some embodiments, the method further comprises selecting an antibody if it not only binds to a VISTA-ECD protein with a K _{D of} 10 ^-7 M, 10 ^-8 M, 10 ^-9 M or less at a pH of 6.5 or lower, but also if it specifically binds to a polypeptide at pH 7.0 or pH 7.4. In some embodiments, test Abs are selected if they specifically bind VISTA-ECD protein under acidic conditions, such as pH 6.5 or less, and also specifically bind VISTA-ECD protein at neutral and/or alkaline pH with similar affinity ( i.e. they are universal binding agents). For example, some such Abs can bind to the VISTA-ECD protein with a K _{D of} 10 ^-7 M, 10 ^-8 M, 10 ^-9 M or less at either pH 6.5, pH 7.0 or pH 7.4 (at a constant temperature, for example at 25°C or at 37°C), such that KD at pH 6.5 is 1.5 times greater than KD at pH 7.0.

Some Abs may be selected if they specifically bind to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or less, with higher affinity than at neutral or alkaline pH (pH-sensitive binders or pH-sensitive antibodies). For example, in some embodiments, the Ab may bind to the VISTA-ECD protein with a K _{D of} 10 ^-8 M or less at pH 6.5 and with a K D greater than 10 ^-8 M at pH 7.0 or pH 7.4. In some such embodiments, the Ab may bind to the VISTA-ECD protein with a KD of 10 ^-8 M or less at pH 6.5 and a KD at pH 7.0 or pH 7.4 that is greater than 1.5 times higher, than KD at pH 6.5. In some embodiments, a pH-sensitive Ab is selected if it specifically binds to the VISTA-ECD protein with a KD that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300 times, 500 times, 1000 times or 5000 times lower at pH 6.5 than at pH 7.0 or pH 7.4 (at constant temperature, for example at 25°C or at 37°C). For example, in some cases, an Ab is selected if it binds to the VISTA-ECD protein with a KD that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold, 500 times, 1000 times, or 5000 times less at pH 6.0 than at pH 7.0 or pH 7.4 or higher (at constant temperature, such as 25°C or 37°C).

In some embodiments, the Ab is selected if it specifically binds to the VISTA-ECD protein with a koff that is lower under acidic conditions compared to koff under neutral, physiological, or alkaline conditions. In some embodiments, an Ab is selected if it binds to the VISTA-ECD protein under acidic conditions with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower at pH 6.5 than koff at pH 7.0 or pH 7.4, when measured, for example, at 25°C or at 37°C. For example, in some embodiments, an Ab is selected if it binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower at pH 6.0 compared to pH 7.0 or pH 7.4, when measured at, for example, 25°C or 37°C.

In some embodiments, the Ab is selected if it binds to the VISTA-ECD protein with a ko _n that is higher under acidic conditions compared to neutral or alkaline conditions. In some embodiments, the Ab is selected if it binds to the VISTA-ECD protein under acidic conditions with a ko _n that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher at pH 6.5 , than ko _n at pH 7.0 or pH 7.4, when measured, for example, at 25°C or at 37°C. For example, in some embodiments, an Ab is selected if it binds to the VISTA-ECD protein with a ko _n that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher at pH 6.0. than at pH 7.0 or pH 7.4, when measured at, for example, 25°C or 37°C.

Methods for Altering the pH Sensitivity of VISTA-ECD Binding Antibodies An antibody that binds to the VISTA-ECD protein but does not bind to it at pH 6.5 or less, or does not bind to it with high affinity at pH 6.5 or less, may be modified to increase its binding affinity at pH 6.5 or lower. For example, the antibody paratope may be mutated, for example by replacing one or more amino acid residues. For example, in some embodiments, 1-8, e.g., 1-6, 1-4, 1-3, 1-2, or 1 amino acid residues in the heavy or light chain of an Ab that are VISTA-ECD contact residues (e.g., residues in one or more CDR regions) may be replaced by another amino acid residue. The mutant Ab can then be tested for binding to the VISTA-ECD protein at pH 6.5 or less, and variant Abs that bind with higher affinity than the parent antibody can be selected. If necessary, the steps above can be repeated so that two or more rounds of mutagenesis and selection are performed on the antibodies, and the antibodies that bind with the highest affinity at acidic pH are selected. In some embodiments, such selections may increase the antitumor efficacy of the resulting antibody compared to the parent antibody.

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The above selection method can also be developed in accordance with the previously described general selection of antibodies that specifically bind the VISTA-ECD protein. Namely, in some embodiments, the improved Ab is selected if it binds to the ECD of the VISTA protein with a KD of 10 ^-8 M or less at pH 6.5. In some embodiments, the selection is performed at pH 6.0, or at pH 5.5, or at pH 5.0 instead of pH 6.5. In some embodiments, the VISTA-ECD protein used for the selection process is a complete hVISTA-ECD protein, or is a polypeptide that includes the IgV domain of hVISTA, or is a polypeptide comprising amino acids 20-95 of SEQ ID NO: 2 or amino acids 20-70, 35-95 or 35-70 SEQ ID NO: 2. In some embodiments, the polypeptide also includes amino acid residues 95-105 of SEQ ID NO: 2. In some embodiment, a polypeptide is used including amino acid residues 35-127 of SEQ ID NO: 2.

In some embodiments, a method of improving the binding of an anti-VISTA antibody to a VISTA ECD at an acidic pH includes increasing the number of glutamic acid, aspartic acid and/or histidine residues in one or more VH or VL CDR regions, such as CDR1, CDR2 and CDR3 of VH or only CDR1 and CDR3 VH. In some embodiments, the method includes increasing the amount of glutamic acid, aspartic acid, and/or histidine residues in regions of the antibody that contact hVISTA, as determined, for example, by crystallography.

In some embodiments, the method further includes testing the binding of the selected Ab at neutral, alkaline, or physiological pH, such as pH 7.0 or 7.4. In some embodiments, the method further comprises selecting an antibody if it not only binds to a VISTA-ECD protein with a K _{D of} 10 ^-8 M or less at a pH of 6.5 or lower, but also if it also specifically binds to a polypeptide at pH 7, 0 or 7.4. In some such embodiments, the Ab is selected if it specifically binds to the VISTA-ECD protein under acidic conditions, for example, at pH 6.5 or less, and also specifically binds to the VISTA-ECD protein at neutral and/or alkaline or physiological pH with similar affinity (i.e. they are universal binders). For example, some such Abs can bind to the VISTA-ECD protein with a KD of 10 ^-8 M or less at both pH 6.5 and pH 7.0 (at a constant temperature, e.g., 25°C or 37°C ), such that KD at pH 6.5 is 1.5 times greater than KD at pH 7.0 or at pH 7.4.

Some Abs may be selected if they specifically bind to the VISTA-ECD protein under acidic conditions, such as pH 6.5 or less, with higher affinity than at neutral, physiological, or alkaline pH (pH-sensitive binders or pH-sensitive At). For example, in some embodiments, Abs can bind to the VISTA-ECD protein with a _{KD of} 10 ^-8 M or less at pH 6.5 and with a KD greater than 10 ^-8 M at pH 7.0. In some such embodiments, Abs can bind to the VISTA-ECD protein with a KD of 10 ^-8 M or less at pH 6.5 and a _KD at pH 7.0 that is greater than 1.5 times higher than the _KD at pH 7.0. pH 6.5. In some embodiments, a pH-sensitive Ab is selected if it specifically binds to the VISTA-ECD protein with a K _D that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold , 300 times, 500 times, 1000 times or 5000 times lower at pH 6.5 than at pH 7.0 or pH 7.4 (at a constant temperature, for example at 25°C or at 37°C). For example, in some cases, an Ab is selected if it binds to the VISTA-ECD protein with a K _D that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 300-fold , 500 times, 1000 times or 5000 times less at pH 6.0 compared to pH 7.0 or pH 7.4 or higher (at constant temperature, for example at 25°C or at 37°C).

In some embodiments, the method further includes determining koff at two pH values. In some such embodiments, the Ab is selected if it specifically binds to the VISTA-ECD protein with a koff that is lower under acidic conditions compared to koff under neutral, physiological, or alkaline conditions. In some embodiments, the Ab is selected if it binds to the VISTA-ECD protein under acidic conditions with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower at pH 6.5 than koff at pH 7.0 or pH 7.4, when measured, for example, at 25°C or at 37°C. For example, in some embodiments, an Ab is selected if it binds to the VISTA-ECD protein with a koff that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower at pH 6.0 compared to pH 7.0, measured at 25°C or 37°C, for example.

In some embodiments, the method further includes determining ko _n at two pH values. In some such embodiments, the Ab is selected if it binds to the VISTA-ECD protein with a ko _n that is higher under acidic conditions compared to neutral, physiological, or alkaline conditions. In some embodiments, the Ab is selected if it binds to the VISTA-ECD protein under acidic conditions with a ko _n that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher at pH 6.5 , than ko _n at pH 7.0 or pH 7.4, when measured, for example, at 25°C or at 37°C. For example, in some embodiments, an Ab is selected if it binds to the VISTA-ECD protein with a ko _n that is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher at pH 6.0. than at pH 7.0 or pH 7.4, when measured at, for example, 25°C or 37°C.

Antibodies that selectively bind to huVISTA at acidic pH compared to neutral or physiological pH can be identified by positive screening

- 95 046477 library of antibodies to VISTA or Fab or scFv fragments for binding at acidic pH, for example pH 6.0 or 6.5, and negative screening of the library for lack of binding at neutral pH, for example pH 7.0, or physiological pH, for example pH 7.4. The library can be enriched for glutamic acid, aspartic acid and histidine residues, for example, to select binding domains that may be charged and may be more likely to bind VISTA at acidic pH. Screening may include positive selection at acidic pH and negative selection at neutral or physiological pH. Positive and negative selections can alternate.

Alternatively, an antibody that binds to VISTA at neutral and acidic pH may be modified so that it does not bind at neutral pH while maintaining or even enhancing binding at acidic pH. For example, a library can be created by replacing amino acid residues VH and optionally VL, for example, in one or more CDR regions, and screening the library by positive selection for antibodies that bind hVISTA at acidic pH, and negative selection for antibodies that bind hVISTA at acidic pH, and negative selection for antibodies that which do not bind to VISTA at neutral (or physiological) pH. A similar method can be used to generate anti-VISTA binding antibodies having the desired pH-selective, pH-dependent or pH-independent VISTA binding profile.

Certain embodiments.

Additional embodiments of the present invention include the following.

1. An isolated antibody (Ab) that specifically binds to the extracellular domain (ECD) of human V-domain suppressor of T-cell activation immunoglobulin (hVISTA), where the Ab:

a) Binds to hVISTA-ECD at pH 6.5 or less with an affinity constant (K _D ) of 10 ^-7 M or less;

b) Binds to hVISTA-ECD at pH 6.5 or less with a dissociation rate constant (koff) of 10 ^-3 sec ^-1 or less; or

c) Binds to hVISTA-ECD at pH 6.5 or less with a KD of 10 ^-7 M or less and koff of 10 ^-3 sec ^-1 or less.

2. The isolated Ab according to embodiment 1, wherein the Ab binds to hVISTA-ECD at a pH of 6.5 or less with a KD of 10 ^-8 M or less.

3. The isolated Ab according to embodiment 2, wherein the Ab binds to hVISTA-ECD at a pH of 6.5 or less with a KD of 10 ^-9 M or less.

4. The isolated Ab according to any one of embodiments 1-3, wherein the Ab binds to hVISTAECD at a pH of 6.5 or less with a koff of 10 ^-4 sec ^-1 or less.

5. The isolated Ab according to any one of embodiments 1-4, wherein the Ab binds to hVISTAECD at pH 7.0 or higher with:

a) KD 10 ^-7 M or less;

b) koff 10 ^-3 sec ^-1 or less; or

c) KD 10 ^-7 M or less and koff 10 ^-3 sec ^-1 or less.

6. The isolated Ab according to embodiment 5, wherein the Ab binds to hVISTA-ECD at pH 7.0 or higher with a KD of 10 ^-8 M or less.

7. The isolated Ab according to embodiment 6, wherein the Ab binds to hVISTA-ECD at pH 7.0 or higher with a KD of 10 ^-9 M or less.

8. The isolated Ab according to any one of embodiments 5-7, wherein the Ab binds to hVISTAECD at pH 7.0 or higher with a koff of 10 ^-4 sec ^-1 or less.

9. The isolated Ab according to any one of embodiments 5-8, wherein the Ab binds to hVISTAECD at pH 6.5 with similar affinity as at pH 7.0.

10. The isolated Ab according to embodiment 9, wherein the Ab binds to hVISTA-ECD at pH 6.5 with higher affinity than at pH 7.0.

11. The isolated Ab according to embodiment 10, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a KD that is at least 1.5 times lower than the KD at pH 7.0.

12. The isolated Ab according to embodiment 11, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a KD that is at least 2 times lower than the KD at pH 7.0.

13. The isolated Ab according to embodiment 11, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a _KD that is at least 5 times lower than the _KD at pH 7.0.

14. The isolated Ab according to any one of embodiments 10-13, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a koff that is lower than the koff at pH 7.0.

15. The isolated Ab according to embodiment 14, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a koff that is at least 1.5 times lower than the koff at pH 7.0.

16. The isolated Ab according to embodiment 15, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a koff that is at least 2 times lower than the koff at pH 7.0.

17. The isolated Ab according to embodiment 16, wherein the Ab binds to hVISTA-ECD at pH 6.5 with a _koff that is at least 5 times lower than the _koff at pH 7.0.

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18. The isolated Ab according to any one of embodiments 1-17, wherein the Ab specifically binds to hVISTA-ECD under conditions in which at least one histidine residue of the hVISTA-ECD is protonated.

19. The isolated Ab according to embodiment 18, wherein the Ab binds to the IgV domain of hVISTA-ECD.

20. The isolated Ab of embodiment 19, wherein the Ab binds to the region located between amino acids 20 and 95 of SEQ ID NO: 2.

21. The isolated Ab of embodiment 20, wherein the Ab binds to the region located between amino acids 20 and 70 of SEQ ID NO: 2.

22. The isolated Ab of embodiment 21, wherein the Ab binds to the region located between amino acids 35 and 70 of SEQ ID NO: 2.

23. The isolated Ab according to any one of embodiments 20-22, wherein the Ab also binds to another region of the hVISTA ECD.

24. The isolated Ab of embodiment 23, wherein the other region is located between amino acids 95 and 105 of SEQ ID NO: 2.

25. The isolated Ab according to any one of embodiments 18-24, wherein binding is determined using hydrogen-deuterium exchange mass spectrometry (HDX-MS).

26. The isolated Ab according to any one of embodiments 1-25, wherein the Ab inhibits the binding of hVISTA to a cell to which hVISTA would otherwise bind.

27. The isolated Ab according to any one of embodiments 1-26, wherein the Ab induces or enhances an immune response in a tumor model.

28. The isolated Ab of embodiment 27, wherein the Ab induces or enhances T cell activity in a tumor model.

29. The isolated Ab according to any one of embodiments 1-28, wherein the Ab inhibits tumor growth in a tumor model.

30. The isolated Ab according to any one of embodiments 1-29, wherein the Ab inhibits the binding of hVISTA to T cells.

31. The Ab of Embodiment 30, wherein the Ab more potently inhibits hVISTA binding to T cells at pH 6.5 or less than at pH 7.0 or higher, for example, wherein the antibody more potently inhibits hVISTA binding to T cells at pH 6.5 than pH 7.0.

32. A composition comprising an isolated Ab according to any of embodiments 1-31 and a pharmaceutically acceptable carrier.

33. A method of treating a subject having cancer, comprising administering to the subject a composition according to embodiment 32.

34. A method for identifying an Ab that binds to hVISTA-ECD at a pH of 6.5 or less with a K _{D of} 10 ^-7 M or less, comprising contacting the test Ab or a plurality of test Abs with a polypeptide comprising hVISTA-ECD or a fragment thereof comprising IgV hVISTA-ECD domain or comprising amino acids 20-95, 20-70 or 35-70 SEQ ID NO: 2, at a pH of 6.5 or less, and selecting a test Ab or Ab that binds to a polypeptide with a K _{D of} 10 ^-7 M or less.

35. A method for identifying an Ab that binds to hVISTA-ECD at pH 6.5 or less with a koff of 10 ^-3 sec ^-1 or less, comprising contacting the test Ab or a plurality of test Abs with a polypeptide comprising the hVISTA-ECD or a fragment thereof comprising IgV domain hVISTA-ECD or comprising amino acids 20-95, 20-70 or 35-70 SEQ ID NO: 2, at a pH of 6.5 or less, and selecting a test Ab or Ab that binds to the polypeptide with a koff of 10 ^-3 sec ^-1 or less.

36. A method for identifying an Ab that specifically binds to hVISTA-ECD at pH 6.5 with similar affinity at pH 7.0, comprising:

a) contact of a test Ab or a plurality of test Abs at pH 6.5 with a polypeptide comprising hVISTA-ECD or a fragment thereof comprising the IgV domain of hVISTA-ECD or comprising amino acids 20-95, 20-70 or 35-70 SEQ ID NO: 2 ;

b) contacting the test Ab or a plurality of test Abs at pH 7.0 with polypeptide (a); And

c) selecting a test Ab if it binds to a polypeptide with a K _{D of} 10 ^-7 M or less at pH 6.5 and at pH 7.0.

37. A method for identifying an Ab that binds with higher affinity to hVISTA-ECD at pH 6.5 than at pH 7.0, comprising:

a) contacting the test Ab or multiple test Abs at pH 6.5 with a polypeptide comprising hVISTA-ECD or a fragment thereof comprising the IgV domain of hVISTA-ECD or comprising amino acids 20-95, 20-70 or 35-70 SEQ ID NO: 2 ;

b) contacting the test Ab or multiple test Abs at pH 7.0 with polypeptide (a); And

c) selecting a test Ab if it binds to a polypeptide with a Kd that is at least 2 times lower at pH 6.5 than at pH 7.0.

38. A method for identifying an Ab that specifically binds to hVISTA-ECD for use in the treatment of cancer, comprising:

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a) identifying an Ab that specifically binds to hVISTA-ECD at a pH of 6.5 or less, for example, according to the methods of embodiments 32-35; And

b) selecting Ab(s) that induce or enhance an immune response in a tumor model or inhibit tumor growth at a pH of 6.5 or less.

39. The method according to embodiment 38, wherein step (b) includes measuring T cell activity.

40. The method according to embodiment 38 or 39, further comprising measuring the antitumor effect of Ab.

41. A method for increasing the antitumor efficacy of an Ab that binds to hVISTA-ECD, including:

a) producing an Ab that binds to hVISTA-ECD at pH 6.5 or less with an affinity that is less than the desired value, for example with a KD of 10 ^-7 M or more, for example 10 ^-6 M, 10 ^-5 M or more, and/or koff 10 ^-2 sec ^-1 or more;

b) replacing 1-5 amino acid residues in the heavy or light chain of the Ab with another amino acid residue, where 1-5 amino acid residues are contact residues with hVISTA-ECD;

c) determining whether the Ab obtained in (b) has a higher affinity for hVISTA-ECD at pH 6.5 or less compared to the Ab from (a); And

d) repeating steps (a)-(c) in a number of rounds sufficient to obtain an Ab that binds to hVISTA-ECD at a pH of 6.5 or less with a KD of 10 ^-7 M or less.

42. A method for increasing the antitumor efficacy of an Ab that binds to hVISTA-ECD, including:

a) producing an Ab that binds to hVISTA-ECD at pH 6.5 or less with an affinity that is less than the desired value, for example with a KD of 10 ^-7 M or more, for example 10 ^-6 M, 10 ^-5 M or more and/or koff 10 ^-2 sec ^-1 or more;

b) obtaining a library of Ab variants (a), where each variant includes the replacement of 1-5 amino acid residues in the heavy or light chain of the Ab with another amino acid residue, where 1-5 amino acid residues are residues of contact with hVISTA-ECD;

c) selecting antibodies from the library of variants (b) that bind to hVISTA-ECD at a pH of 6.5 or less with a KD of 10 ^-7 M or less; and optionally

d) Testing the antitumor efficacy of antibodies (c) in a tumor model.

43. A method of improving the pharmacokinetics of an antibody that binds to human VISTA ECD, comprising increasing the ability of the antibody to bind to human VISTA under acidic conditions, for example, equal to or lower than pH 6.5.

44. A method for selecting an antibody that binds to human VISTA and has an extended half-life (good pharmacokinetic properties), wherein the method comprises selecting an antibody that binds to human VISTA under acidic conditions, for example, equal to or lower than pH 6.5.

Other exemplary embodiments are presented in the claims below.

Examples

The examples discussed below are intended solely to illustrate the invention and should in no way be construed as limiting the invention. The examples are not intended to demonstrate that the experiments below are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numerical values used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1: The extracellular domain of VISTA is exceptionally rich in histidines.

This example shows that the extracellular domain of VISTA is exceptionally rich in histidine residues, that these histidine residues are evolutionarily conserved, and that they can facilitate receptor-ligand interactions involving VISTA.

Amino acid sequences of extracellular domains (ECDs) of immunoglobulin domain-containing proteins were obtained from the uniprot and swiss-prot databases and analyzed for histidine content. In fig. Figure 1A presents the results of this analysis in graphical form. For each protein, the frequency of histidine residues as a percentage of all extracellular domain amino acid residues is plotted on the y-axis, and the total number of extracellular domain amino acid residues is plotted on the x-axis. The diameter of each data point corresponds to the total number of histidine residues in the extracellular domain of each protein. VISTA (marked) contains an exceptionally high frequency of histidine residues in the extracellular domain.

The evolutionary conservation of histidine residues in VISTA was then assessed. In fig. 1B shows the human, cynomolgus and mouse VISTA reference amino acid sequences that were aligned, excluding signal peptides (Sig), transmembrane domains (TMDs), and intracellular domains. Histidine residues, conserved for all three species, are isolated

- 98 046477 we are in bold and underlined. Histidine residues conserved in human and cynomolgus VISTA are shown in bold without underlining. Many histidine residues in the extracellular domain of VISTA are evolutionarily conserved, suggesting an important biological role for the high histidine content in VISTA.

A three-dimensional model of the IgV domain of hVISTA was created based on sequence homology analysis for the available established structures in the PDB database. The model shown in FIG. 1C indicates that many of the histidines in ECD VISTA are exposed on the surface of the molecule, where they may play some role in ligand binding as well as antibody recognition. Histidine residues are shown as balls and rods.

Example 2 Histidine protonation may regulate VISTA receptor ligand binding and immunosuppressive activity in tumors and other acidic microenvironments.

This example describes histidine protonation in response to physiologically relevant acidic pH, as well as a model in which VISTA extracellular domain histidines confer counterreceptor or ligand selectivity at acidic pH rather than physiological pH.

In fig. Figure 2A shows the equilibrium between the absence and presence of protonation of the pyrrol ammonium group (NH) on the histidine residue. The pKa value of histidine in solution is 6.5, indicating that histidine residues will be protonated at pH 6.5 and below and thus be more likely to be positively charged than at higher pH. Increasing the positive charge on the surface of VISTA ECD as a result of protonation may affect receptor or ligand binding and the structure and/or function of VISTA. Thus, changes in pH may also modify the epitopes to which antibodies bind and/or may lead to changes in antibody affinity.

In fig. 2B shows a model in which VISTA selectively interacts with PSGL-1 or other counterreceptors and ligands (VISTA-R) at acidic pH. At physiological pH, such as in blood, the histidine residues on the VISTA ECD are not expected to be protonated. As a result, binding of VISTA to PSGL-1 or other counterreceptors and ligands is negligible at physiological pH. In contrast, in areas with acidic extracellular pH, such as tumor microenvironments or sites of inflammation, acidic pH may partially or completely regulate histidine protonation in the ECD of VISTA and thus mediate the interaction of VISTA with PSGL-1 or other counterreceptors and ligands. Thus, antibodies that bind strongly to VISTA-ECD proteins in acidic pH ranges may more effectively inhibit VISTA activity in tumors.

Example 3 VISTA is expressed by myelomonocytic cells in tumors.

This Example shows that VISTA in tumors is often expressed by myelomonocytic cells, including macrophages, dendritic cells and granulocytes.

Surgically resected non-small cell lung carcinoma, renal clear cell carcinoma, melanoma, colorectal carcinoma, and other tumor specimens were washed in ice-cold PBS, cut into approximately 15 mm ³ pieces, and suspended in ice-cold RPMI-1640 (Fisher Scientific part number 11875093) supplemented 2% heat-inactivated FBS and 2 mM EDTA (Fisher Scientific 15575020). Each sample was transferred to a wide-gap glass Dounce homogenizer (Tenbroeck Tissue Grinders) and crushed until tissue fragments were visually separated from each other. The suspensions were filtered through a nylon filter with 70 μm mesh and centrifuged. Supernatants were discarded and cell pellets were resuspended in PBS at room temperature supplemented with 0.1% bovine serum albumin and 250 mg/ml filter-sterilized DNase 1 (class II, from bovine pancreas, Roche catalog number 10104159001) for 3 min at room temperature. Cells were then washed with ice-cold RPMI with supplements and resuspended in ice-cold PBS. A dye was added to assess cell viability and the cells were incubated on ice in the dark. After 20 min, nonspecific antibody staining was blocked by the addition of 4% normal rat serum, 4% normal mouse serum, 20% human AB plasma serum, and a 1:125 dilution of Human TruStain FcX™ (Biolegend catalog number 422302). Cells were stained with fluorophore-conjugated antibodies against HLA-DR (BD Biosciences part number 564040), CD8 (Fisher Scientific part number 46-0087-42), CD14 (Biolegend part number 325620), CD45 (Biolegend part number 304017) , CD4 (BD Biosciences part number 563875), CD11c (BD Biosciences part number 744439), CD15 (BD Biosciences part number 563142), PD-1 (BD Biosciences part number 565299), CD3 (BD Biosciences part number 565299) 565515), CD56 (Fisher Scientific part number 610567-42), CD19 (BD Biosciences part number 564977) and VISTA (VISTA 3 antibody conjugated to AlexaFluor™ 647, Fisher Scientific part number A20186) suspended in Brilliant Stain Buffer (BD Biosciences part number 562794) for 30 minutes on ice in the dark. Stained cells were washed with ice-cold PBS, fixed (Fisher Scientific part number 00-552300), and recorded on a flow cytometer. Data were analyzed using the program

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FlowJo™ (BD Biosciences). As shown in FIG. 3, cell surface expression of VISTA was highest on macrophages and granulocytes, moderate on dendritic cells, and low on T cells, NK cells, and B cells.

Example 4: Binding of VISTA to cells demonstrates selectivity with respect to acidic pH.

This example demonstrates that multimerized human VISTA ECD binds more efficiently to stimulated human CD4+ T cells and human peripheral blood mononuclear cells at acidic pH than at neutral or physiological pH, and that such binding can be blocked by a blocking anti-human VISTA antibody. Binding of the acidic pH-selective, dimerized murine ECD VISTA to murine splenocytes has also been demonstrated.

Human CD4+ T cells were enriched from the blood of healthy donors using RosetteSep™ (Stemcell part number 15062) and stimulated in vitro for approximately four days using Human T-Activator CD3/CD28 Dynabeads™ (Fisher Scientific part number 111.32D) and recombinant human IL-2 (Peprotech part number 200-02) in RPMI-1640 supplemented with 10% heat-inactivated FBS, Glutamax™ (Fisher Scientific part number 35050061), nonessential amino acids (Fisher Scientific 11140050), sodium pyruvate (part number Fisher Scientific catalog 11360070) and 2-mercaptoethanol (Fisher Scientific 21985023). Activated CD4+ T cells were stained with monobiotinylated hVISTA ECD molecules (Phe 33-Ala 194 (reg. no. AAH20568)-polyhistidine; AcroBiosystems, Inc. B75-H82F3) loaded at a molar ratio of 28:1 onto phycoerythrin (PE)-conjugated streptavidin dextramers ( part number DX01-PE) diluted in Hank's Buffered Saline Solution (HBSS, Fisher Scientific part number 14025134) acidified to various pH values using mM MES (Sigma, 1317-100 ml) for 30 min at room temperature. As a control, activated CD4+ T cells were stained with PE-conjugated streptavidin dextramers that were not loaded with hVISTA. Stained cells were washed with HBSS+MES and recorded on a flow cytometer. Data were analyzed using FlowJo™ software (BD Biosciences). The results presented in Fig. 4A show that hVISTA did not bind CD4+ T cells better than control at pH >6.5. In contrast, hVISTA exhibited increasingly strong binding to CD4+ T cells at pH <6.5. On the left, from darker to lighter gray, the shaded histograms show binding at pH 7.0, 6.5, 6.4, 6.3, 6.1, and 6.0. Some bar graphs indicate the corresponding pH values. Binding of control multimers without VISTA at pH 6.0 is shown as an open histogram. On the right, the graph shows the mean fluorescence intensity (MFI) of PE CD4+ T cells stained with hVISTA-loaded dextramers (circles) or unloaded dextramers (triangles) at different pH.

Peripheral blood mononuclear cells (PBMCs) were enriched from the blood of healthy donors by Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Life Sciences catalog number 17144003) and stained with hVISTA-loaded dextramers (also called multimers) and fluorophore-conjugated ones diluted in HBSS buffers. +MES as described above. In fig. 4B shows shaded histograms that show, from darker to lighter gray, binding at pH 6.0 to CD19+ B cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD14+ monocytes. Open histograms with solid and dotted boundaries show binding at pH 7.4 to total PBMCs of lymphocytes and monocytes, respectively. In fig. 4F and 4G show that VISTA binds to both monocytes and neutrophils, with stronger binding at pH 6.0 than at pH 7.4. The results show that hVISTA can bind many leukocytes at acidic pH, but not significantly at physiological pH.

Activated human CD4+ T cells were stained with hVISTA multimers at pH 6 in the presence of a titrated anti-human VISTA antibody or an isotype-matched nonspecific VISTA antibody. The results, presented as a graph in Fig. 4C shows the MFI of the VISTA multimer relative to antibody concentration. An anti-hVISTA antibody (anti-VISTA 3; squares), but not a VISTA-specific control antibody (circles), blocked hVISTA binding to activated CD4+ T cells in a concentration-dependent manner. PE MFI values of CD4+ T cells that were not stained with hVISTA-loaded multimers are included as a control (one triangle).

In fig. 4D shows representative 2D flow cytometry plots for VISTA multimer staining at pH 6.0 with heparan sulfate mutant Chinese hamster ovary (CHO) cells (line pGSD-677, American Type Culture Collection) that were transfected to express full-length human PSGL-1 ( SEQ ID NO: 3; nucleic acid NM_003006.4). Staining was performed in the presence or absence of titrated blocking anti-VISTA antibody (mAb 3). Cells left unstained with VISTA multimers are shown as controls. Anti-PSGL-1 antibody staining (BD Biosciences catalog no. 562758) is plotted on the y-axis, and VISTA multimer staining is plotted on the x-axis.

Splenocytes were collected from C57BL6/J mice (Jackson Laboratory catalog number 000664) and stained

- 100 046477 added chimeric mVISTA ECD/Human IgG Fc fusion proteins (crystallizing fragment) followed by fluorophore-conjugated anti-human Fc IgG secondary antibodies (Jackson Immunoresearch catalog number 109-065-098) at pH 6.0 or 7, 4. The results are presented as a histogram in Fig. 4E show that mVISTA binds murine splenocytes more effectively at pH 6.0 than at physiological pH (approximately pH 7.4). From darker gray to lighter shaded histograms indicate binding at pH 6.0 to CD8+ T cells, CD11b+ myeloid cells, and CD4+ T cells. The open histogram shows binding at pH 7.4 to total splenocytes.

Example 5 VISTA mediates cell:cell adhesion and immune suppression selectively at acidic pH.

This Example demonstrates that VISTA mediates cell:cell adhesion and more potently suppresses T cell activation at acidic pH than at neutral or physiological pH.

A flow cytometry-based cell/cell conjugate assay compatible with acidic pH was developed. 293T cells (immortalized human embryonic kidney cell line, ATCC catalog number CRL-3216) ectopically expressing full-length human VISTA or vector were labeled with CFSE (carboxyfluorescein succinimidyl ester; Fisher Scientific catalog number C34554). CHO cells were labeled with CellTrace™ Far Red (Fisher Scientific catalog number C34564). Vector- or VISTA-containing 293T cells were then mixed in a 1:1 ratio with CHO cells in pH 7.0 or pH 6.0 buffers and incubated for 1 hour at room temperature. The formation of cell/cell conjugates of CHO and 293T was assessed using flow cytometry. The results shown in FIG. 5A-B demonstrate that VISTA-expressing 293T cells preferentially adhered to CHO cells at acidic pH, and that inclusion of a blocking anti-VISTA antibody (VISTA mAb 3; white bars) inhibited VISTA-mediated cell/cell adhesion.

An acid pH compatible T cell suppression assay was developed. Jurkat cells (immortalized human T cell line, ATCC catalog no. TIB-152), expressing a luciferase reporter under the control of the NFkB promoter, were co-cultured in HBSS+MES buffers with different pH with 293T cells (immortalized human embryonic kidney cell line, no. ATCC catalog CRL-3216), ectopically expressing full-length human VISTA and a single-chain variable fragment clone of an agonistic anti-human T-cell receptor antibody OKT3 at a Jurkat:293T cell ratio of 10:1. A blocking anti-VISTA antibody (VISTA mAb 3) or an isotype-matched non-VISTA-specific control antibody was added to the cocultures at a concentration of 10 μg/ml. After incubation, Jurkat T cell activation was quantified by measuring luciferase activity (1 s interval, Promega catalog number G7940). The results are shown in Fig. 5C-D. In fig. Figure 5C shows a curve of luciferase units in Jurkat cells treated with anti-VISTA antibody (squares) or control antibody (circles) at different pH. In fig. 5D shows a plot of the luciferase signal in cocultures treated with anti-VISTA antibody divided by the luciferase signal in cocultures treated with control antibody at each pH tested. The results indicate that VISTA-mediated T cell suppression is most effective at acidic pH.

Example 6: VISTA migrates through intracellular recycling endosomes.

This Example demonstrates that VISTA can be found in intracellular endosomes, especially Rab11+ recycling endosomes, and can recycle to and from the cell surface through endosome migration. The strength with which anti-VISTA antibody binds VISTA at acidic pH influences its ability to remain bound to VISTA during endosome migration.

Monocytes were isolated from PBMCs using magnetic field-activated cell sorting. Monocytes and 293T cells were then fixed in 4% paraformaldehyde and intracellularly stained for Rab5, Rab7, or Rab11 and anti-VISTA antibody or control antibody. A control antibody (cAb) that is not a non-VISTA binding antibody of the same isotype as the anti-VISTA antibody does not exhibit detectable binding to monocytes or 293T cells expressing human VISTA. Antibodies against VISTA and control antibodies were directly labeled with Alexa488. Rab antibodies were detected using secondary Alexa594-conjugated anti-rabbit Ig antibodies. Hoescht 33342 staining was performed to identify cell nuclei. Images were obtained using a spinning disk confocal microscope. In fig. 6A shows the colocalization of VISTA, Rab5 (an early endosome marker), Rab7 (a late endosome marker), and Rab11 (a recycling endosome marker) in 293T cells expressing human VISTA. In fig. 6B shows the colocalization of VISTA and Rab11 in human monocytes. Intracellular VISTA colocalizes with Rab11+ recycling endosomes.

To evaluate the ability of VISTA to recycle through endosomes, an endolysosomal-dependent killing assay was performed with an antibody-drug conjugate with three anti-hVISTA antibodies (VISTA mAbs 1, 2, and 3) with different VISTA binding properties at physiological and acidic pH. First, SPR analysis was performed to compare the hVISTA binding profiles of all three ans.

- 101 046477 antibody to VISTA at pH 7.4, 6.7 and 6.0. Antibodies to VISTA were bound to a Biacore® T100 (GE Healthcare) CM5 biosensor containing immobilized protein A, then 100 nM hVISTA-ECD (amino acids 32-193 SEQ ID NO: 1 with a 7xHis tail, i.e. AFKVATPYSL YVCPEGQNVT LTCRLLGPVD KGHDVTFYKT WYRSSRGEVQ TC SERRPIRN LTFQDLHLHH GGHQAANTSH SEQ ID NO: 325) were run in PBST running buffer at the indicated pH, at 37°C. Sensograms with subtraction of reference values were normalized to the recorded 'binding' point and plotted. Anti-VISTA 3, mAb 3 (Fig. 6C, top) showed the greatest degree of impairment of VISTA binding at acidic pH, followed by anti-VISTA 2, mAb 2 (Fig. 6C, middle), which only moderately interfered with binding. The anti-VISTA 1 antibody, mAb 1, maintained strong VISTA binding under acidic and physiological pH conditions (Fig. 6C, bottom).

The endolysosomal-dependent killing assay of the antibody-drug conjugate was performed as follows. AML3 cells (immortalized human monocyte cell line, ATCC CRL-9589), which endogenously express human VISTA, were cultured with titrated anti-VISTA antibodies or non-VISTA-specific control antibodies and secondary anti-human IgG antibodies that were conjugated to a cathepsin B-sensitive linker and cytotoxic payload tubulisin. Because cathepsin B is predominantly active in late endosomes and lysosomes, anti-VISTA antibodies that recycle VISTA through early endosomes and recycling endosomes will exhibit low levels of linker cleavage and, as a result, low levels of cytotoxic payload release and cell death. Anti-VISTA antibodies that dissociate from VISTA in acidic endosomes and are sorted into late endosomes and lysosomes will undergo higher levels of linker cleavage. Cell viability was measured using Cell Titer Glo® (Promega part number G7573) after five days of culture. In fig. 6D shows the results of this assay with AML3 (Cell Titer Glo) viability plotted on the y-axis and primary antibody concentrations on the x-axis. Calculated EC50 values for primary antibodies: anti-VISTA 1, inverted triangles, 0.485 μg/ml; anti-VISTA 2 antibody, circles, 0.092 μg/ml; anti-VISTA 3 antibody, squares, 0.006 μg/ml; Control, triangles, 1.085 μg/ml. Antibody activity was inversely related to anti-VISTA antibody binding at acidic pH.

To confirm that binding at acidic pH was responsible for the differences in potency, the anti-VISTA 3 antibody was affinity optimized so that its ability to bind VISTA at acidic pH was improved. In fig. 6E shows a SPR assay comparing the hVISTA antibody binding profiles of anti-VISTA 3 with this variant, anti-VISTA 3c, using the assay conditions described for FIG. 6C. The anti-VISTA 3 antibody again demonstrated impaired VISTA binding at acidic pH, whereas the anti-VISTA 3c antibody variant showed comparable VISTA binding at acidic and physiological pH. In fig. 6F shows the activity of anti-VISTA 3c antibody (diamonds) in the killing assay described for FIG. 6D. The acidic pH optimized version of the anti-VISTA antibody 3 showed 31-fold lower activity than the parent antibody, indicating that disruption of anti-VISTA antibody binding at acidic pH results in loss of antibody binding during VISTA recycling.

Based on these results, a recycling model is proposed in which VISTA is recycled to and from the cell surface through early endosomes and recycling endosomes. This model is shown in Fig. 6G. Antibodies against VISTA can recycle with VISTA through these endosomes, maintaining target binding. However, anti-VISTA antibodies with impaired VISTA binding at acidic pH, especially those with rapid dissociation at acidic pH, can become dissociated from VISTA during recycling and become trapped or degraded within cells, resulting in poor target engagement and continued depletion of circulating antibodies. In contrast, antibodies that bind and remain bound to VISTA at acidic pH may maintain higher levels of target interaction, especially in acidic microenvironments such as tumors, and exhibit longer average retention times in vivo.

Example 7: Superiority of anti-VISTA antibodies that do not bind at physiological pH.

We showed that VISTA is an acidic pH selective immunoreceptor, demonstrating the importance and utility of targeting VISTA with antibodies that bind well at acidic pH. Additionally, antibodies that bind little or no to VISTA at physiological pH are advantageous for several reasons. First, due to the relatively high expression of VISTA on circulating myelomonocytic cells, particularly monocytes and neutrophils, antibodies that bind VISTA at physiological pH are subject to high levels of target-mediated drug distribution (TMDD) in the blood. This effect is complicated by the propensity of VISTA to be recycled through intracellular endosomes, leading to the internalization and degradation of anti-VISTA antibodies. This secondary effect is particularly problematic for antibodies that have impaired binding

- 102 046477 at acidic pH, as can be observed in the case of antibodies that bind the histidine-rich VISTA ligand interface. Both effects result in a decrease in the amount of anti-VISTA antibody in the bloodstream, reducing the amount of antibody that can reach the tumor and, therefore, the expected biological activity of the antibody. Second, antibodies that bind to VISTA at physiological pH and have effector functions such as inducing antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or serve to deliver an immunomodulatory payload will expose circulating myelomonocytic cells to these effector functions. functions, potentially leading to undesirable effects such as depletion or activation of circulating neutrophils. Thus, the inventors found that antibodies that bind to huVISTA at acidic pH, but bind little at physiological pH, have the dual advantage of (1) better targeting relevant sites such as tumors, and (2) having reduced toxicity in cases of antibodies with effector functions such as ADCC, ADCP or delivery of immunomodulatory load. In addition, since VISTA itself is an acidic pH-selective immunoreceptor, blockade of the VISTA ligand interface at physiological pH is likely not required to modulate VISTA receptor-ligand activity. Thus, antibodies that bind to huVISTA at acidic pH but bind negligibly at physiological pH were generated as described below.

Example 8 Isolation of anti-VISTA antibodies that selectively bind to human VISTA at acidic pH compared to physiological pH.

This example describes the generation of antibodies that selectively bind to human VISTA at low (acidic) pH compared to neutral or physiological pH.

A library of antigen-binding fragments of anti-VISTA antibodies was constructed and screened as follows. Antibody libraries were generated using genetic material isolated from HuMab mice immunized with full-length human VISTA (hVISTA). These antibodies were in scFv format and were selected for binding against full-length hVISTA at low pH (pH 6.0) via mRNA display (Xu L et al. (2002) Chemistry & Biology 9: 933; Roberts RW and JW Szostak (1997) Proc Natl Acad Sci USA 94:12297 Kurz et al (2000) Nucleic Acids Res 28(18):E83. The selection results were analyzed by next generation sequencing (NGS) and library members that showed enrichment for binding to VISTA at low pH were identified, reformatted as IgG 1.3 (non-effector IgG1 constant region, consisting of IgG1 Fc with amino acid mutations L234A, L235E and G237A ) and screened for binding to VISTA using SPR.

Surface plasmon resonance (SPR) analysis was performed to measure association rates (defined as k _a or k _on , units 1/Ms), dissociation rates (defined as kd or koff , units s ^-1 ) and affinity constants (defined as KD , units M) for antibodies to VISTA at acidic and physiological pH values using the Biacore® T200 instrument (GE Healthcare). Protein A (Fisher Scientific catalog 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare), aimed at immobilization density of Protein A 6000 RU per flow cell. PNR experiments were performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4 and 6.0. Antibodies were diluted to 20 nM in PBST pH 7.4 and captured in the active flow cells of the biosensor at a flow rate of 5 μL/min for 50 s. A series of concentrations of 50-0.2 nM monovalent hVISTA-ECD (SEQ ID NO: 325) were prepared in pH 7.4 and 6.0 running buffers and passed over bound antibodies at 40 μl/min to measure association and dissociation. Two injections of 10 mM glycine at pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. Rate constants k _a (kon) and kd (koff) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 Evaluation Software v.2.0. For each anti-VISTA antibody, the ratio koff at pH 6/koff at pH 7.4 was calculated to identify antibodies exhibiting a low dissociation rate at acidic pH and a high dissociation rate at physiological pH.

Six antibodies reformatted to IgG1.3 antibodies showed almost equivalent affinities at pH 6 and at pH 7.4. In particular, two antibodies had a lower dissociation rate at pH 6.0 than at pH 7.4 (ie, higher koff at pH 7.4 than at pH 6.0). The variable regions of these two huVISTA antibodies were designated P1-061015 and P1-061029, and antibodies incorporating these variable regions and formatted into IgG1.3 antibodies were designated P1-061015.IgG1.3 and P1061029.IgG1.3, respectively. The koff rates of P1-061015.IgGl.3 and P1-061029.IgG1.3 are presented in Table. 1.

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Table 1 koff of selected antibodies at pH 6.0 and pH 7.0

Antibody name _kOff at pH 6 (s' ¹ ) koff at pH 7 (s' ¹ ) koff pH 6/рН 7 Pl-061015.IgG1.3 1.4χ10' ³ 2.3x10' ³ 0.6 Pl-061029.IgG1.3 4.8χ10' ³ 9.1χ10' ³ 0.5

The sequences of CDR1, CDR2 and CDR3 of the heavy and light chains of P1-061015 and P1-061029 are presented in table. 2 below, and are also shown in the sequence table following the Examples section of this specification.

table 2

Amino acid sequences of anti-huVISTA antibodies selectively binding to huVISTA at pH 6.0 compared to pH 7.4

Pl ID VH gene VHCDR1 VHCDR2 VHCDR3 Pl-061015.IgG1.3 3-33 GFTFSSYAMH (SEQ ID NO: 95 residues 26-35) IIWYDGSNKYYAD SVKG (SEQ ID NO: 95 residues 50-66) DSGFYSSYYFDY (SEQ ID NO: 95 residues 99-110) Pl-061029.IgG1.3 3-09 GFTLDDYAMH (SEQ ID NO: 67 residues 26-35) GINWNSANIGYAD SVKG (SEQ ID NO: 67 residues 50-66) VPGYSGGWIDAF D.V. (SEQ ID NO: 67 residues 99-112) VL gene VLCDR1 VLCDR2 VLCDR3 Pl-061015.IgG1.3 L6 RASQSVSSSYLA (SEQ ID NO: 96 residues 24-35) DASNRAT (SEQ ID NO: 96 residues 51-57) QQYNSYPYT (SEQ ID NO: 96 residues 90-98) Pl-061029.IgG1.3 A27 RASQSVSSSYLA (SEQ ID NO: 68 residues 24-35) GASSRAT (SEQ ID NO: 68 residues 51-57) QQYGSSPFT (SEQ ID NO: 68 residues 90-98)

Example 9. Further construction of antibodies P1-061015 and P1-061029 against VISTA in order to create antibodies selective for acidic pH.

This Example describes further engineering of the P1-061015 and P1061029 variable regions identified in Example 2 to obtain anti-huVISTA variable regions that have a higher binding k _off ratio at pH 6.0 compared to pH 7.4.

Two libraries were created by introducing specific mutations into the VH CDR regions P1061015 and P1-061029, respectively. The libraries only allowed amino acid substitutions that were most likely to improve binding at low pH, i.e., aspartate, glutamate, and histidine. The library also allowed single and double amino acid substitutions in each CDR and recombination between CDR regions (maximum of 6 amino acid substitutions per chain). In fig. 7A shows mutations that were introduced into the heavy chain CDR3 amino acid sequences of P1061029 to produce library P1-061029. The figure shows that certain sequences were excluded to avoid introducing biases (eg, DG).

The '029 and '015 libraries were screened using multiple rounds of binding to full-length hVISTA at pH 6.0 via yeast surface display. Further rounds of selection were performed by switching between positive (binding to huVISTA at pH 6.0) and negative (binding to huVISTA at pH 7.4) (shown in Fig. 7B) selection when library members that did not bind to VISTA at pH 7.4, were collected in negative selection rounds. The selection results were studied using NGS. Members of the '029 library that bound to huVISTA at pH 6.0 after round 9 of selection were screened for binding to human VISTA at pH 6.0 and pH 7.4 by flow cytometry. In fig. Figure 7C shows typical 2D flow cytometry plots 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 are shown. The results demonstrated very strong pH 6-selective binding to human VISTA, especially at 20 nM.

Additional '029 daughter clones were isolated from the '029 library using a different method. Some clones were the same as the clones identified by the first method, and nine additional clones were isolated.

clones isolated from the '029 library selected for further analysis were reformatted as IgG1.3 antibodies. The amino acid differences in the heavy chain CDR regions of these clones compared to the VH '029 CDR regions are shown in Table. 5.

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Table 5

Amino acid sequences of CDR1, CDR2 and CDR3 VH (separated by underscore) of antibodies derived from the parent antibody '029

NAME CDR1 CDR2 CDR3 SEQ ID NO (gender 26-35) (gender 50-66) (gender 99-110)

Р1-061029 GFTLDDYAMH GINWNSANIGYADSVKG VPGYSGGWIDAFDV 67

Р1-068757 ----HER---_---- - HER---------_------- ----E-D 71 Р1-068759 ----HER- —_—D- — E----------_- ----E-D 87 Р1-068761 ----HER- —---- - HER---------_- ----n- ----E— 51 Р1-068763 ----E--- —_—D- — E----------_- ----n- ----E — 91 Р1-068765 ---DE--- _ - HER---------_- — ----E-D 63 Р1-068767 ----E--- —_—D- — E----------_- ----E-D 55 Р1-068769 ----HER- —DH---------_- ----E-D 83 Р1-068771 ----HER- _ - ^NOT ---------_ ----E-D 75 Р1-068773 ----E--- —_—D- — D----------_- ----E-D 59 Р1-068775 ----HER- —_—D- - HER---------_- ----n- ----E-D 79 Р1-069059 ----E--- —DH---------- ----E-D eleven

P1-069061 ----E-----_-------E---------_-----------E-D 15

P1-069063 ----E----------_-------E---------_-----------D-E 19

P1-069065 ----EE----DD----------------------- 23

P1-069067 ----------_------EE---------_-----------D-E27

Р1-069069 ----------_------EE---------_-----------D—31

P1-069071 ----E-E---_-------D---------_-----E 35

P1-069073 ----E-----—D---D---------------------E-D 39

P1-069075 ----E-----_----D—E---------_-----N-----E—43

P1-069077 ----EE----DE--------- 47

The binding of multiple preparations of each of the '029 daughter clones and the parent '029 antibodies formatted into IgG1.3 antibodies to human VISTA at pH 6.0 and 7.4 was measured using surface plasmon resonance (SPR). SPR analysis was performed to measure the binding affinity of koff and KD antibodies to VISTA at acidic and neutral pH using a Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare) aimed at immobilization density protein A 2000 RU per flow cell. SPR experiments were performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween® 20) at pH 7.4 and 6.0. Antibodies were diluted to 25 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μL/min for 60 s. A series of concentrations of 50-5 nM monovalent hVISTA-ECD (SEQ ID NO: 325) were prepared in pH 7.4 and 6.0 running buffers and passed over bound antibodies at 40 μl/min to measure association and dissociation. Two injections of 10 mM glycine at pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. Rate constants k _a (ko _n ) and kd (k _off ) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 Evaluation Software v.2.0. The affinity constant, K _D , was calculated as the ratio of the rate constants k _off /k _on for each anti-VISTA antibody.

The maximum (or magnitude) human VISTA binding response was determined as the response at the recorded 'binding' point minus the reference value at the end of injection of 50 nM VISTA for each antibody and expressed in response units (RU). The maximum binding response of human VISTA (RU) with each antibody is presented in FIG. 7D. The average binding response (two to four replicate antibodies) is plotted and the error bars represent the standard deviation. The results show that the selected daughter clones '029 bind to hVISTA at pH 6.0 but do not bind at pH 7.4 (all open circles representing binding at pH 7.4 are located at the bottom of the graph, with the exception of the parent clone ' 029).

The k _off rates at pH 6.0 for '029 and its daughter clones were determined by SPR using the method described above and are presented in FIG. 7E. The dotted line in the figure represents the koff rate _of '029, and the clones to the left of the dotted line have a lower _koff rate at pH 6.0 compared to the parent antibody '029, while the clones to the right have a higher _koff rate

- 105 046477 at pH 6.0 compared to the parent antibody '029.

Representative SPR sensorgrams of hVISTA binding to antibodies '029, '761, and '767 at neutral and acidic pH are shown in FIG. 7F. Sensograms for 50 and 5 nM huVISTA with subtraction of reference values are presented in the form of curves. At neutral pH, a VISTA binding signal of <10 RU was observed for '761 and '767, so to properly measure and compare koff and KD for '761 and '767 with '029, SPR kinetics analysis was required using µM concentrations of VISTA at physiological pH.

For this analysis, '029, '761, and '767 were reformatted to the hIgG1f isotype and expressed in both standard hIgG1f and afucosylated hIgG1f format for comparison with hIgG1.3f Fc. SPR kinetics analysis was performed to measure binding affinities k _off and K _D for anti-VISTA antibodies at acidic and physiological pH using a Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare) aimed at immobilization density protein A 2000 RU per flow cell. SPR experiments were performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4 and 6.0. Antibodies were diluted to 25 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μL/min for 45 s. Concentration series of 1600-0.78 nM (pH 7.4) and 100-0.78 nM (pH 6.0) monovalent hVISTA-ECD (SEQ ID NO: 325) were prepared in running buffer and passed over bound antibodies at speed 40 µl/min to measure association and dissociation. Two injections of 10 mM glycine at pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. Rate constants k _a (ko _n ) and kd (koff) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 Evaluation Software v.2.0. The affinity constant, K _D , was calculated as the ratio of the koff/kon rate constants for each anti-VISTA antibody. The koff and KD ratios at pH 7.4/pH 6.0 were calculated to compare dissociation rates and affinity improvements at acidic pH compared to physiological pH. Although binding rate constants at neutral pH could not previously be determined for '761 and '767 using 50 nM hVISTA (Figures 7D and 7F), increasing the concentration range of VISTA at neutral pH to 1.6 μM resulted in binding responses (>10 RU ) for these clones, which follow a 1:1 binding model. Kinetics data for these acid-selective antibodies to VISTA are shown in Table. 6. The parent antibody '029 shows equivalent koff at both pHs, while '761 and '767 show more than 10-fold selectivity at pH 6 over pH 7.4 for koff and more than 2000-fold selectivity at pH 6 for compared to pH 7.4 according to KD. Human VISTA binding rate constants are conserved for the hIgG1.3f, hIgG1f, and afucosylated hIgG1f isotype variants.

Table 6

Binding characteristics of anti-VISTA antibodies determined by SPR

Antibody Isotype pH 7.4 pH 6.0 kd ratio (7.4/6) KD ratio (7.4/6) ka (1/Mc) kd(1/c) KD(M) ka (1/Mc) kd(1/c) KD(M) Р1-061029 h!gG1.3f l,6E+05 6.8E-03 4.2E-08 Ι,ΙΕ+06 7.9E-03 7.2E-09 0.9 5.8 hlgGlf l,7E+05 7.4E-03 4.2E-08 Ι,ΙΕ+06 8.0E-03 7DE-09 0.9 5.9 hlgGlf afucosylated l,7E+05 7.2E-03 4DE-08 Ι,ΙΕ+06 7.8E-03 6.9E-09 0.9 5.9 Р1-068761 h!gG1.3f 3.8E+03 4.2E-02 Ι,ΙΕ-05 3.7E+05 l,6E-03 4.3E-09 26.3 2558.1 hlgGlf l,2E+03 4.2E-02 3.5E-05 3.6E+05 l.5E-03 4.2E-09 28.0 8333.3 hlgGlf afucosylated 5DE+03 4.2E-02 8.2E-06 3.7E+05 l.5E-03 4DE-09 28.0 2000.0 Р1-068767 h!gG1.3f l,9E+03 3.6E-02 l,9E-05 3.3E+05 2.6E-03 7.8E-09 13.8 2435.9 hlgGlf l.5E+03 3.2E-02 2.2E-05 3.2E+05 2.6E-03 8.0E-09 12.3 2750.0 hlgGlf afucosylated l,3E+03 3.3E-02 2.4E-05 3.3E+05 2.6E-03 7.9E-09 12.7 3038.0 a-VISTA acidic pH sensitive h!gG1.3f 2.2E+05 7.8E-04 3.6E-09 2.8E+06 9.0E-02 3.2E-08 0.01 0.1

The binding kinetics of human VISTA antibodies P1-061029 ('029), P1-068761 ('761) and P1-068767 ('767) (as IgG1.3 antibodies) were measured at pH values from 7.4 to pH 6.0 , i.e. at pH 6.9 and pH 6.45, using the Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare) aimed at immobilization density protein A 2000 RU per flow cell. The assay was performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4, 6.9, 6.45 and 6.0. Antibodies

- 106 046477 was diluted to 25 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μl/min for 45 s. A series of concentrations of 100-0.78 nM monovalent hVISTAECD (SEQ ID NO: 325) were prepared at pH 7.4, 6.9, 6.45 and 6.0 in working buffers and passed over bound antibodies at 40 μl/min for measurement associations and dissociations. Two injections of 10 mM glycine at pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. Rate constants k _a (k _on ) and kd (k _off ) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 Evaluation Software v.2.0. The affinity constant, K _D , was calculated as the ratio of the rate constants k _off /k _on for each anti-VISTA antibody. The ratios of k _off and KD at each pH in comparison with pH 6.0 were calculated to assess the change in VISTA k _off and KD when the pH of the buffer was changed to physiological, and are shown in table. 7. The parent antibody '029 showed a constant k _off at each pH tested, while the daughter clones '761 and '767 showed at least a 10-fold higher VISTA k- _off rate and a 100-fold weaker VISTA KD at pH 6. 9 compared to pH 6.0. When the pH of the buffer was changed from acidic to physiological, VISTA k _off and KD weakened for '761 and '767. Data at physiological pH for comparison between '761 and '767 are taken from table. 7 and are marked with an asterisk (*).

Table 7

Kinetic characteristics of binding of antibodies '029, '761 and '767 at different pH values

Antibody pH ka (1/Ms) kd (1/s) KD (M) Koff ratio with pH 6.0 KD ratio with pH 6.0 Р1-061029 (original) 6.0 2.9E+06 5.7E-03 2.0E-09 1.0 1.0 6.45 7.4E+05 4.0E-03 5,ZE-09 0.7 2.7 6.9 4DE+05 5.7E-03 1.4E-08 1.0 7.1 7.4 2.5E+05 6.4E-03 2.6E-08 1.1 13.2 Р1-068761 6.0 6.0E+05 6.6E-04 1.1E-09 1.0 1.0 6.45 Ι,ΙΕ+05 2.1E-03 2.0E-08 3.2 18.4 6.9 4.8E+04 8.9E-03 1.9E-07 13.4 170 7.4* 3.8E+03 4.2E-02 1.1E-05 -63.6 -10000 Р1-068768 6.0 5.6E+05 1.9E-03 3.4E-09 1.0 1.0 6.45 1,ЗЭ+05 4.8E-03 3.8E-08 2.5 and about 6.9 7.4E+04 2.9E-02 4.0E-07 15.3 115.1 7.4* 1.9E+03 3.6E-02 1.9E-05 -19.0 -5000

Data in table. 7 indicates at least 10-fold lower binding affinity of '761 and '767 to hVISTA at pH 6.45 compared to pH 6.0; at least 100-fold lower binding affinity of '761 and '767 to hVISTA at pH 6.9 compared to pH 6.0; and at least 1000-fold lower binding affinity of '761 and '767 to hVISTA at pH 7.4 compared to pH 6.0.

The '015 library also showed a slight preference for pH 6 selective binding to VISTA. Differences in the amino acid sequence of the daughter clones relative to the CDR regions of VH '015 are shown in table. 8. Binding of multiple preparations of each of the '015 daughter clones and the parent '015 antibody (all as IgG1.3 antibodies) to human VISTA at pH 6.0 and 7.4 was measured by SPR using an identical method to that described for the assay '029 above, and shown in table. 8.

- 107 046477

Table 8

Amino acid sequences of CDR1, CDR2 and CDR3 of VH antibodies (separated by underscore) derived from the parent antibody '015

NAME CDR1 (roses 26-35) CDR2 (roses 50-66) CDR3 (rose 99-110) SEQ ID NO Р1-061015 GFTFSSYAMH_ IIWYDGSNKYYADSVKG _DSGFYSSYYFDY 95 Р1-068736 -----E----_ -D-------D------- _-----D-----□ 107 Р1-068738 -----E--N-_ ---D---------Н-------- _-----ED----- 131 Р1-068740 -----D----_ ------- _{D_D -------} _-----D-----D 115 Р1-068742 -----D----_ ------- _{D_D -------} _-----ED----- 119 Р1-068744 -----E----_ Η---------E------ _-----E-----E 103 Р1-068746 — -------- _nn ------- _-----D------ 123 Р1-068748 -----NN---_ -------- _DD ------- _-----D------ 99 Р1-068750 -----D-D—_ _E—D-----------_ -EE--------- 127 Р1-068752 — _E-------D------- _-----D-----E 111 Р1-068754 -----D-D--_ _E—D------------ _----H-D----- 135

Summary of the binding kinetics of '029 and '015 and their daughter clones with huVISTA at pH 6.0 and 7.4, determined by SPR, are shown in table. 9 and 10.

Table 9

Summary of huVISTA kinetics and CDR sequences of VH clone '029 and their daughter clones

ID Avg 7.4 ka (1/Mc) Average 7.4 kd (1/s) Average 7.4 KD (M) Avg 6.0 ka (1/Ms) Avg 6.0 kd (1/s) Medium 6.0 KD (M) CDR 1 VH (floor 26-35) CDR2 VH (floor 50-66) CDR3 VH (floor 99-110) SEQ ID NO Pl-069077 6.0E+04 1.9E-03 3.1E-08 6,ЗЭ+05 1.2E-04 1.9E-10 ...HER... ......DE......... .............. 47 Pl-069065 5.9E+04 2, ZE-OZ 3.9E-08 5.7E+05 2.2E-04 3.8E-10 ...HER... ......DD......... .............. 23 Pl-069075 l,3E+05 2, ZE-OZ 1.8E-08 1,ЗЭ+06 2.9E-04 2.2E-10 ...E..... ...D..E......... .....NOT.. 43 Pl-069071 4.3E+04 4.0E-03 9,ZE-08 7.0E+05 5.1E-04 7,ZE-10 ...HER... .......D......... .....E........ 35 Pl-069061 Weak, fast kd 4,ЗЭ+05 Ι,ΙΕ-03 2.5E-09 ...E..... .......E......... ..........E.D. 15 Pl-069069 9.0E+04 7.5E-03 8.4E-08 1.4E+06 1.2E-03 8.6E-10 .......... ......HER......... ..........D.. 31 Pl-068761 Weak, fast kd 3.8E+05 1.4E-03 3.8E-09 ...HER... ......HER......... .....NOT.. 51 Pl-069059 Weak, fast kd 3.4E+05 1.6E-03 4.8E-09 ...E..... ......DH......... ..........E.D. AND Pl-068767 Weak, fast kd 3.4E+05 2.6E-03 7.6E-09 ...E..... ..D...E......... ..........E.D. 55 Pl-068773 Weak, fast kd 3.0E+05 2.9E-03 9.4E-09 ...E..... ..D...D......... ..........E.D. 59 Pl-069063 1.2E+05 2.7E-02 2,ZE-07 1.9E+06 4.4E-03 2.4E-09 ...E..... .......E......... ..........D.E. 19 Pl-069067 1.0E+05 2.7E-02 2.9E-07 1.7E+06 4.5E-03 2.7E-09 .......... ......HER......... ..........D.E. 27 Pl-069073 Pl-061029 Weak, fast kd 2.9E+05 5.6E-03 1.9E-08 6.1E+05 1.6E+06 5.8E-03 5.8E-03 9.4E-09 3.6E-09 ...HER... GFTLDDYA MN .......E......... GINWNSANIGYA DSVKG ..........E.. VPGYSGGWID AFDV 39 67 Pl-068765 No binding 3.7E+05 7.0E-03 1.9E-08 ...DE..... ......HER......... ..........E.D. 63 Pl-068757 No binding 8.9E+05 1.7E-02 1.9E-08 ...HER... ......HER......... ..........E.D. 71 Pl-068771 No binding 7.6E+05 1.8E-02 2.5E-08 ...HER... ......NOT......... ..........E.D. 75 Pl-068769 No binding 8.1E+05 4.0E-02 5.5E-08 ...HER... ......DH......... ..........E.D. 83 Pl-068775 No binding 1.8E+06 4.7E-02 2,ZE-08 ...HER... ..D...EE......... .....H.....E.D 79 Pl-068759 No binding 1,ЗЭ+06 8.0E-02 6.0E-08 ...HER... ..D...E......... ..........E.D. 87

- 108 046477

Table 10

Summary. huVISTA kinetics and CDR sequences of VH clone '015 and their daughter clones

Clone Avg 7.4 ka (1/Mc) Average 7.4 kd (1/s) Avg 7.4 KD (M) Avg 6.0 ka (1/Mc) Avg 6.0 kd (1/s) Medium 6.0 KD (M) HCDR sequence (gender 26-35) (gender 50-66) (gender 99-110) SEQ ID NO Pl-061015 2.3E+05 2.0E-03 8.8E-09 l,8E+06 9.5E-04 5.4E-10 GFTFSSYAMHIIWYDGSNKYYADSVKGDSGF YSSYYFDY 95 Pl-068748 No binding l,4E+06 1.5E-03 1.0E-09 .....NN..._........DD......._.....D...... 99 Р1-068744 l,3E+06 1.8E-03 1,ZE-09 .....E....N.........E......_.....E.....E 103 Pl-068736 8.4E+06 9.5E-03 1DE-09 .....E.....D......D......._.....D.....D 107 Pl-068752 6DE+06 3.4E-02 5.6E-09 .........._E........D......._.....D.....E 111 Pl-068740 Too fast 4.7E-02 ND .....D........D.D....... .....D.....D 115 Pl-068742 Too fast >1E-02 ND .....D...._.......D.D......._.....ED..... 119 Pl-068746 Too fast >1E-02 ND .........._........НН......._.....D...... 123 Pl-068750 Weak .....D.D..E..D............. .EE......... 127

Thus, several daughter clones of '029 were identified that either retained or had improved _koff for VISTA-ECD at pH 6.0 compared to the original '029, and also exhibited weaker koff or loss of binding to VISTA at physiological pH . The '015 daughter clones showed selective binding to VISTA-ECD at acidic pH with no detectable binding to VISTA at neutral pH, but all '015 daughter clones analyzed gave a higher _koff at pH 6.0 compared to the parent '015.

Example 10 Acid pH Selective '029 Daughter Clones Exhibit Acid pH Dependent Cell Binding and Effector Function with Retained VISTA Blocking Activity.

The pH-dependent binding of clones '761 and '767 to Raji cells modified to ectopically express full-length human VISTA (SEQ ID NO: 1 with D187E substitution) was measured. For this experiment, '761 and '767 were formatted into IgG1.3 antibodies and binding was measured using a secondary anti-human immunoglobulin IgG antibody (Jackson Immunoresearch catalog number 109-065-098). The results shown in FIG. 8A-8B indicate that clones '761 (FIG. 8A) and '767 (FIG. 8B) bind poorly at pH 7.2 and 8.1, but bind better at acidic pH, particularly at pH 6.0. 6.1, 6.2 and 6.4. Binding MFI values are plotted on the y-axis and primary antibody concentrations are plotted on the x-axis on a log scale. Nonlinear regressions are also shown.

In fig. 8C shows data from the experiment described in FIG. 8A-B, which measured the binding of P1-068767 (circles) and an isotype-matched nonspecific control antibody (triangles) to Raji cells expressing human VISTA at a concentration of 3125 ng/ml at different pH. pH50, the pH at which 50% of P1-068767 binding is lost, is approximately 6.6. The binding of MFIs is prepared on the Y axis and the pH buffer is prepared on the X axis. Nonlinear regressions are also shown.

In fig. 8D shows the MFI of the isotype-matched nonspecific control antibody (filled and open circles for pH 7.0 and 6.0, respectively), mAb against VISTA 2 (control, see Fig. 6C, filled and open squares at pH 7.0 and 6, 0, respectively), P1-068761 (filled and open triangles for pH 7.0 and 6.0, respectively), and P1-068767 (filled and open inverted triangles for pH 7.0 and 6.0, respectively) when binding to human monocytes. Binding was detected as described in FIG. 8A-B. A non-pH-selective control antibody to VISTA (mAb 2) bound monocytes at both pHs. Both engineered acid pH-selective antibodies bound monocytes well at pH 6.0 but did not bind better than the nonspecific isotype-matched control at pH 7.0. Thus, clones '761 and '767 have little or no binding to VISTA at neutral pH, and instead selectively bind VISTA to cells at acidic pH.

In fig. Figure 8E shows comparable blocking of recombinant VISTA multimer binding to activated human CD4+ T cells at pH 6.0 by clones '029 (squares), '761 (triangles) and '767 (inverted triangles), whereas a non-VISTA-specific control antibody (circles) did not block VISTA binding. This blocking assay was performed as described in Example 4. These data demonstrate that the engineered acid pH-selective anti-VISTA antibodies are still able to block VISTA receptor-ligand binding at acidic pH.

Specific NK cell lysis of target cells (the same Raji cells expressing human VISTA described in Fig. 8A-B) by antibody-dependent cellular cytotoxicity (ADCC) at physiological pH was measured for antibodies P1-061029.IgG1f, P1-068761. IgG1f, P1068767.IgG1f, a non-VISTA specific antibody, and a negative control non-VISTA specific antibody, which were expressed as afucosylated IgG1 antibodies. NK cells were enriched from PBMCs through negative selection using beads (StemCell Technologies part number 19055) and cultured overnight in Myelocult™ medium (StemCell Tech part number

- 109 046477 nologies 05150) with the addition of 1 μM hydrocortisone (StemCell Technologies catalog number 07904) and 500 U/ml recombinant human IL-2 (Peprotech catalog number 200-02). On the day of analysis, Raji cells ectopically expressing human VISTA (described in Fig. 8A-B) were labeled with Calcein AM (Life Technologies catalog number C3100MP) and cocultured with cultured NK cells at a NK:target cell ratio of 10:1 and with antibodies P1-061029.IgG1f, P1068761.IgG1f, P1-068767.IgG1f, a non-specific VISTA antibody and a non-specific VISTA negative control antibody for 2 hours at physiological pH. Specific lysis was interpolated from the fluorescence signal of the supernatant (EnVision™ plate spectrophotometer). The spontaneous lysis signal obtained from the coculture without antibodies and the maximum lysis signal were determined by lysing target cells using Delfia® lysis buffer (PerkinElmer catalog number 4005-0010). Antibody-specific lysis was calculated as the percentage of observed lysis divided by (maximum lysis signal minus spontaneous lysis signal).

The results presented in Fig. 8F show reduced activity of P1-068761.IgG1f and P1068767.IgG1f compared to P1-061029 and the positive control in mediating antibody-dependent cellular cytotoxicity (ADCC) at physiological pH.

Example 11. PK of antibodies to VISTA in Cynomolgus.

Cynomolgus macaques naïve to human antibodies were injected intravenously with a single dose of 5 mg/kg of anti-VISTA antibody, which bound comparably at acidic and neutral pH (control; mAb2), and anti-VISTA antibodies with reduced binding at acidic pH (sensitive to acidic pH, mAb3) or acid pH-selective antibody '767 to determine the PK of these antibodies in cynomolgus.

The kinetics of binding of SPR antibodies used in this Example are presented in table. 11 and was determined as follows. Cynomolgus VISTA cross-reactivity for acid pH-selective and control anti-VISTA antibodies was assessed at acidic and neutral pH. Binding affinity measurements for VISTA Abs were performed using a Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare) aimed at immobilization density protein 2000 RU per flow cell. The assay was performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4 and 6.0. Antibodies (formatted as IgG1.3 antibodies) were diluted to 25 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μL/min for 45 s. Concentration series of 1600-0.78 nM (pH 7.4) and 100-0.78 nM (pH 6.0) monovalent hVISTAECD (SEQ ID NO: 325) and VISTA-ECD cynomolgus (AFKVATLYSL YVCPEGQNVT LTCRVFGPVD KGHDVTFYKT WYRSSRGEVQ TCSERRPIRN LTFQ DLHLHH GGHQAANTSH DLAQRHGLES ASDHHGNFSI TMRNLTLLDS GLYCCLVVEIRHHHSEHRVH GAMELQVQTG KDAPSSCVAY PSSSQESENI TANNNNNNNN; (SEQ ID NO: 326) was prepared in running buffer and passed over bound antibodies at a rate of 40 μl/min to measure association and dissociation. Two injections of 10 mM glycine at pH 1.5 to 15 s used to regenerate the capture surface with Protein A between assay runs. Rate constants k _a (kon) and kd (koff) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 software Evaluation Software v.2.0 The affinity constant, K _D , was calculated as the ratio of the rate constants ko _ff /ko _n for each VISTA antibody.The ratios k _off and K _D at pH 7.4/pH 6.0 were calculated to compare differences in dissociation rates and affinities at acidic pH compared to neutral pH and are shown in Table. 11. All anti-VISTA antibodies tested showed comparable (within a 2-fold difference) binding kinetics to human VISTA and cynomolgus VISTA at both pH levels, confirming cross-reactivity with cynomolgus VISTA. Both VISTA control antibodies showed improved kd and stronger K _D at physiological pH compared to acidic pH, and the acid pH sensitive control showed faster VISTA kd at acidic pH compared to the control antibody.

- 110 046477

Table 11

Kinetics of binding of SPR antibodies to VISTA with VISTA of cynomolgus

Antibody VISTA pH 7.4 pH 6.0 kd ratio (7.4/6) KD ratio (7.4/6) ka (1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (M) Р1-061029 Human 1.2E+05 7.5E-03 6.2E-08 9.8E+05 6.6E-03 6.8E-09 1.1 9.1 cynomolgu with 1.4E+05 6.7E-03 4.7E-08 6.2E+05 6.2E-03 1.0E-08 1.1 4.7 Р1-068761 Human 4,ZE+OZ 3.7E-02 8.7E-06 3.5E+05 1.4E-03 4DE-09 26.4 2122.0 cynomolgu with 6.5E+03 3.6E-02 5.5E-06 2DE+05 1.7E-03 7.9E-09 21.2 696.2 Р1-068767 Human 1.6E+03 3.5E-02 2,ZE-05 3.2E+05 2.4E-03 7.5E-09 14.6 3066.7 cynomolgu with 1,ZE+OZ 3.4E-02 2.6E-05 1.9E+05 2.5E-03 1,ZE-08 13.6 2000.0 a-VISTA control (mAb 3) Human 4.4E+05 1, ZE-OZ 3.0E-09 9.6E+05 6.0E-03 6.2E-09 0.2 0.5 cynomolgu with 4.9E+05 1.7E-03 3.4E-09 5.5E+05 7DE-03 1,ZE-08 0.2 o, s a-VISTA acidic pH-sensitive (mAb 2) Human 1.8E+05 7.8E-04 4,ZE-09 1.8E+06 5.0E-02 2.8E-08 0.02 0.2 cynomolgu with 1.9E+05 6.8E-04 3.5E-09 1.2E+06 5.2E-02 4.4E-08 0.01 0.08

Human antibody-naïve cynomolgus monkeys were intravenously administered a single dose of 5 mg/kg VISTA mAb 2 (control), VISTA mAb 3 (acidic pH-sensitive), or P1-068767.IgG1.3. The serum concentration of each antibody after injection is shown in FIG. 9. The average retention times for P1-068767.IgG1.3 and the control anti-VISTA antibody were 717 and 22 hours, respectively, indicating that acid pH selectivity significantly reduced the target-mediated drug distribution (TMDD) of the VISTA antibody. Although the control antibody (mAb 2) and the acidic pH-sensitive antibody (mAb 3) bind VISTA comparably at physiological pH, the acidic pH-sensitive antibody had a lower average retention time of 7.6 h, demonstrating the importance of binding at acidic pH to recycle the anti-VISTA antibody as described in Examples 6 and 7. The results indicate that the acidic pH selective antibodies have superior PK and thus more readily bind target in tumors or other microenvironments.

Example 12: pH-selective '029 daughter clones do not bind nonspecifically to proteins having a high pI.

The binding specificity of clones '029, '761 and '767 to VISTA and other high pI proteins was assessed by NIR at neutral and acidic pH using a Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the CM3 biosensor flow cells according to the manufacturer's suggested amine coupling protocol (GE Healthcare) aimed at immobilization density protein A 800 RU per flow cell. SPR experiments were performed at 25°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4 and 6.0. Antibodies (formatted as IgG1.3 antibodies) were diluted to 50 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μL/min for 60 s. A series of 100-10 nM concentrations of monovalent hVISTA-ECD (SEQ ID NO: 325), avidin (ThermoFisher Scientific part number 21128), cytochrome C (Sigma part number C2867), BSA (Calbiochem part number 126593) and monovalent control antigen (Ag) was prepared in pH 7.4 and 6.0 working buffers and passed over bound antibodies at 50 μL/min to assess binding specificity. Two injections of 10 mM glycine, pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. The reference flow cell and 0 nM blank subtracted sensorgrams were examined using Biacore® T200 Evaluation Software v.2.0. The results are presented in table. 12.

Table 12 ___________Binding of VISTA clones to proteins having a high pI___________

Sample Isoel. dot (pl) '029 '761 '767 Anti-Ag PBS (without At) pH 6 pH 7.4 pH 6 pH 7.4 pH 6 pH 7.4 pH 6 pH 7.4 pH 6 pH 7.4 huVISTA-His 6.9 Avidin 10 Cytochrome C 10.7 B.S.A. 4.7 Ag-His 6.5 ί«ί:¥:¥ίίίή¥?ί: I·:·:·:·;··:·:·:·:·:·:·:·:·:·:··:·:

In table 12 specific binding, defined as PPR binding responses >10 RU at the end

- 111 046477 sample input, shown as filled gray rectangles. Anti-Ag is a control antibody that binds VISTA. Clone '029 was specific for VISTA at acidic and neutral pH. The daughter clones of '029, '761 and '767, were specific for VISTA at acidic pH, while the control antibody also retained antigen specificity. Nonspecific binding (NSB) of pI control proteins to the protein A reference surface was not observed in this assay.

Thus, charged amino acids introduced into the VH 761 and '767 CDR regions do not result in electrostatic binding of these antibodies to other high pI proteins such as avidin and cytochrome C or low pI proteins such as BSA.

Example 13. Inhibition of T cell activation by antibodies '761 and '767.

This example describes an assay that can be performed to determine the ability of antibodies '761 and '767 to block hVISTA inhibition of Jurkat T cell activation.

The same assay as in Example 5 was used. Briefly, Jurkat cells (a human T cell line) expressing the NFkB reporter luciferase were cocultured at various pH values with 293T cells expressing human VISTA and a single chain variable fragment of an anti-T-agonist antibody. human cell receptor OKT3. Antibodies against VISTA '761 and '767 or isotype-matched non-VISTA-specific control antibodies were added to cocultured cells. Jurkat activation is shown as luciferase units and fold increase in luciferase signal upon treatment with anti-VISTA antibodies compared to control.

Example 14 Mutational analysis identified key residues conferring pH-dependent binding properties to anti-VISTA antibodies.

Antibodies P1-068761.IgG1.3 and P1-068767.IgG1.3 contain 5-6 mutations from P1-061029 (Table 7). Mutational analysis was performed to identify key residues important for conferring the pH-dependent properties of anti-VISTA antibodies. Thus, a panel of N-1 (reversion of 1 amino acid to P1-061029) and N-2 (reversion of 2 amino acids to P1-061029) variants P1-068761 and P1-068767 were synthesized, expressed in IgG1.3 format and analyzed for their binding with huVISTA at pH 6, pH 6.7 and pH 7.4.

Binding kinetics were measured using a Biacore® T100 instrument (GE Healthcare). Protein A (ThermoFisher Scientific catalog number 21181) was diluted to 20 μg/ml in 10 mM sodium acetate, pH 4.5 and immobilized on the flow cells of the CM5 biosensor according to the manufacturer's proposed amine coupling protocol (GE Healthcare) aimed at immobilization density protein A 2000 RU per flow cell. The assay was performed at 37°C using PBST running buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20) at pH 7.4, 6.7 and 6.0. Antibodies were diluted to 25 nM in PBST pH 7.4 and bound to the active flow cells of the biosensor at 5 μL/min for 40 s. Concentration series of 100-10 nM monovalent hVISTA-ECD (SEQ ID NO: 325) were prepared in pH 7.4, 6.7, and 6.0 running buffers and passed over bound antibodies at 40 μL/min to measure association and dissociation . Two injections of 10 mM glycine at pH 1.5 for 15 s were used to regenerate the protein A capture surface between assay runs. Rate constants k _a (k _on ) and kd (k _off ) were obtained from the reference flow cell and sensorgrams with the subtraction of 0 nM blank and fitted to a 1:1 binding model in Biacore® T200 Evaluation Software v.2.0.

The affinity constant, KD, was calculated as the ratio of the kof/kon rate constants for each anti-VISTA antibody. % Rmax was calculated to compare how pH affects the binding ability of an anti-VISTA antibody and represents the measured maximum VISTA binding response versus the expected maximum VISTA binding response. % Rmax is defined as the ratio of the response at the recorded 'binding' point subtracting the reference value at the end of injection of 100 nM VISTA for each antibody (Rmax) relative to the expected VISTA binding response (Rexp). Rexp is calculated as Rexp=[(molecular weight of VISTA-ECD/molecular weight of mAb)x(response at the registered mAb 'capture' point (RU)]x2 binding site per mAb.

The SPR results obtained for the reverse variants of P1-068761 are shown in FIG. 10A, which are ranked by pH 6.0 _koff , from slowest to fastest. In this table, antibodies that showed a weak or no binding response (<10 RU) to 100 nM hVISTA were categorized as non-binding (NB). The results indicate that E at positions 32 (i.e., amino acid residue 7 in CDR1 VH clone '761) and 100f (amino acid residue 12 in CDR3 VH clone '761) are required to maintain acid pH selectivity, since these back mutations in the original P1-061029 sequences provided significant binding of hVISTA at physiological pH (% Rmax >10). Subsequent analysis showed that E55A reversion variants (amino acid residue 6 in CDR2 VH clone '761) retained acidic pH selectivity and exhibited comparable binding kinetics within a 2-fold of P1-068761. In contrast, while the H100G, E56N, and E30D reversion variants (amino acid residues 12 in CDR3 VH, 7 in CDR2 VH, and 4 in CDR1 VH clone 761, respectively) retained acid pH selectivity, these mAbs also exhibited ~3-fold faster koff at acidic pH values compared to P1-068761, which brings the dissociation rate of these mutants at acidic pH closer to

- 112 046477 to the original P1-061029. Thus, the addition of the G100H, N56E, and/or D30E mutations to the parent clone P1-061029 contributed to the increased affinity for VISTA at acidic pH observed in the acid-selective clone P1-068761.

The research results obtained for the reverse versions of P1-068767 are shown in Fig. 10B, which are ranked by pH 6.0 koff, from slowest to fastest. Results indicate that D at position 102 (amino acid residue 14 in CDR3 VH clone '767) was required to maintain acid pH selectivity, and reversion of D102V back to the original sequence P1-061029 allowed significant binding of hVISTA at neutral pH (%Rmax > 10). Further analysis showed that variants with E30D, D52N and E55A reversions (amino acid residues 4 in CDR1 VH, 3 in CDR2 VH and 6 in CDR3 VH clone '767, respectively) retained selectivity for acidic pH and showed kinetics comparable to pH 6.0 binding within 2-fold values of P1-068767. In contrast, variants with the E100fF reversion (amino acid residue 12 in CDR3 VH clone 767) retained acid pH selectivity, albeit with a more than 3-fold faster koff at acid pH compared to P1-068767. It is noteworthy that variants with E100fF reversion showed even faster k _off at acidic pH compared to the parent mAb P1-061029.

Corroborating binding kinetics data were obtained (using the procedure described in Example 9) with some of the reverse mutants, the data are shown in table. 22 and 23.

Thus, the summary indicators of the reverse mutants P1-068761 and P1-068767 (selective for acid pH) compared to P1-061029 (pH-tolerant) are presented below in Table. 13 (HCDR1, HCDR2 and HCDR3 are separated by an underscore).

Table 13 Seq Id.

Pl-061029 GFTLDDYAMH_GINWNSANIGYADSVKG_VPGYSGGWIDAFDV 67

Pl-068761. ......EE........._.....H.....E.. 51

Pl-068767 ....E....._..D...E.........._...........E.D 55 floor. ak 26-35 50-66 99-110

The VH CDR sequences presented above for P1-061029, P1-068761 and P1-068767 in Table. 13 are indicated by amino acids (pol. aa) 26-35, 50-66 and 99-110 SEQ ID NO: 67, 51, 55, respectively. Key mutations required for acid pH selectivity are highlighted in bold, and mutations with a greater than 3-fold effect on k _d at pH 6.0 compared to P1-068761 and P1068767 are underlined.

The F100fE, V102D, and Y32E mutations were then introduced into '029 individually or together to determine whether these amino acid substitutions were sufficient to make '029 pH-selective. The following antibodies were generated: P1-061029_F100fE_V102D (P1-072000); P1-061029_F100fE (P1-072002); P1061029_V102D (P1-072004); P1-061029_Y32E (P1-072006) and P1-061029_Y32E_F100fE (P1-072008), and their binding kinetics were determined as described in Example 9. The amino acid sequences of their CDR regions are shown in table. 23. The results shown in table. 22 indicate that V102D is sufficient to provide pH selectivity of '029, but binding kinetics are improved if F100fE is also present. However, F100fE or Y32E by themselves do not make '029 pH selective.

Example 15. Epitope mapping of antibodies to VISTA.

The epitopes of hVISTA antibodies '015, '029, '761 and '767, formatted as IgG1.3 antibodies, were determined by 2 different methods: competitive BLI (biolayer interferometry) and surface yeast display.

Competitive BLI epitope binning assays were performed to evaluate whether the acid pH-selective antibodies to VISTA, P1-068761 and P1-068767, retained similar or overlapping epitopes on VISTA compared to the parent clone P1-061029, P1-061015, and the corresponding control antibodies. to VISTA 1, 2 and 3. Sandwich and tandem binning analyzes were performed on an OctetRed384 BLI instrument (PALL/ForteBio). All assay steps were performed at 30°C and shaking speed of 1000 rpm, using acidic (pH 6.0) or neutral (pH 7.4) PBST buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20). For the sandwich format, anti-human IgG Fc sensors (ANS, PALL/ForteBio) were first captured with a panel of anti-VISTA antibodies at pH 7.4, then the anti-human immunoglobulin capture sensors were blocked with total human IgG (Jackson 009-000-002). Human VISTA-ECD was then captured at pH 6.0, and finally competition was assessed for all possible antibody combinations at pH 6.0. In a tandem format assay, streptavidin-coated biosensors (SAX, PALL/ForteBio) first captured biotinylated hVISTA-ECD at pH 7.4, then captured the full panel of anti-VISTA antibodies on the sensors at pH 6.0, ensuring fully saturated binding of each antibody to VISTA followed by evaluation of competition with all possible antibody combinations at pH 6.0.

The results of competitive BLI epitope binning assays are represented by the competition matrix,

- 113 046477 fig. 11A. In this figure, the first captured antibody is shown in a row, and its binding or blocking activity to (second) competing antibodies is shown in each column. The competition matrix was identical for both analysis formats. For the sandwich assay, binding (light gray) of the competing antibody was detected by a signal ranging from 0.4 to 1.2 nM, and blocked antibodies (black) showed a no-binding signal of <0.1 nM. For the tandem assay, binding of the competing antibody was determined by a signal ranging from 0.3 to 0.8 nM, and blocked antibodies showed a no-binding signal of <0.2 nM. Although 'VISTA mAb 3' exhibits rapid dissociation in acidic conditions from hVISTA-ECD in the SPR at 37°C (Figure 6C), it does not dissociate rapidly in either BLI assay format (performed at 30°C). These competition assays showed that P1-061015, P1-061029, the acidic pH-selective antibodies P1-068761 and P1-068767, and antibodies to VISTA 2 and 3 compete with each other for similar or overlapping epitopes on VISTA. However, the anti-VISTA 1 antibody binds to a separate and distinct epitope. Thus, the charged amino acid mutations introduced into the VH CDR region of P1-061029 to create the acid-selective clones P1-068761 and P1-068767 did not lead to a significant change in the VISTA binding epitope.

Epitopes of antibodies '029, '015, '761 and '767 were also mapped using yeast surface display and NGS according to the method of Chao et al. (2004) J. Mol. Biol. 342:539-550, Oliphantetal. (2006) J. Virol. 80:12149-12159, and Kowalsky et al. (2015) J. Biol. Chem.290:26457–26470. Briefly, a saturation mutagenesis library of VISTA ECD single-point mutants was prepared and exposed to the surface of yeast. VISTA mutants that lost binding to the mapping antibody but retained binding to the non-blocking antibody (mAb1) were sorted and sequenced. Because they retained binding to mAb1, these mutants were likely correctly folded, and the loss of binding observed for the mapped antibody was likely due to the loss of an energetically important contact residue. The positions of these mutations were identified as energetically important residues in the antibody epitope and are shown in Table. 14.

Table 14

huVISTA residues that have been identified as epitope residues of the anti-VISTA mAb

mat T Y and to and T N Ύ 41 R m T II F U Q W L and n m L IT n n and F IT L 1" V pt 1 11* n 111 n 1ы S 114 E 1Y R 12T P1 061015 To 1 1 X X X To X X X To X X X Р1061029 X X X X To X X X X X X X X X X X X Р1068761 X To X X To X X X X X X X X X X X X PI068767 To X X X To X X To X X X X X X X X X

Table 15 includes details from Table 1. 14 and lists hVISTA amino acid residues that are likely to reduce binding of each antibody listed, based on residue frequency observed in the yeast surface display/NGS method

Table 15 VISTA Amino Acid Substitutions That Are Likely to Reduce Binding of the Antibodies Listed

Р1-061015 pH 6 Р1-061015 pH 7 Pl-061029 pH 6 Pl-061029 pH 7 Pl-068761 pH 6 Pl-068767 pH 6 T35 P, Y, w Y37 P, G, A, S, T, K, R, Η, N, D, E, Q P, G, S, N, D, EQ Y, S, T, V, L, I, M, K, R, N, D, Q P, G, S, Τ, V, L, I, M, K, R, N, D, E, Q P, G, A, S, T, V, L, I, Μ, K, R, N, D, E, Q G, T, V, L, I, M, K, R, N, Q KZ 8 P, G, A, S, V T39 G, M, R, N, F, Y, W, N, D, E, Q M, K, R, N, F, Y, W, D, E, Q G, A, S, M, Y, W, N, D, E, Q G, A, S, V, L, M, R, H, F, Y, W, N, D, E, Q G, Y, D, E G, A, S, Η, Y, W, N, D, E, Q Y41 A, S, T, I, M P, I, M, H

- 114 046477

Pl-061015 pH 6 Pl-061015 pH 7 Pl-061029 pH 6 Pl-061029 pH 7 Pl-068761 pH 6 Pl-068767 pH 6 R54 L, M, F, Y, E M,E P, A, T, V, I, M, F, Y, N, D, E, Q P, G, A, S, T, V, L, I, M, H, F, Y, W, N, D, E, Q A, T, V, L, I, Μ, K, F, Y, E, Q P, A, S, T, V, L, I, M, F, Y, W, D, E, Q T61 G, L, R, H, F, Y, D, E, Q V, L, K, R, H, F, Y G, V, Η, Y, D L, R, H, F, Y, D, E F62 G, A, S, M, K, R, N, D, E, Q G, K, R, D, E, Q P, G, A, S, T, V, I, M, H, Y, W, D, E, Q P, G, A, S, T, V, L, I, M, K, R, Η, Y, W, N, D, E, Q P, G, A, S, T, V, M, H, Y, W, D, E, Q P, G, A, S, T, V, L, M, Η, Y, W, N, D, E, Q Q63 G, R, W, D, E W,D,E G, A, S, T, V, K, R, Η, Y, W, N, D, E P, G, S, T, L, M, K, R, H, F, Y, W, N, D, E G, S, T, K, H, Y, N, D, E P, G, A, S, T, V, L, I, M, K, H, F, Y, W, N, D, E L65 P, G, A, S, T, K, R, H, W, N, D, E, Q P, G, S, K, W, D, E, Q G, T, Y, D, E, Q P, G, A, S, T, Η, Y, W, N, D, E, Q, P, G, S, H, D, E, Q H66 P, T, V, L, I, Μ, K, R, F, Y, w P, T, V, L, I, Μ, K, R, F, Y, w T, V, L, I, Y, D, E, Q G, S, T, V, L, I, M, K, R, W, N, D, E, Q T, I, K, W, D T, V, I, K, w, D, E L67 G, A H68 L, I, M, F, E L, I, E G, T, V, L, I, Y, W, D, E, Q F97 G,D,E L115 R,W A, T, K, N, Q A, T, K, F, N, Q A, T, M, K, F, N A, T, K, F, N, Q V117 M, K, N, D M, K, R, W, E T, M, K, R, W, E T, L, I, M, K, R, W, E T, I, M, K, w T, L, I, M, K, R, W, E 1119 F,P P,N P, M, E P, M, E M,H P, M, H, F, N, E H121 V, E, Q H122 P, Y, N, D S124 P, V, L, I, K, F, D, E L, I, M, H, W, Q L, I, M, H, W, Q L, I, M, Q L,I,M E125 A, S, T, L, M, K, H, Y, D A, T, V, I, M, K, H, F, Y, W, N, D T, V, I, M, H, F, Y, W G, T, K, H, Y, W, N, D V, I, Η, N T, V, I, F, Y, W, N R127 S, V, M, H P, S, V, M, K, H, N P, V, M, N P, S, V, M, H, N

In fig. 11B and FIG. 11C shows a representation of the epitope spanning all residues for the hVISTA blocking antibody as listed in Table 1. 14 (Fig. 11B), compared with the epitope of a non-blocking hVISTA antibody (mAb1; Fig. 11C). Amino acid residues 66(H) and 162(A) are indicated to indicate the orientation of the molecule. Histidine residues are shown in gray and epitope residues are shown in black. Specifically, all anti-VISTA blocking mAbs occupied the same epitope region, consistent with Octet binning data (showing that they competed with each other), with minor residue differences among the antibodies studied. In contrast, the non-blocking hVISTA antibody (mAb1) occupies a distinct epitope region on the hVISTA molecule, and this is also supported by the Octet binning data, showing that none of the blocking mAbs competed with mAb1.

Example 16. Biophysical properties of '761 and '767.

The physical and chemical properties of P1-068761 and P1-068767 were compared with those of the parent P1061029 (all with IgG1.3 constant region) using the following analytical and biophysical methods.

SEC analytical data were obtained using an Agilent 1260 HPLC instrument with a Shodex™ KW403-4F column (4.6 mm id x 300 mm dl) in a buffer containing 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3 (filtered through 0.2 µm), at a flow rate of 0.30 ml/min. Data were recorded using an Agilent 1260 Infinity diode array detector set at 280 nm and analyzed using Agilent Chemstation software (Agilent, Santa Clara, CA).

Image-guided capillary isoelectric focusing (icIEF) data were acquired using a ProteinSimple iCE3 instrument with an Alcott 720NV autosampler. Antibody samples were mixed with the separation mixture, resulting in final antibody concentrations of 0.2 mg/ml, 0.35% methylcellulose, 2.0 M urea, 1% v/v pharmalyte pI 5-8 and 3% v/v pharmalyte pI 8- 10.5. These samples were analyzed using a prefocusing time of 1 min at 1500 V and a focusing time of 10 min at 3000 V in a ProteinSimple cIEF FC-coated cartridge (product 101701). Data were analyzed using iCE CFR Software V4.3.1.5352.

- 115 046477

The hydrodynamic size of the antibodies was determined by dynamic light scattering (DLS), and thermal stability was examined by fluorescence spectroscopy and static light scattering (SLS) using an UNcle molecular characterization instrument (Unchained Labs). Antibodies P1-061029, P1-068761 and P1-068767 were prepared at a concentration of 2 mg/ml in 1x PBS buffer and then diluted 1:1 with either 40 mM Tris in 1x PBS or 40 mM citrate in 1x PBS, at different pH levels, to obtain final samples of 1 mg/ml antibody in 20 mM Tris/1x PBS or 20 mM citrate/1x PBS, at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0. These samples were loaded into a UNi cuvette cartridge and analyzed within 1 h of dilution in different pH formulations. DLS data were collected at 25°C using 4 data acquisitions of 5 s each. Intensity autocorrelation functions were fitted using UNcle analysis software version V2.0. Thermal denaturation data were obtained by scanning samples from 25°C to 90°C with a scan speed of 0.5°/min and excitation at 266 nm and 473 nm. Fluorescence data were recorded in the range of 250–720 nm. Fluorescence and SLS data were analyzed using UNcle analysis software version V2.0.

The apparent viscosity of antibodies P1-061029, P1-068761 and P1-068767 was measured on a UNcle molecular characterization instrument (Unchained Labs) using the sphere-based DLS method, which measures the diffusion rate of polystyrene spheres in the presence of prepared antibody solutions, according to the recommended protocol Unchained Labs. Briefly, a 10% solution of 100 nm polystyrene beads (Thermo Scientific, part number 3100A) was prepared in buffer to formulate containing 0.5% Tween 80. 3 μl of this polystyrene bead mixture was added to 30 μl of the prepared antibody solution ( different concentrations of Ab in 20 mM histidine, 260 mM sucrose, pH 6.0) and applied the resulting protein/sphere mixture to 3 separate lanes of a UNi cuvette cartridge (9 μL per lane) for analysis in triplicate. Data were analyzed using UNcle analysis software version V2.0 using a reference viscosity of 1.3 cP.

The physical stability of antibodies P1-061029, P1-068761 and P1-068767 was studied under accelerated stress conditions by preparing antibody samples with a concentration of 50 mg/ml in 20 mM histidine, 260 mM sucrose, pH 6.0 and exposure to thermal stress at 40°C within 4 weeks. Aliquots were collected immediately before incubation at 40°C (zero time point t0), and after 1 week (1 wk) and 4 weeks (4 wk) of thermal stress, after which samples were diluted to 2 mg/ml using formulation buffer and investigated using aSEC. aSEC data were obtained on an Agilent 1260 HPLC system using a Shodex KW403-4F column (id 4.6 mm x 300 mm) in a buffer containing 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3 (filtration through 0.2 µm), at a flow rate of 0.30 ml/min. Data were recorded using an Agilent 1260 Infinity diode array detector set at 280 nm and analyzed using Agilent Chemstation software (Agilent, Santa Clara, CA).

The following results were obtained. Analytical size exclusion chromatography (aSEC) data showed that all three antibodies could be purified to high purity, with each antibody sample consisting of more than 99.3% monomer (major peak), less than 0.7% high molecular weight species (HMW). and undetectable levels of low molecular weight compounds (LMC), table. 16.

Table 16

Analytical SEC data for anti-VISTA antibodies indicating the percentage of high molecular weight compounds (%HMW), the percentage of monomeric/basic compounds (%Basic) and the percentage of low molecular weight compounds (%LMW)

Sample name %vmm %Basic %nmm Р1-061029 0.4 99.6 0.0 Р1-068761 0.6 99.4 0.0 Р1-068767 0.5 99.5 0.0

The charged variant profile determined by image-guided capillary isoelectric focusing (icIEF) for antibody P1-061029 showed the presence of basic compounds (69.4%) with an isoelectric point (pI) of 8.56 and 30.6% acidic compounds (Fig. . 12AC). P1-068761 showed basic compounds (66.4%) with a pI of 6.69 and 33.6% acidic compounds. P1-068767 showed basic compounds (61.4%) with a pI of 6.63 and 38.6% acidic compounds. Thus, the distribution of acidic, alkaline, and basic compounds is similar for the three antibodies, but the engineered antibodies P1-068761 and P1-068767 have a significantly lower isoelectric point than the parent antibody P1-061029.

The oligomeric state of P1-061029, P1-068761 and P1-068767 was determined in the pH range 3-9 using dynamic light scattering (DLS) in buffers with different pH. All values of the hydrodynamic radius (Rh) for each antibody were in the range of 4.8-5.7 nM, which is typical for samples of monomeric antibodies, table. 17. This indicates that these antibodies do not form detectable levels of high molecular weight aggregates at a concentration of 1 mg/ml within the first hour after dilution to formulate pH 3-9.

- 116 046477

Table 17

Hydrodynamic radius determined by DLS for 1 mg/mL anti-VISTA antibody samples in the pH range 3-9

Rh (nm) Rh (nm) Rh (nm) pH Buffer Pl-061029 Pl-068761 Pl-068767 9 20 mM Tris/1 xPBS 5.2 4.8 5.2 8 20 mM Tris/lxPBS 5.2 5.2 5.2 7 20 mM Tris/1 xPBS 4.8 5.2 5.2 7 20 mM citrate/1 xPBS 4.8 5.2 5.7 6 20 mM citrate/1 xPBS 5.2 5.2 4.8 5 20 mM citrate/1 xPBS 5.2 4.8 5.2 4 20 mM citrate/1 xPBS 4.8 4.8 5.2 3 20 mM citrate/1 xPBS 5.2 5.2 5.2

The thermal stability of P1-061029, P1-068761 and P1-068767 was measured in the pH range 3–9 by monitoring fluorescence and static light scattering as a function of temperature in buffers of different pH. The first thermal transition of denaturation (Tm1), which typically represents denaturation of the CH2 domain of IgG1 antibodies, was determined by fluorescence and is shown in Table 1. 18, and the onset of aggregation (Tagg), which typically represents denaturation of the FAB domain of IgG1 antibodies, was measured by static light scattering and is shown in Table. 19. At neutral pH (pH 7.0) in Tris/PBS, the Tm1 values for the three antibodies were as follows: P1-061029 (67.4°C), P1-068761 (67.0°C) and P1-068767 (65.3°C), with Tagg values P1-061029 (67.8°C), P1-068761 (67.5°C) and P1-068767 (65.8°C). All Tm1 values for each antibody in the citrate/PBS formulation at the same neutral pH 7.0 or slightly more acidic pH 6.0 were within 0.7° of the pH 7.0 Tris/PBS values. However, Tm1 values were slightly lower (0.3-1.1° lower) at the more alkaline pH 8-9 and significantly lower at the more acidic pH 3-5 for each antibody. Compared to neutral pH, Tagg for P1-061029 was within 0.1° of pH 7.0 at a more alkaline pH of 8.0-9.0, was 1.0° lower at pH 5.0 and much lower (6.1°-19.6° lower) in the most acidic conditions at pH pH 3.0-4.0. Tagg for P1-068761 and P1-068767 were also significantly lower at pH 3.0-4.0. However, at pH 5.0, Tagg for P1-068761 was only 0.2° lower than Tagg at pH 6.0, while Tagg for P1-068767 was 2.2° lower at pH 5.0 than at pH 6.0, showing some differences in Tagg for each antibody, table. 19.

Table 18 Thermal stability (Tm1 values) for P1-061029, P1-068761, P1-068767 in the pH range 3-9, as determined by fluorescence spectroscopy

Tml (°C) Tml (°C) Tml (°C) rn Buffer Pl-061029 Pl-068761 Pl-068767 9 20 mM Tris/lxPBS 66.6 65.9 65.0 8 20 mM Tris/lxPBS 67.0 66.5 64.8 7 20 mM Tris/lxPBS 67.4 67.0 65.3 7 20 mM citrate/1 xPBS 67.2 66.9 64.8 6 20 mM citrate/1 xPBS 67.6 67.5 65.0 5 20 mM citrate/1 xPBS 64.4 64.7 62.1 4 20 mM citrate/1 xPBS 51.8 52.0 50.8 3 20 mM citrate/1 xPBS 30.7 28.1 28.7

Table 19

Thermal stability (Tagg values) for P1-061029, P1-068761, P1-068767 in the pH range 3-9, as determined by static light scattering

Tagg (°C) Tagg (°C) Tagg (°C) pH Buffer Pl-061029 Pl-068761 Pl-068767 9 20 mM Tris/l xPBS 67.7 67.1 66.0 8 20 mM Tris/l xPBS 67.8 67.5 65.8 7 20 mM Tris/l xPBS 67.8 68.2 65.9 7 20 mM citrate/l xPBS 67.8 68.1 65.7 6 20 mM citrate/l xPBS 68.1 68.9 65.6 5 20 mM citrate/l xPBS 66.8 68.7 63.7 4 20 mM citrate/l xPBS 61.7 63.6 56.9 3 20 mM citrate/l xPBS 48.2 48.8 41.0

The apparent viscosity of P1-061029, P1-068761 and P1-068767 was measured using the sphere-based DLS method, which measures the diffusion rate of polystyrene spheres in the presence of

- 117 046477 prepared antibody solutions. Comparison of all three antibodies at 44 mg/ml shows similar viscosity for both engineered antibodies as the parent antibody under these conditions, Table. 20. In a second study, additional protein material for P1-068761 and P1-068767 was concentrated to higher concentrations for viscosity analysis at 136 mg/mL, 100 mg/mL, and 50 mg/mL. These data showed increased apparent viscosity at higher antibody concentrations with maximum apparent viscosities of 5.7 ± 0.7 for P1-068761 and 5.3 ± 0.6 for P1-068767 at 136 mg/mL.

Table 20 Apparent viscosity (in cP) for antibodies in 20 mM histidine, 260 mM sucrose, pH 6.0, at 25°C, as determined by the sphere-based DLS method. Values represent the mean and standard deviation of data from three UNi tracks

Antibody Apparent Apparent Apparent Apparent viscosity viscosity viscosity viscosity (cP) at 136 (cP) at 100 (cP) at 50 (cP) at 44 mg/ml mg/ml mg/ml mg/ml Р1-061029 1.6±0.1 Р1-068761 5.7±0.7 3.1±0.0 1.4±0.2 1.5±0.4 Р1-068767 5.3±0.6 3.0±0.3 1.7±0.2 1.6±0.2

The physical stability of 50 mg/ml samples P1-061029, P1-068761 and P1-068767 in 20 mM histidine, 260 mM sucrose, pH 6.0, was studied under accelerated stress conditions at 40°C for 4 weeks. The oligomeric state of antibodies was monitored using aSEC for samples immediately before incubation at 40°C (zero time point=10), and after 1 week (1 wk) and 4 weeks (4 wk) of stress at 40°C. These data show that all three antibodies remain more than 96% monomeric after 4 weeks at 40°C, with low levels of HMM compounds (<1.6% HMM) and low levels of HMM compounds (<2.0% HMM), table 21.

Table 21

aSEC data for anti-VISTA antibody samples under accelerated aging conditions, showing the percentage of high molecular weight compounds (%HMW), the percentage of monomeric/basic compounds (%Basic) and the percentage of _______low molecular weight compounds (%LMW), for t0, 1 week and 4 week samples.

Antibody Sample % VMM % Main % nmm Р1-061029 to 0.4 99.7 0.0 1week 0.5 99.4 0.2 4weeks 0.8 97.2 2.0 Р1-068761 to 0.6 99.4 oh oh 1week 0.9 98.8 o, s 4weeks 1.6 96.4 2.0 Р1-068767 to 0.5 99.5 oh oh 1week 0.8 98.9 o, s 4weeks 1.6 96.4 2.0

Example 17. Production of anti-VISTA antibodies with germline substitutions in the heavy chain framework region.

Antibodies against VISTA P1-061029 or their daughter clones, in particular P1-068761, P1-068767, P1068761_E55A (P1-070868), P1-068767_D52N (P1-070906), P1-068767_E55A (P1-070908) and P1 52DB Р1070916), in which the variable heavy chain framework regions have been modified by one or both of the K16R and T84A substitutions (in other words, antibody P1-070868 has the VH and VL amino acid sequences of antibody P1-068761_E55A, but with the K16R and T84A substitutions in the heavy chain framework region.) These substitutions were made to make the antibodies more closely match the germline heavy chain framework sequence, which contains residues 16R and 84A. In fig. 14 is an alignment showing the location of each of these amino acid residues relative to VH P1-068761 and its CDR sequence. The K16R and T84A substitutions in VH P1-068761 and other antibodies are shown in the sequence table.

These substitutions change the amino acid residues at those two positions so that they contain the same amino acids as in the germline from which the '029 heavy chain was derived. Residues 16R and 84A are also present in the framework regions of VH P1-61015 (see SEQ ID NO: 95).

The binding of these antibodies to hVISTA was measured as described in Example 9. The results are presented in table. 22, and amino acid substitutions of antibodies are shown in table. 23. The results indicate that K16R and T84A do not significantly affect the binding kinetics of the '029 daughter clones.

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Therefore, any of the anti-hVISTA antibodies described herein may include K16R and/or T84A. Antibodies P1-061029_F100fE_V102D (P1-072000); Р1-061029_Р100ж (P1-072002); P1061029_V102D (P1-072004); P1-061029_Y32E (P1-072006) and P1-061029_Y32E_F100fE (P1-072008) with K16R and/or T84A will be constructed and their binding tested as described herein.

Table 22

Binding kinetics of selected antibodies to hVISTA

ID Description pH 7.4 ka (1/Ms) pH 7.4 kd (1/s) pH 7.4 KD (M) pH 6.0 ka (1/Ms) pH 6.0 kd (1/s) pH 6.0 KD (M) Pl-071757 P1 -061029 HCK16RT84A (FW revertant) 1,ЗЭ+05 5.9E-03 4.5E-08 8DE+05 5.9E-03 7,ZE-09 Pl-071759 Pl-061029_HC_K16R (FW revertant) 1,ЗЭ+05 6.0E-03 4.6E-08 8.0E+05 5.9E-03 7.4E-09 Pl-071761 P1-061029 HCT84A (FW revertant) 1.5E+05 6.0E-03 4.0E-08 8.7E+05 6.0E-03 6.9E-09 Pl-071763 P1 -068761_HC_K 16RT84A (FW revertant) No binding at 100 nM 2.9E+05 1.4E-03 4.9E-09 Pl-071765 P1-068761 HCK16R (FW revertant) No binding at 100 nM 2.8E+05 1.4E-03 5.0E-09 Pl-071767 Pl -068761 HCT84A (FW revertant) No binding at 100 nM HERE+05 1.6E-03 5.1E-09 Pl-071769 P1-068767 HCK16RT84A (FW revertant) No binding at 100 nM 2.4E+05 2.6E-03 1.1E-08 Pl-071771 P1-068767 HCK16R (FW revertant) No binding at 100 nM 2.5E+05 2.6E-03 1.1E-08 Pl-071773 Pl-068767_HC_T84A (FW revertant) No binding at 100 nM 2.7E+05 2.6E-03 9.8E-09 Pl-071775 P1-070868 HCK16RT84A (FW revertant) No binding at 100 nM 2.5E+05 1.7E-03 6.9E-09 Pl-071777 P1-070868 HCK16R (FW revertant) No binding at 100 nM 2.7E+05 1.7E-03 6.4E-09 Pl-071779 P1-070868 HCT84A (FW revertant) No binding at 100 nM 2.6E+05 1.8E-03 7.0E-09 Pl-071781 P1-070906 HCK16RT84A (FW revertant) No binding at 100 nM 2.2E+05 1.7E-03 7.7E-09 Pl-071783 P1-070906 HCK16R (FW revertant) No binding at 100 nM 2.2E+05 1.7E-03 7.6E-09 Pl-071785 P1-070906 HCT84A (FW revertant) No binding at 100 nM 2.5E+05 1.7E-03 6.7E-09 Pl-071787 P1-070908 HCK16RT84A (FW revertant) No binding at 100 nM 2DE+05 2.7E-03 1,ZE-08 Pl-071789 P1-070908 HCK16R (FW revertant) No binding at 100 nM 2DE+05 2.6E-03 1,ZE-08 Pl-071791 P1-070908 HCT84A (FW revertant) No binding at 100 nM 2.4E+05 2.7E-03 1.1E-08 Pl-071793 P1 -070916 HC-K16RT84A (FW revertant) No binding at 100 nM 2DE+05 1.7E-03 8.1E-09 Pl-071795 P1-070916 HCK16R (FW revertant) No binding at 100 nM 2DE+05 1.7E-03 8.1E-09 Pl-071797 P1-070916 HCT84A (FW revertant) No binding at 100 nM 2.2E+05 1.7E-03 7.7E-09 Pl-072000 Pl-061029 FlOOffi V102D No binding at 100 nM 2.0E+05 2,ZE-OZ 1.2E-08 Pl-072002 Pl-061029 FlOOffi 4.4E+04 8.5E-03 1.9E-07 6.5E+05 1.6E-03 2.5E-09 Pl-072004 Pl-061029 V102D No binding at 100 nM 1.4E+05 2.5E-02 1.8E-07 Pl-072006 Pl-061029 Y32E 1.2E+04 6,ZE-OZ 5.4E-07 3.4E+05 1.2E-03 3.5E-09 Pl-072008 Pl-061029 Y32E FlOOffi No binding at 100 nM 2.7E+05 2.4E-02 8.8E-08 Pl-070916 Pl-068767 D52N E55A No binding at 100 nM 2,ЗЭ+05 1.7E-03 7.4E-09 Pl-070908 Pl-068767 E55A No binding at 100 nM 2.2E+05 2.5E-03 1.1E-08 Pl-070906 Pl-068767 D52N No binding at 100 nM 2.4E+05 1.6E-03 6.7E-09 Pl-070868 Pl-068761 E55A No binding at 100 nM 2.6E+05 1.8E-03 7.0E-09 Pl-0687677 acidic pH-selective child clone '767 No binding at 100 nM 2.6E+05 2.6E-03 1.0E-08 Pl-0687617 acidic pH-selective child clone '761 No binding at 100 nM 2.8E+05 1.5E-03 5.5E-09 Pl-0610296 GI original 1.8E+05 6.4E-03 3.6E-08 7.8E+05 5.8E-03 7.4E-09

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Table 23

Amino acid sequences of the CDR regions of VH antibodies from the table. 22

ID Description HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Pl-071757 Pl- 06 1029 HCK16RT84A (FW revertant) Pl-071759 P1-061029 HCK16R (FW revertant) Pl-071761 P1-061029 HCT84A (FW revertant) Pl-071763 Pl- 06876 l-HCK 16RT84A (FW revertant) ....HER... ......HER......... .....NOT.. Pl-071765 P1-068761 HCK16R (FW revertant) ....HER... ......HER......... .....NOT.. Pl-071767 Pl -068761 HCT84A (FW revertant) ....HER... ......HER......... .....NOT.. Pl-071769 Pl- 068767_HC_K16R_T84A (FW revertant) ....E..... ..D...E......... ..........E.D. Pl-071771 P1-068767 HCK16R (FW revertant) ....E..... ..D...E......... ..........E.D. Pl-071773 P1-068767 HCT84A (FW revertant) ....E..... ..D...E......... ..........E.D. Pl-071775 Pl- 070868_HC_K16R_T84A (FW revertant) ....HER... .......E......... .....NOT.. Pl-071777 P1-070868 HCK16R (FW revertant) ....HER... .......E......... .....NOT.. Pl-071779 P1-070868 HCT84A (FW revertant) ....HER... .......E......... .....NOT.. Pl-071781 Pl- 070906_HC_K16R_T84A (FW revertant) ....E..... ......E......... ..........E.D. Pl-071783 P1-070906 HCK16R (FW revertant) ....E..... ......E......... ..........E.D. Pl-071785 P1-070906 HCT84A (FW revertant) ....E..... ......E......... ..........E.D. Pl-071787 Pl- 070908 HCK16RT84A (FW revertant) ....E..... ..D............. ..........E.D. Pl-071789 P1-070908 HCK16R (FW revertant) ....E..... ..D............. ..........E.D. Pl-071791 P1-070908 HCT84A (FW revertant) ....E..... ..D............. ..........E.D. Pl-071793 Pl- 0709 16-HCK16RT84A (FW revertant) ....E..... ..........E.D. Pl-071795 P1-070916 HCK16R (FW revertant) ....E..... ..........E.D. Pl-071797 P1-070916 HCT84A (FW revertant) ....E..... ..........E.D. Pl-072000 Pl- 061029 FloOffi V102D ..........E.D. Pl-072002 Pl-061029_F100ffi ..........E.. Pl-072004 Pl-061029_V102D .............D Pl-072006 Pl-061029 Y32E ......E... Pl-072008 Pl-061029 Y32E FlOOfE ......E... ..........E.. Pl-070916 Pl-068767 D52N E55A .....E..... ..........E.D. Pl-070908 Pl-068767 E55A .....E..... ..D............. ..........E.D. Pl-070906 Pl-068767_D52N .....E..... ......E......... ..........E.D. Pl-070868 Pl-068761 E55A .....HER... .......E......... ....NOT.. Pl-0687677 acidic pH-selective daughter clone '767 .....E..... ..D...E......... ..........E.D. Pl-0687617 acidic pH-selective daughter clone '761 .....HER... ......HER......... ....NOT.. Pl-0610296 GI original GFTLDDYA MN GINWNSANIGYADS VKG VPGYSGGWIDA FDV RASQSVSSSY LA GASSRA T QQYGSSPFT

Thus, in this example, anti-human VISTA antibodies were identified that bind to human VISTA with 200- to 10,000-fold greater affinity at acidic pH than at physiological pH. In cell-binding assays, these acidic pH-selective anti-VISTA antibodies exhibited an inflection point in binding intensity at approximately pH 6.5, similar to what was observed for VISTA binding to T cells.

Example 18 Anti-VISTA antibody and anti-PD-1 antibody act synergistically to induce tumor rejection.

To investigate the effects of blocking the acidic pH-selective contact region of the VISTA ligand in tumors, a surrogate mouse antibody, VISTA, was generated. 10, which blocks communication

- 120 046477 binding of mouse VISTA to murine T cells at acidic pH (VISTA. 10 also binds mVISTA at physiological pH). To avoid interaction with the Fc receptor and any downstream effector functions, VISTA. 10 was converted to the IgG1 isotype with a point mutation, D265A, to avoid Fc receptor interaction and effector functions {Clynes, 2000}. MC38 tumors were subcutaneously implanted into mice, and when the tumors reached approximately 70 mm ² , the mice were given the following therapy every three days: Group 1: 4 doses of anti-KLH antibody mIgG1-D265A at a dose of 30 mg/kg; Group 2: two doses of anti-PD-1 antibody mIgG1-D265A at a dose of 5 mg/kg; Group 3: 4 doses of anti-VISTA mIgG1-D265A antibody at a dose of 30 mg/kg; and Group 4: combination of anti-PD-1 antibody+anti-VISTA antibody. Combination therapy VISTA. 10 and a PD-1 blocking antibody induced tumor rejection in the majority of mice implanted with MC38 colorectal adenocarcinoma tumors (FIG. 15A-D), treatment with anti-PD-1 antibody alone and VISTA alone. 10 moderately delayed but did not prevent tumor progression (Fig. 15A-D).

Consistent with these results, ex vivo analysis of tumors from treated mice showed a 5- and 10-fold increase in the frequency of tumor-infiltrating CD8+ T cells and CD4+ T cells, respectively, in VISTA-treated mice. 10 and anti-PD-1 antibody (FIG. 15E-F). Other leukocyte subsets were generally not affected. Combination therapy also resulted in significantly lower expression of PD-1, LAG-3, and TIM-3, all markers of T cell exhaustion and dysfunction, on tumor-infiltrating CD8+ T cells (Figure 15E). Treatment with anti-PD-1 or anti-VISTA antibodies alone had only a modest effect on T cell frequency and phenotype (Figure 15E-F). The frequencies of intratumoral myeloid cell subsets, including macrophages, monocytic myeloid suppressor cells (MSCs), and granulocytic MSCs, were largely unaffected by treatment with anti-VISTA antibodies.

To examine the activity of anti-VISTA antibodies, VISTA knockout mice were implanted with MC38 tumors and treated with PD-1 blocking or control antibodies. As shown in FIG. 15I, in control treatment groups, MC38 tumors grew commensurately in VISTA knockout mice and their wild-type littermates. VISTA knockout mice exhibited increased sensitivity to anti-PD1 antibody, reminiscent of the effectiveness of VISTA and PD-1 combination. This sensitivity again correlated with increases in intratumoral CD4+ and CD8+ T cells. These data indicate that antibodies that block VISTA binding at acidic pH are sufficient to cause regression of VISTA-mediated immunosuppression.

Because VISTA functions selectively at acidic pH, it has been proposed that VISTA-mediated suppression of antitumor responses occurs predominantly in the tumor bed itself. We tested the activity of an acidic pH-selective blocking antibody against human VISTA, P1-068767 ('767), and its pH-nonselective parent antibody, P1-061029 ('029), in transgenic mice expressing the human VISTA extracellular domain instead of the endogenous VISTA extracellular domain (human VISTA knockin mice, genOway). Consistent with the combinations and VISTA knockout experiments described above, P1-061029 and P1-068767 demonstrated comparable efficacy when combined with a blocking mouse anti-PD-1 antibody (FIG. 15J-M).

The half-life of P1-068767 and P1-061029 in human VISTA knockin mice was measured. As shown in FIG. 15N, P1-068767 showed almost 20 times longer mean retention time (MRT) than P1-061029, indicating weak binding to VISTA at pH 7.4 and therefore a reduced TMDD (71 h and 4.1 h respectively).

To evaluate the interaction of antibodies with peripheral VISTA in a non-transgenic model, cynomolgus monkeys were administered P1-068767 and a neutral pH-selective antibody designated VISTA.4 (a separate family of patent applications by the same applicants provides additional description of VISTA.4) as follows. VISTA.4 and P1-068767 were assessed following 10-minute intravenous infusions in protein-naïve cynomolgus monkeys at a dose of 5 mg/kg (n=1 per antibody). Serial blood samples were collected at 0.17, 0.5, 2, 4, 6, 24, 48, 72, 168, 216, 240, 336 hours after infusion. Serum samples were then obtained for antibody concentration analysis using a ligand binding assay that used recombinant VISTA as capture agent and anti-human IgG Fc mAb as detection agent. The lower limit of quantitation for the assay was 1 ng/mL. Mean retention time was estimated by compartmentalized analysis of serum mAb concentration versus time data using Kinetica software (version 5.0, Thermo Fisher Scientific). The results show that P1-068767 again exhibited a much longer MRT (717 hours and 7.6 hours, respectively, FIG. 60). These results indicate that VISTA blockade in the tumor microenvironment, rather than in the blood and non-acidic tissues, enhances antitumor efficacy.

Example 19 VISTA.4 inhibits the binding of VISTA to PSGL-1.

The immunoreceptor glycoprotein ligand P-selectin 1 (PSGL-1) was previously identified as a VISTA ligand (see WO 2018132476). PSGL-1 is a receptor for selectins, particularly P-selectin, and binding to its major ligand, P-selectin, is a well-studied factor promoting adhesive interactions between leukocytes, platelets and endothelial cells (Carlow, D.A., et al., PSGL-1 function in immunity and steady state homeostasis. Immunol Rev,

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2009. 230(1): p. 75-96, and Abadier, M. and K. Ley, P-selectin glycoprotein ligand-1 in T cells. Curr Opin Hematol, 2017. 24(3): p. 265-273. 18). PSGL-1 has also been identified as a negative regulator of T cell response in relation to chronic viral infection, immunity against malignancy, and some autoimmune diseases (Angiari, S., et al., Regulatory T cells suppress the late phase of the immune response in lymph nodes through P-selectin glycoprotein ligand-1. J Immunol, 2013. 191(11): pp. 5489-500; Matsumoto, M., M. Miyasaka, and T. Hirata, P-selectin glycoprotein ligand-1 negatively regulates T-cells. immune responses. J Immunol, 2009. 183(11): p. 7204-11; Nunez-Andrade, N., et al., P-selectin glycoprotein ligand-1 modulates immune inflammatory responses in the enteric lamina propria. J Pathol, 2011. 224(2): p. 212-21; Perez-Frias, A., et al., Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice. Arthritis Rheumatol , 2014. 66(11): p.3178-89; Tinoco, R., et al., PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion. Immunity, 2016. 44(5): p. 1190-203). This immunosuppressive function appears to be independent of known PSGL-1 ligands (Tinoco, R., et al., PSGL-1: A New Player in the Immune Checkpoint Landscape. Trends Immunol, 2017. 38(5): p . 323-335).

In cell-based assays, recombinant PSGL-1 and recombinant P-selectin were shown to be able to block binding of VISTA multimer to activated human CD4+ T cells. Removal of PSGL-1 from activated CD4+ T cells by CRISPR also abolishes VISTA multimer binding. In addition, ectopic expression of PSGL-1 was shown to be sufficient to mediate VISTA binding to CHO cells at acidic pH, and expression of VISTA was sufficient to mediate PSGL-1 binding to 293T cells at acidic pH.

This Example shows that PSGL-1 bound P-selectin commensurately at acidic and physiological pH, but bound VISTA only at acidic pH (Fig. 17A). The experiment was carried out using Octet biosensor assays with VISTA, P-selectin and the minimal glycopeptide PSGL-1 (amino acids 1-19, with post-translational modifications of sulfothyrosine and sialyl-Lewis X carbohydrates), which was previously shown to retain high affinity binding of P- selectin (Sako, D., et al., A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding. Cell, 1995. 83(2): p. 323-319).

The PSGL-1 ligand contact region relies on post-translational modifications of negatively charged sulfothyrosines and sialyl-Lewis-X to bind P-selectin with high affinity (Sako et al. 1995 Cell 83(2): p. 323-319), and therefore naïve T cells that express PSGL-1 unmodified by sialyl-Lewis-X are unable to effectively interact with Pselectin. Modified sialyl-Lewis-X PSGL-1 is constitutively expressed on circulating monocytes and neutrophils and inducibly expressed on activated T cells, consistent with the strong binding of VISTA to these cell types at acidic pH. However, VISTA was found to bind to both naïve and activated T cells, suggesting that, unlike P-selectin, VISTA binds PSGL-1 independent of the presence of sialyl-Lewis-X. In additional assays on the Octet biosensor, it was found that while VISTA and P-selectin bound preferentially to PSGL-1 glycopeptides with sialyl-Lewis-X modification, only VISTA bound PSGL-1 glycopeptides without sialyl-Lewis-X. In addition, PSGL-1, produced in cells that do not express the enzymes glucosaminyl (N-acetyl) transferase (GCNT1) and alpha(1,3)-fucosyltransferase-7 (FUT7), does not have sialyl-Lewis-X modifications and binds poorly with Rselectin (Fig. 25A). In contrast, VISTA binds PSGL-1 independently of sialyl-Lewis-X (Fig. 25A). This result is consistent with the fact that VISTA, but not P-selectin, binds to naïve T cells lacking sialyl-Lewis-X.

Also similar to P-selectin, VISTA binds moderately to heparan sulfate at acidic pH.

In addition, anti-PSGL-1 antibodies that block P-selectin binding did not block VISTA binding. These data indicate that VISTA binds a contact region of PSGL-1 that is similar to, but distinct from, the contact region bound by P-selectin.

This example also shows that antibodies P1-061029, P1-068761, P1-068767 and VISTA.4, which block VISTA binding to T cells, also blocked VISTA binding to PSGL-1 glycopeptide.

Octet competition assays were performed to evaluate whether the acid pH-selective antibodies to α-VISTA, P1-068761 and P1-068767, the pH-independent parent antibody P1-061029, and the acid pH-sensitive VISTA.4 blocked VISTA binding to PSGL1. Binding assays were performed on an OctetRed384 biolayer interferometry (BLI) instrument (PALL/ForteBio). All assay steps were performed at 30°C with shaking at 1000 rpm using PBST buffer, pH 6.0 (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, 0.05% Tween 20 ). Human VISTA-Fc (R&D Systems 7126-B7) was diluted to 400 nM in PBST pH 6.0 and premixed for 30 min with titration series of 0 nM, 40 nM and 400 nM P1-068761, P1-068767, P1-061029 and VISTA.4. Human PSGL1 19-mer-huFc protein, consisting of the 19 N-terminal amino acids of mature PSGL1 fused to human Fc, was coupled to anti-human IgG-Fc antibody sensors (ANS, PALL/ForteBio). Anti-human Fc sensors were then blocked with total human IgG (Jackson

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009-000-002). Binding of captured PSGL1 to the VISTA-Fc/a-VISTA antibody mixture was then measured to assess whether anti-α-VISTA antibodies interfered with the binding of VISTA to PSGL1. For each antibody titration series, the magnitude of VISTA binding to PSGL1 (shift in nm) was normalized to the 0 nM unblocked VISTA:PSGL1 response set to 100%. The results of this analysis are presented in Fig. 17V. In this assay, P1-061029, P1-068761, P1-068767, and VISTA.4 all showed blocking activity at 400 nM. Binding of the VISTA-Fc protein to the captured human PSGL1-19-mer-huFc protein was prevented, as indicated by the observed reduced VISTA binding.

In addition, it was demonstrated that the binding of VISTA to CHO-PSGL-I cells was blocked by both VISTA.4 and the P-selectin blocking antibody to PSGL-1, KPL-1 (Fig. 17C). In PSGL-1 antibody blocking assays, cells were preincubated with the indicated KPL1 (BD Biosciences or Biolegend) or PL2 (MBL) antibodies before labeling with 32 nM VISTA multimers or VISTA-Fc chimeric proteins. VISTA-Fc binding was detected using anti-IgG (Jackson ImmunoResearch) or anti-6™his (Columbia Biosciences) antibodies. Cells were imaged using flow cytometry or homogeneous time-resolved fluorescence (HTRF).

Example 20: Crystal structure of P1-068767 bound to hVISTA

To study the structure of VISTA and the molecular determinants of binding of antibodies to VISTA, a cocrystal of the IgV domain of hVISTA was obtained with the antigen-binding fragment (Fab) P1-068767. The structure of the resulting complex was determined with a resolution of 1.6 A (Fig. 18). In general, the IgV VISTA domain is characteristic of this family and has some similarity to PD-L1 (Fig. 18B). However, unlike PD-L1 and most other members of the B7 family or immunoglobulin superfamily, the two C-terminal β-chains of the IgV VISTA domain contain many additional residues, resulting in an unusually elongated and histidine-rich central β-sheet (Fig. 18B). . The blocking antibody P1-068767 binds VISTA in this β-sheet extension (Fig. 18C), whereas the non-blocking antibody VISTA. 5 connects another region (Fig. 18E). The VISTA β-sheet extension is capped by three histidine residues: H121, H122, and H123. Residues P1-068767, E110 and D112, form hydrogen bonds with VISTA residues H121 and H122, respectively (Fig. 18D). These interactions are in good agreement with the data described in the previous Examples, according to which residues P1-068767, E110 and D112, are necessary and sufficient for selectivity with respect to acidic pH. In the cocrystal, the H123 VISTA residue interacts with a sulfate molecule from the precipitating reagent and forms a salt bridge with the E1 residue in P1-068767; although it is possible that H123 VISTA can form a strong hydrogen bond with P1-068767 in the absence of sulfate (Figure 18D). Additional interactions are presented in Table. 24 These data indicate that the unusual histidine-rich β-sheet extension in the IgV domain of VISTA is a key component of the acidic pH-selective VISTA receptor-ligand interface region.

In table 24 details the distances in angstroms (A) between the HC '767 Fab atoms within 4 A of the VISTA atoms.

Table 24

## 1 '767 Fab ns | Dist. VISTA 1 1 eleven H:GLU 1 [OE1] | 3, 1 V:HIS 123 [ΝΞ2] 2 1 H:VAL 2 [N]| 3.2 V:HIS 123 [0] 3 1 H:GLY 26 [Oil 3, 1 V:GLU 125 [N] 4 1 H:GLU thirty toil 3, 3 V:ARG 54 [NH2] 5 1 H:GLU thirty [OE1] | 3, 8 V:ARG 127 [NE] 6 1 H:GLU thirty [OE1] | 3, 2 V:ARG 127 [NH2] Ί 1 H:GLU thirty [OE2] | 3, 4 V:ARG 127 [NH1] 8 1 H:GLU thirty [OE2] | 3, 5 V:ARG 127 [NH2] 9 | H:ASP 31 [GDI] | 2, 8 V:ARG 54 [NH1] 10 | H:ASP 31 [OD1] | 2.7 V:ARG 54 [NH2] 11 | H:ASP 31 [OD1] | 2, 8 V:ARG 127 [NH1] 12 | H:ASP 31 [OD2] | 3, 8 V:ARG 127 [NE] 13 | H:ASP 31 [OD2] | 3, 1 V:ARG 127 [NH1] 14 | H:TYR 32 [OH] 1 2, 6 V:GLU 125 [OE1] 15 | H:GLU 110 [OE2] | 2, 8 V:HIS 122 [N] 16 | H:GLU 110 [OE1] | 2.7 V:HIS 121 [ND1] 17 | H:GLU 110 [OE2] | 3, 8 V:HIS 121 [ND1] 18 | H:GLU 110 [OE2] | 3, 5 V:HIS 122 [ND1] 19 | H:ASP 111 [OD1] | 3, 6 V:HIS 122 [NE2] 20 | < H:ASP 112 [OD1] | T 3, 5 V:HIS 122 [ND1]

Example 21: Epitope Mapping VISTA.4.

VISTA.4 was used in the BLI epitope binning competition assay described previously in Example 15. The results indicate that VISTA.4 competes for binding of human VISTA with the antibodies described above, P1-061015, P1-061029, P1-068761 and P1-068767 , and thus belongs to the same

- 123 046477 epitope group, like these antibodies (Group A). VISTA.4 does not compete for binding of human VISTA to VISTA mAb 1 (see Fig. 11A).

The VISTA.4 epitope was also mapped using yeast surface display and NGS as described in Example 15 for antibodies P1-061015, P1-061029, P1-068761 and P1-068767. VISTA mutants that lost binding to the mapping antibody but retained binding to the non-blocking antibody (mAb1) were sorted and sequenced. Because they retained binding to mAb1, these mutants appeared to be correctly folded, and the loss of binding observed for the mapped antibody was likely caused by the loss of an energetically important contact residue. The positions of the mutations that resulted in loss of binding and that were identified as energetically important residues in the antibody epitope are shown in Table 1. 31, together with the energetically important contact residues of antibodies P1-061015, P1-061029, P1-068761 and P1-068767 (which are also shown in Table 14 above).

Table 31

Energetically important VISTA.4 contact residues of antibodies '029, '015, '761 and '767'

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to examine the binding epitopes of human VISTA to mAb VISTA.4. HDX-MS probes protein structure and conformational dynamics in solution by monitoring the rate and extent of exchange of deuterium atoms with hydrogen atoms of skeletal amide groups (Huang and Chen (2014) Analytical and Bioanalytical Chemistry 406, 6541-6558; Wei, et al. Drug Discovery Today (2014) 19, 95-102). The level of HDX depends on the accessibility of the hydrogen atoms of the skeletal amide groups and the hydrogen bonds of the protein to the solvent. The increase in protein mass in HDX can be accurately measured using MS. By combining this method with enzymatic digestion, structural features can be determined at the peptide level, allowing one to distinguish surface-exposed peptides from peptides folded inward or from peptides isolated at the protein:protein complex interface. Typically, deuterium labeling experiments followed by stopping the reaction are performed followed by enzymatic digestion, peptide separation, and MS analysis.

Before epitope mapping experiments, experiments were performed without deuteration to obtain a list of common peptides for recombinant human VISTA (15 μM) and VISTA protein complexes with mAb VISTA.4 (1:1 molar ratio). For the HDX-MS experiment, 5 μL of each sample (VISTA or VISTA with mAb VISTA.4) was diluted in 55 μL of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0) to initiate labeling reactions. Reactions were carried out for different periods of time: 1 min, 10 min and 240 min. At the end of each labeling reaction period, the reaction was stopped by adding stopping buffer (100 mM phosphate buffer with 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v/v) and 50 μl of the sample after stopping the reaction was injected into Waters HDX-MS system for analysis. Deuterium uptake levels of conventional peptides were monitored in the absence/presence of VISTA.4. The resulting sequence coverage was 82%.

HDX-MS experiments provided 85% coverage of human VISTA sequences. As shown in FIG. 19, analysis of HDX-MS data for VISTA.4 in human VISTA shows that the VISTA.4 epitope consists of three human VISTA regions, with region 2 being the major epitope (residue numbers correspond to the native human VISTA sequence, FIG. 20).

Region 1: 57LGPVDKGHDVTF ⁶⁸ (SEQ ID NO: 566).

Region 2: 86RRPIRNLTFQDL ⁹⁷ (SEQ ID NO: 567).

Region 3: ¹⁴⁸ VVEIRHHHSEHRVHGAME ¹⁶⁵ (SEQ ID NO: 568).

The VISTA.4 antibody binds equally well at acidic and neutral pH. Subsequent rounds of selection yielded a variant that bound VISTA with 200-fold higher affinity at pH 6.0 than at pH 7.4. Similar efforts with VISTA blocking antibodies resulted in variants with selectivity up to 10,000-fold for pH 6.0 over pH 7.4. These antibodies were used to map the VISTA receptor-ligand binding region at acidic and neutral pH. pH-independent, neutral-pH-selective, and acid-pH-selective blocking antibodies to VISTA bound nearly identical epitopes, suggesting that histidine protonation alone, without detectable conformational changes, regulates the ability of VISTA to interact with its counterreceptor under acidic conditions. pH (VISTA.4 is also described in another family of applications by the same applicants).

Example 22 VISTA.4 inhibits VISTA binding to T cells at acidic pH.

This example shows that VISTA.4 and other antibodies in the epitope group A blocked the binding

- 124 046477 VISTA binding to T cells at acidic pH, whereas VISTA.5 (mAb1) and other antibodies in the B epitope group did not block such binding (Fig. 21A and B).

This example was carried out essentially in the same way as described in Example 4.

Example 23 VISTA.4 increases T cell proliferation and IFN-γ production.

This example shows that VISTA blocking antibodies (Epitope Group A) increased T cell proliferation and IFN-γ production (FIG. 22). VISTA.5 (mAb1), which is not a blocking antibody, did not increase T cell proliferation or IFN-γ production. VISTA.4 had no effect when T cells were co-cultured with 293T-OKT3 cells, which did not express VISTA.

This experiment was performed by adding anti-VISTA antibodies to CD4+ T cells co-cultured with 293T cells modified to express human VISTA and a single-chain variable fragment of an anti-T cell receptor OKT3 agonist antibody (293TOKT3-VISTA).

Example 24 VISTA inhibits T-cell receptor-mediated NF-kB signaling.

This example was conducted to further evaluate the effect of pH on VISTA function and showed that VISTA more effectively suppressed T cell receptor-mediated NFkB signaling at acidic pH than at neutral pH (Figure 23A). Maximum suppression was achieved at pH below 6.5, similar to VISTA:T cell binding (Figure 23A and Figure 4A).

NF-kB signaling was measured using Jurkat NFkB reporter T cells essentially as described in Examples 5 and 13. Jurkat cells were modified to express luciferase under the control of an NF-kB inducible promoter. These NFkB-luciferase Jurkat cells were cocultured with non-irradiated 293T-OKT3-VISTA cells at a 4:1 ratio in HBSS (ThermoFisher) acidified to various pH values using MES and human anti-human VISTA antibodies for 4 h. Activation Jurkat cells were measured using a luciferase substrate assay (Promega).

VISTA-mediated suppression was strongest at acidic pH, although moderate levels of activity persisted at pH 7.0 and above. Likewise, recombinant VISTA suppressed NFkB phosphorylation of T cells at acidic pH more than at pH 7.4 (Fig. 23B). These results indicate that VISTA inhibits T cells preferentially at acidic pH and that this activity is abolished by antibodies that block T cell binding at acidic pH.

Thus, these data indicate that blockade of the acidic pH-selective VISTA receptor-ligand interface may relieve immunosuppression.

Example 25 The binding specificity of VISTA:PSGL-1 is determined by histidine and sulfothyrosine residues.

To analyze the binding specificity of PSGL-1, in addition to the specificity provided by sialyl-Lewis X (described in Example 19), post-translational modifications of tyrosine sulfation that promote P-selectin binding were examined. To test the role of tyrosine sulfation, PSGL-1 glycopeptides were fractionated into sulfothyrosine-rich (>90%) and sulfothyrosine-poor (<1%) peaks using anion-exchange liquid chromatography. Neither VISTA nor P-selectin bound at a detectable level to sulfothyrosine-poor PSGL-1 (Fig. 25B). VISTA also failed to bind PSGL-1 glycopeptides in which tyrosines were replaced by alanines (not shown in the figures). These results indicate that sulfothyrosine residues are key mediators of PSGL-1 binding to VISTA.

It was further proposed that VISTA binding specificity is mediated by the same histidine residues found in the VISTA blocking antibody epitope: H153, H154 and H155 (see Example 20). Replacement of these histidine residues with uncharged alanine or negatively charged aspartic acid significantly reduced VISTA binding to recombinant PSGL-1 and CHO-PSGL-I cells (Figure 25C-D). VISTA-Fc proteins with mutation of residues H153, H154 and H155 to alanine, aspartic acid or arginine were obtained by transient transfection of Expi293 cells. These mutants were also unable to induce functional T cell suppression (not shown in the figures). In contrast, substitution with positively charged arginine residues preserved VTSTA binding and function (Figure 25C-D). The blocking antibodies VISTA.4 and P1-068767 bound well to the alanine and arginine mutant of VISTA, but poorly bound to the VISTA mutant containing an aspartic acid substitution (not shown). The nonblocking antibody VISTA.5 bound commensurately to wild-type and mutant VISTA proteins (not shown).

The established structures of PSGL-1 bound to P-selectin and VISTA bound to Fab P1068767 (Figure 18) were then used to develop a computer model of the 19-mer glycopeptide PSGL-1 linked to VISTA (Figure 25E). In this model, the PSGL-1 tyrosine residues, Y46 and Y48, make ionic interactions with the VISTA histidine residues, H153 and H154 (distances 2.5-3.0 A). Residue Y51 of PSGL-1 is further removed from VISTA (~4.5 A) but can interact significantly with VISTA H100. Residue E56 of PSGL-1 also forms ionic interactions with VISTA H98 and H100. Hydroxyl group

- 125 046477

T57 PSGL-1, which can be modified by sialyl-Lewis X, is directed away from VISTA, consistent with the negligible effect of sialyl-Lewis X on VISTA:PSGL-1 binding. Taken together, these data and modeling suggest that the binding of VISTA to PSGL-1 at acidic pH is due primarily to the histidine residues VISTA and H153, H154, and H155, as well as the sulfated tyrosine residues Y46 and Y48 of PSGL-1.

Sequence table

Below is a table of some of the sequences provided in the present invention. In SEQ ID NO: 2, amino acid position 187 may contain either D or E.

In the antibody sequences below, the CDR1, CDR2 and CDR3 VH sequences are located at amino acid positions including amino acids 26-35, 50-66 and 99-110, respectively, and the CDR1, CDR2 and CDR3 VL sequences are located at amino acid positions including amino acids 24- 35, 51-57 and 90-98, respectively. CDR1 VH numbering corresponds to AbM (AA 26-35; Abhinandan and Martin (2008) Mol. Immunol. 45:3832-3839; Swindells et al. (2017) J. Mol. Biol. 429: 356-364), and all others CDRs (CDR2 VH, CDR3 VH, CDR1-3 VL) are numbered according to Kabat. The CDR sequences of specific antibodies are in bold and underlined below on their VH and VL sequences.

SEQ ID NO Name Subsequence 1 hVISTA (with leader sequence) MGVPTALEAG SWRWGSLLFA LFLAASLGPV AAFKVATPYS LYVCPEGONV TLTCRLLGPV DKGHDVTFYK TWYRSSRGEV OTCSERRPIR NLTFODLHLH HGGHOAANTS HDLAORHGLE SASDHHGNFS ITMRNLTLLD SGLYCCLVVE IRHHHSi:i IRV 1IGAMI I OVQT GKI)APSXC\ V YPSSSQDSEN ITAAALATGA CIVGILCLPL illlvykqrq aasnrraqel vrmdsniqgi ENPGFEASPP AQGIPEAKVR HPLSYVAQRQ PSESGRHLLS EPSTPLSPPGPGDVFFPSLD PVPDSPNFEV I 2 hVISTA (without leader sequence) FKVATPYSLY VCPEGONVTL TCRLLGPVDK GHDVTFYKTW YRSSRGEVOT CSERRPIRNL TFODLHLHHG GHOAANTSHD LAORHGLESA SDHHGNFSIT MRNLTLLDSG LYCCLVVEIR HHHSEHRVHG AMELOVQTGKDAPSNCVVYP SSSOID/EISENIT AAALATGACI VGILCLPLILLLVYKQRQAA SNRRAQELVR

- 126 046477

MDSNIQGIEN PGFEASPPAQ GIPEAKVRHP LSYVAQRQPS ESGRHLLSEP STPLSPPGPGDVFFPSLDPV PDSPNFEVI 3 Human PSGL-1 isoform 2 precursor, with signal peptide MPLQLLLLLILLGPGNSLQL WDTWADEAEK ALGPLLARDR RQATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS TGLDAGGAVT ELTTELANMGNLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE AQTTRLTATE AQTTPLAATE AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQT TAPAAME AQTTPPAAME AQTTQTTAME AQTTAPEATE AQTTQPTATE AQTTPLAAME ALSTEPSATE ALSMEPTTKR GLFIPFSVSS VTHKGIPMAA SNLSVNYPVG APDHISVKQC LLAILILALV ATIFFVCTVV LAVRLSRKGH MYPVRNYSPT EMVCISSLLP DGGEGPSATA NGGLSKAKSP GLTPEPREDR EGDDLTLHSF LP 4 Human PSGL-1 isoform 2, no signal peptide LQL WDTWADEAEK ALGPLLARDR RQATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS TGLDAGGAVT ELTTELANMG NLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE AQTTRLTATE AQTTPLAATE AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQTTAPAAME AQTTPPAAME AQTTQTTAME AQTTAPEATE AQTTQPTATE AQTTPLAAME ALSTEPSATE ALSMEPTTKR GLFIPFSVSS VTHKGIPMAA SNLSVNYPVG APDHISVKQC LL AILILALV ATIFFVCTVV LAVRLSRKGH MYPVRNYSPT EMVCISSLLP DGGEGPSATA NGGLSKAKSP GLTPEPREDR EGDDLTLHSF LP 5 ECD of human PSGL1 isoform 2, with signal peptide MPLQLLLLLI LLGPGNSLQL WDTWADEAEK ALGPLLARDR RQATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS TGLDAGGAVT ELTTELANMGNLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE AQTTRLTATE AQTTPLAATE AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQ TTAPAAME AQTTPPAAME AQTTQT 6 ECD of human PSGL1 isoform 2, without signal peptide A QTTQT 7 ECD of human PSGL-1 (N-terminal positions 42-295 of full-length human PSGL-1, trans. QATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS TGLDAGGAVT ELTTELANMGNLSTDSAAME IQTTQPAATE AQTTPLAATE AQTTRLTATE AQTTPLAATE

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number AAC50061) AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQTTAPAAME AQTTPPAAME AQTTQTTAME AQTTAPEATE AQTTQPTATE AQTTPLAAME ALSTEPSATE ALSMEPTTKR GLFIPFSVSS VTHKGIPMAA SNLSV 8 HumPSGL1 isoform 1 precursor, with signal peptide ΝΡ_001193538 MAVGASGLEG DKMAGAMPLQ LLLLLILLGP GNSLQLWDTW ADEAEKALGP LLARDRRQAT EYEYLDYDFL PETEPPEMLR NSTDTTPLTG PGTPESTTVE PAARRSTGLD AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT PLAATEAQTT RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEA QTT APAAMEAQTT APAAMEAQTT PPAAMEAQTT QTTAMEAQTT APEATEAQTT QPTATEAQTT PLAAMEALST EPSATEALSM EPTTKRGLFI PFSVSSVTHK GIPMAASNLS VNYPVGAPDH ISVKQCLLAILALVATIF FVCTVVLAVR LSRKGHMYPV RNYSPTEMVC ISSLLPDGGE GPSATANGGL SKAKSPGLTP EPREDREGDD LTLHSFLP 9 Human PSGL-1, no signal peptide LQLWDTW ADEAEKALGP LLARDRRQAT EYEYLDYDFL PETEPPEMLR NSTDTTPLTG PGTPESTTVE PAARRSTGLD AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT PLAATEAQTT RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEAQTT APAAMEAQTT APAAMEAQTT PPAAMEAQTT QTTAMEAQTT APEATEAQTT QPTATEAQTT PLAAMEALST EPSATEALSM EPTTKRGLFI PFSVSSVTHK GIPMAASNLS VNYPVGAPDH ISVKQCLLAI LILALVATIF FVCTVVLAVR LSRKGHMYPV RNYSPTEMVC ISSLLPDGGE GPSATANGGL SKAKSPGLTP EPREDREGDD LTLHSFLP 10 Human PSGL-1 ECD, with signal peptide MAVGASGLEG DKMAGAMPLQ LLLLLILLGP GNSLQLWDTW ADEAEKALGP LLARDRRQAT EYEYLDYDFL PETEPPEMLR NSTDTTPLTG PGTPESTTVE PAARRSTGLD AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT PLAATEAQTT RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEA QTT APAAMEAQTT APAAMEAQTT PPAAMEAQTT QT AND Pl-069059 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSDHIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 12 Р1-069059 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 13 Pl-069059 IgG1.3 NS EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW

- 128 046477

VRQAPGKGLEWVSGINWNSDHIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTH TCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 14 Pl-069059 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 15 Pl-069061 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 16 Pl-069061 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYC0OYGSSPFTFGPGTKVDIK 17 Pl-069061 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 18 Pl-069061 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 19 Pl-069063 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDADDEWGQGTMVTVSS 20 Pl-069063 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS

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RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 21 Pl-069063 IgG1.3 NS EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA DDEWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 22 Pl-069063 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 23 Pl-069065 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSDDIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAFDVWGQGTMVTVSS 24 Pl-069065 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 25 Pl-069065 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSDDIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 26 Pl-069065 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 27 P1-069067 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDADDEWGQGTMVTVSS

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28 P1-069067 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 29 P1-069067 IgG1.3 NS EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA DDEWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG thirty Pl-069067 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 31 P1-069069 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW ID ADD VWGQGTMVT VS S 32 P1-069069 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 33 P1-069069 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA DDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 34 Pl-069069 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 35 Pl-069071 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVRQAPGKGLEWVSGINWNSADIGYADSVKGRFTIS

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RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSEGW IDAFDVWGQGTMVTVSS 36 Pl-069071 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 37 Pl-069071 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSADIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSEGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 38 Pl-069071 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 39 Pl-069073 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 40 Pl-069073 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 41 Pl-069073 IgGL3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 42 Pl-069073 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC

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43 Pl-069075 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWDSAEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 44 Pl-069075 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 45 Pl-069075 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWDSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 46 Pl-069075 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 47 P1-069077 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSDEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 48 P1-069077 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 49 P1 -069077 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSDEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 50 Pl-069077 LC EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

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NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 51 Pl-068761 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 52 Pl-068761 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 53 Pl-068761 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 54 Pl-068761 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 55 Pl-068767 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 56 P1-068767 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 57 Pl-068767 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 58 Pl-068767 LC EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS

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RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 59 Pl-068773 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSDNIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 60 Pl-068773 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 61 Pl-068773 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSDNIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 62 Pl-068773 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 63 Pl-068765 VH EVQLVESGGGLVQPGKSLRLSCAASGFTDEDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 64 Pl-068765 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 65 Pl-068765 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTDEDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG

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66 Pl-068765 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 67 Pl-061029 VH EVOLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSANIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAFDVWGQGTMVTVSS 68 P1-061029 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 69 P1-061029 IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 70 Pl-061029 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 71 Pl-068757 VH EVOLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 72 Pl-068757 VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 73 Pl-068757 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD

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SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 74 Pl-068757 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 75 Pl-068771 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSHEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 76 Pl-068771 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 77 Pl-068771 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSHEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 78 Pl-068771 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 79 Pl-068775 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGIDWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDDWGQGTMVTVSS 80 Pl-068775 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 81 Pl-068775 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGIDWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

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NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 82 Pl-068775 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 83 Pl-068769 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSDHIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 84 P1-068769 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 85 Pl-068769 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSDHIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 86 Pl-068769 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 87 Pl-068759 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 88 Pl-068759 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 89 Pl-068759 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM

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ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH Y TQKSLSLSPG 90 Pl-068759 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 91 Pl-068763 VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 92 Pl-068763 VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 93 Pl-068763 IgG1.3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 94 Pl-068763 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 95 Pl-061015 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHW VRQAPGKGLEWVAIIWYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYSSYYF DYWGQGTLVTVSS 96 Pl-061015 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 97 Pl-061015 IgG1.3HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHW VRQAPGKGLEWVAIIWYDGSNKYYADSVKGRRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYSSYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

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L YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 98 Pl-061015 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 99 Pl-068748 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSHHAMH WVROAPGKGLEWVAIIWYDGSNDDYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYY FDYWGQGTLVTVSS 100 Pl-068748 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIK 101 Pl-068748 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSHHAMHW VRQAPGKGLEWVAIIWYDGSNDDYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 102 Pl-068748 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 103 P1-068744 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSEYAMH WVRQAPGKGLEWVAHIWYDGSNKYEADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARDSGFYESY YFDEWGQGTL VT VS S 104 P1-068744 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 105 P1-068744 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGTFFSEYAMHW VRQAPGKGLEWVAHIWYDGSNKYEADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYESYYF

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DEWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT K PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 106 Ρ1-068744 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 107 Pl-068736 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSEYAMH WVROAPGKGLEWVAIDWYDGSNKDYADSVKGRFTI SRDNSKNTLYLOMNSLRAEDTAVYYCARDSGFYDSY YFDDWGQGTL VT VS S 108 Pl-068736 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 109 Pl-068736 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSEYAMHW VRQAPGI<GLEWVAIDWYDGSNI<DYADSVI<GRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 110 Pl-068736 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 111 Pl-068752 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHW VRQAPGKGLEWVAEIWYDGSNKDYADSVKGRFTIS RDNSKNTLYLOMNSLRAEDTAVYYCARDSGFYDSYY FDEWGQGTLVTVSS 112 Pl-068752 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK FROM Pl-068752 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFFSSYAMHW

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VRQAPGI<GLEWVAEIWYDGSNI<DYADSVI<GRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DEWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVD KRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 114 Pl-068752 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 115 Pl-068740 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSDYAMH WVROAPGKGLEWVAIIWYDGSDKDYADSVKGRFTI SRDNSKNTLYLOMNSLRAEDTAVYYCARDSGFYDSY YFDDWGQGTL VT VS S 116 P1-068740 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 117 Pl-068740 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYAMHW VRQAPGKGLEWVAIIWYDGSDKDYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S V VTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 118 Pl-068740 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 119 P1-068742 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSDYAMH WVROAPGKGLEWVAIIWYDGSDKDYADSVKGRFTI SRDNSKNTLYLOMNSLRAEDTAVYYCARDSGFYEDY YFD YWGQGTL VT VS S 120 P1-068742 VL EIVLTOSPATLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS

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SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 121 P1-068742 IgG1.3 NS QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYAMHW VRQAPGKGLEWVAIIWYDGSDKDYADSVKGRRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYEDYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S V VTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 122 Pl-068742 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 123 Pl-068746 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHW VRQAPGKGLEWVAIIWYDGSNHHYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DYWGQGTLVTVSS 124 P1-068746 VL EIVLTQ SPATLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 125 Pl-068746 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHW VRQAPGKGLEWVAIIWYDGSNHHYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFYDSYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S V VTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 126 Pl-068746 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 127 Pl-068750 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSDYDMH WVROAPGKGLEWVAEIWDDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARDEEFYSSY YFD YWGQGTL VT VS S

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128 Pl-068750 VL EIVLTQ SPATLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIK 129 Pl-068750 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYDMHW VRQAPGI<GLEWVAEIWDDGSNI<YYADSVI<GRFTISR DNSKNTL YLQMNSLRAEDT AVY YC ARDEEF YS S YYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L Y SLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 130 Pl-068750 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 131 Pl-068738 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSEYAHHW VRQAPGKGLEWVAIIWDDGSNHYYADSVKGRFTISR DNSKNTL YLQMNSLRAEDTAVYYCARDSGFYEDYY FDYWGQGTLVTVSS 132 Pl-068738 VL EIVLTQ SPATLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYC90YNSYPYTFGQGTKLEIK 133 Pl-068738 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSEYAHHW VRQAPGKGLEWVAIIWDDGSNHYYADSVKGRFTISR DNSKNTL YLQMNSLRAEDTAVYYCARDSGFYED YYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S V VTVP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 134 Pl-068738 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 135 Pl-068754 VH OVOLVESGGGVVOPGRSLRLSCAASGFTFSDYDMH WVRQAPGKGLEWVAEIWDDGSNKYYADSVKGRFTI

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SRDNSKNTLYLQMNSLRAEDTAVYYCARDSGFHSDY YFD YWGQGTL VT VS S 136 Pl-068754 VL EIVLTQ SPATLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQOYNSYPYTFGQGTKLEIK 137 Pl-068754 IgG1.3 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYDMHW VRQAPGKGLEWVAEIWDDGSNKYYADSVKGRRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDSGFHSDYYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L YSLS S VVT VP S S SLGTQT YICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVE VEIN ACT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 138 Pl-068754 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC 139 Pl-069293 VH (Pl-06876LIgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 140 Pl-069293 VL (Pl-06876LIgGlf) EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 141 Pl-069293 HC (Pl-06876LIgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 142 Pl-069293 LC (Pl-06876LIgGlf) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC

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143 Pl-069298 VH (Pl-068767.IgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 144 Pl-069298 VL (Pl-068767.IgGlf) EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 145 Pl-069298 HC (Pl-068767.IgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 146 Pl-069298 LC (Pl-068767.IgGlf) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 147 Pl-069302 VH (Pl-061029.IgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSANIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAFDVWGQGTMVTVSS 148 P1-0693 02 VL (Pl-061029.IgGlf) EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 149 Pl-069302 HC (Pl-061029.IgGlf) EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 150 Pl-069302 LC (Pl-061029.IgGlf) EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

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NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 151 Pl-069312 VH (Pl-068761. IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 152 Pl-069312 VL (Pl-068761.IgGlf afucosylated) EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 153 Pl-069312 HC (Pl-068761.IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 154 Pl-069312 LC (Pl-068761.IgGlf afucosylated) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 155 Pl-069309 VH (Pl-068767.IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 156 P1-0693 09 VL (Pl-068767.IgGlf afucosylated) EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 157 Pl-069309 HC (Pl-068767.IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 158 Pl-069309 LC (Pl-068767.IgGlf EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS

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afucosylated) RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 159 P1-069307VH (Pl-061029.IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAFDVWGQGTMVTVSS 160 P1-069307VL (Pl-061029.IgGlf afucosylated) EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 161 P1-069307NS (Pl-061029.IgGlf afucosylated) EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 162 Pl-069307 LC (Pl-061029.IgGlf afucosylated) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 163 IgG1.3 (or!gG1.3f) heavy chain constant region (L234A, L235E, G237A) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 164 Approximate light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SP VTKSFNRGEC 165 Human VISTA NP_071436.1 (Fig. 1B) See FIG. I.B. 166 Soup VISTA ХР 005565644.1 (Fig. 1B) See FIG. I.B. 167 Mouse VISTA NP 083008.1 See FIG. I.B.

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(Fig. IB) 168 Pl-061029 HDR3 chip oligo (Figure 7A) XPGYSGGWIDAFDV 169 Pl-061029 HDR3 chip oligo (Figure 7A) XXGYSGGWIDAFDV 170 P1-061029 HDR3 chip oligo (Fig. 7A) XPXYSGGWIDAFDV 171 P1-061029 HDR3 chip oligo (Fig. 7A) XPGXSGGWIDAFDV 172 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYXGGWIDAFDV 173 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSXGWIDAFDV 174 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGXWIDAFDV 175 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGXIDAFDV 176 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWXDAFDV 177 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWIXAFDV 178 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWIDXFDV 179 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWIDAXDV 180 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWIDAFXV 181 P1-061029 HDR3 chip oligo (Fig. 7A) XPGYSGGWIDAFDX 182 IgGIf (human wild-type f allotype) heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

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EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 183 IgGl.lf Heavy chain constant region (L234A, L235E, G237A, A330S, P331S) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 184 IgGlfa.P238K (or IgGl.P238K) heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 185 Р1-070864 P1-068761 E30D VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 186 Р1-070864 P1-068761 E30D VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 187 P1-070864 P1-068761 E30D IgG1.3 NS EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 188 P1-070864 P1-068761 E30D LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 189 Р1-070866 Р1-068761 E32Y VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS

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190 Pl-070866 Pl-068761-E32Y VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 191 Pl-070866 Pl-068761-E32Y IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 192 Pl-070866 Pl-068761-E32Y L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 193 Pl-070868 Pl-068761_E55A VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 194 Pl-070868 Pl-068761_E55A VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 195 Pl-070868 Pl-068761_E55A IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 196 Pl-070868 Pl-068761_E55A LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 197 Pl-070870 Pl-068761 E56N EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVRQAPGKGLEWVSGINWNSENIGYADSVKGRFTIS

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VH RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 198 Pl-070870 Pl-068761_E56N VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 199 Pl-070870 Pl-068761_E56N IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 200 Pl-070870 Pl-068761_E56N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 201 Pl-070872 Pl-068761H100G VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 202 Pl-070872 Pl-068761H100G VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 203 Pl-070872 Pl-068761H100G IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 204 Pl-070872 Pl-068761H100G L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC

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205 Pl-070874 Pl-06876 lElOOfF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAFDVWGQGTMVTVSS 206 Pl-070874 Pl-06876 lElOOfF VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO OKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 207 Pl-070874 Pl-06876 lElOOfF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 208 Pl-070874 Pl-06876 lElOOfF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 209 Pl-070876 P1-068761 E30DE32Y VH EVOLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 210 Pl-070876 P1-068761 E30DE32Y VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 211 Pl-070876 P1-068761 E30DE32Y IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 212 Pl-070876 P1-068761 E30DE32Y L.C. EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

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NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 213 Pl-070878 Ρ1-068761_Ε55Α_Ε56Ν VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 214 Pl-070878 Pl-068761_E55A_E56N VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 215 Pl-070878 Pl-068761_E55A_E56N IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSLS S V VT VP S S SLGTQT YICNVNHKP SNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 216 Pl-070878 Pl-068761_E55A_E56N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 217 Pl-070880 Pl- 068761_H100G_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 218 Pl-070880 Pl- 068761_H100G_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCOQYGSSPFTFGPGTKVDIK 219 Pl-070880 Pl- 068761_H100G_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 220 Pl-070880 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ

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Pl- 068761_H100G_E100fF L.C. QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 221 Pl-070882 Pl-068761_E32Y_E55A VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 222 Pl-070882 Pl-068761_E32Y_E55A VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 223 Pl-070882 Pl-068761_E32Y_E55A IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 224 Pl-070882 Pl-068761_E32Y_E55A L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 225 Pl-070884 Pl-068761_E32Y_E56N VH EVOLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 226 Pl-070884 Pl-068761_E32Y_E56N VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 227 Pl-070884 Pl-068761_E32Y_E56N IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

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YTQKSLSLSPG 228 Pl-070884 Pl-068761_E32Y_E56N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 229 Pl-070886 Pl-068761 E32YH100G VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 230 Pl-070886 Pl-068761 E32YH100G VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 231 Pl-070886 Pl-068761 E32YH100G IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 232 Pl-070886 Pl-068761 E32YH100G L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 233 Pl-070888 Pl-068761_E32Y_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAFDVWGQGTMVTVSS 234 Pl-070888 Pl-068761_E32Y_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 235 Pl-070888 Pl-068761_E32Y_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ

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VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 236 Pl-070888 Pl-068761_E32Y_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 237 Pl-070890 P1-068761 E30DE55A VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 238 Pl-070890 P1-068761 E30DE55A VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 239 Pl-070890 P1-068761 E30DE55A IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 240 Pl-070890 P1-068761 E30DE55A L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 241 Pl-070892 Pl-068761_E30D_E56N VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 242 Pl-070892 Pl-068761_E30D_E56N VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 243 Pl-070892 Pl-068761_E30D_E56N IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS SV VT VP S S SL GTQT YICNVNHKP SNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

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TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 244 Pl-070892 Pl-068761_E30D_E56N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 245 Pl-070894 P1-068761 E30DH100G VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMH WVROAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 246 Pl-070894 P1-068761 E30DH100G VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 247 Pl-070894 P1-068761 E30DH100G IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 248 Pl-070894 P1-068761 E30DH100G L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 249 Pl-070896 Pl-068761_E30D_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMH WVRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAEDVWGQGTMVTVSS 250 Pl-070896 Pl-068761_E30D_E100fF VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 251 Pl-070896 Pl-068761_E30D_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR

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VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 252 Pl-070896 Pl-068761_E30D_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 253 Pl-070898 Pl-068761_E55A_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSAEIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAFDVWGQGTMVTVSS 254 Pl-070898 Pl-068761_E55A_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 255 Pl-070898 Pl-068761_E55A_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 256 Pl-070898 Pl-068761_E55A_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 257 Pl-070900 Pl-068761 E56NH100G VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 258 Pl-070900 Pl-068761 E56NH100G VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 259 Pl-070900 Pl-068761 E56NH100G IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA

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ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 260 Pl-070900 Pl-068761 E56NH100G L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 261 Pl-070902 Pl-068761_E56N_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGW IDAFDVWGQGTMVTVSS 262 Pl-070902 Pl-068761_E56N_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 263 Pl-070902 Pl-068761_E56N_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 264 Pl-070902 Pl-068761_E56N_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 265 Pl-070904 P1-068767 E30D VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 266 Pl-070904 P1-068767 E30D VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 267 Pl-070904 Pl-068767 E30D EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD

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IgG1.3 NS NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 268 Pl-070904 P1-068767 E30D L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 269 Pl-070906 Pl-068767_D52N VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 270 Pl-070906 Pl-068767_D52N VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 271 Pl-070906 Pl-068767_D52N IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 272 Pl-070906 Pl-068767_D52N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 273 Pl-070908 Pl-068767_E55A VH EVOLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSANIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 274 Pl-070908 Pl-068767_E55A VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK

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275 Pl-070908 Ρ1-068767_Ε55Α IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 276 Pl-070908 Pl-068767_E55A LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 277 Pl-070910 Pl-068767_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAFDDWGQGTMVTVSS 278 Pl-070910 P1-068767 E1001F VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 279 Pl-070910 Pl-068767_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 280 Pl-070910 Pl-068767_E100fF LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 281 Pl-070912 Pl-068767_D102V VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 282 Pl-070912 EIVLTQSPGTLSLSPGERATLSCRASOSVSSSYLAWYQ

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Pl-068767_D102V VL OKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 283 Pl-070912 Pl-068767_D102V IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 284 Pl-070912 Pl-068767_D102V LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 285 Pl-070914 P1 -068767_E3 0DD52N VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 286 Pl-070914 P1 -068767_E3 0DD52N VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 287 Pl-070914 P1 -068767_E3 0DD52N IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 288 Pl-070914 P1 -068767_E3 0DD52N L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 289 Pl-070916 P1 -068767_D52N_E5 5 A VH EVOLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW

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IDAEDDWGQGTMVTVSS 290 Pl-070916 Pl-068767_D52N_E55A VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 291 Pl-070916 Pl-068767_D52N_E55A IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 292 Pl-070916 Pl-068767_D52N_E55A L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 293 Pl-070918 Pl-068767_E55A_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAFDDWGQGTMVTVSS 294 Pl-070918 Pl-068767_E55A_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 295 Pl-070918 Pl-068767_E55A_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 296 Pl-070918 Pl-068767_E55A_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 297 Pl-070920 EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH

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Pl- 068767_E100fF_D102V VH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAFDVWGQGTMVTVSS 298 Pl-070920 Pl- 068767_E100fF_D102V VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCOQYGSSPFTFGPGTKVDIK 299 Pl-070920 Pl- 068767_E100fF_D102V IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 300 Pl-070920 Pl- 068767_E100fF_D102V L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 301 Pl-070922 Pl-068767_E30D_E55A VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGIDWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDDWGQGTMVTVSS 302 Pl-070922 Pl-068767_E30D_E55A VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 303 Pl-070922 Pl-068767_E30D_E55A IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGIDWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 304 Pl-070922 Pl-068767_E30D_E55A L.C. EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

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NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 305 Pl-070924 Pl-068767_E30D_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAFDDWGQGTMVTVSS 306 Pl-070924 Pl-068767_E30D_E100fF VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 307 Pl-070924 Pl-068767_E30D_E100fF IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 308 Pl-070924 Pl-068767_E30D_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 309 Pl-070926 P1 -068767_E3 ODD 102 V VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMH WVROAPGKGLEWVSGIDWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 310 Pl-070926 P1-068767 E3 ODD 102 V VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIK 311 Pl-070926 P1 -068767_E3 ODD 102 V IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 312 Pl-070926 Pl-068767 E30D D102V EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS

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L.C. RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 313 Pl-070928 Pl- 068767_D52N_E100fF VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLOMNSLRTEDTALYYCAKVPGYSGGW IDAFDDWGQGTMVTVSS 314 Pl-070928 Pl- 068767_D52N_E100fF VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCOQYGSSPFTFGPGTKVDIK 315 Pl-070928 Pl- 068767_D52N_E100fF IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 316 Pl-070928 Pl- 068767_D52N_E100fF L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 317 Pl-070930 P1 -068767_D52N_D 102 V VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGINWNSENIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 318 Pl-070930 P1 -068767_D52N_D 102 V VL EIVLTQ SPGTLLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 319 Pl-070930 P1 -068767_D52N_D 102 V IgG1.3HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

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YTQKSLSLSPG 320 Pl-070930 P1 -068767_D52N_D 102 V L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 321 Pl-070932 P1 -068767_E5 5 AD 102 V VH EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMH WVROAPGKGLEWVSGIDWNSANIGYADSVKGRFTIS RDNAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGW IDAEDVWGQGTMVTVSS 322 Pl-070932 P1-068767-E5 5 AD 102 V VL EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYO QKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQOYGSSPFTFGPGTKVDIK 323 Pl-070932 P1-068767-E5 5 AD 102 V IgGL3 HC EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 324 Pl-070932 P1-068767-E5 5 AD 102 V L.C. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK H KVYACEVTHQGLSSPVTKSFNRGEC 325 hVISTA-ECD 6-Hi s-mark AFKVATPYSL YVCPEGQNVT LTCRLLGPVD KGHDVTFYKT WYRSSRGEVQ TCSERRPIRN LTFQDLHLHH GGHQAANTSH DLAQRHGLES ASDHHGNFSI TMRNLTLLDS GLYCCLVVEI RHHHSEHRVH GAMELQVQTG KDAPSNCVVY PSSSQESENI TAHHHHHHH 326 Cyno VISTA-ECD 6-Hi S-Mark AFKVATLYSL YVCPEGQNVT LTCRVFGPVD KGHDVTFYKT WYRSSRGEVQ TCSERRPIRN LTFQDLHLHH GGHQAANTSH DLAQRHGLES ASDHHGNFSI TMRNLTLLDS GLYCCLVVEIRHHHSEHRVH GAMELQVQTG KDAPSSCVAY PSSSQESENI TAHHHHHHH 327 PI-069059 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGACCATATAGGCT

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ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTGTATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 328 P1-069059 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 329 P1-069061 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAAATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 330 Pl-069061 VL№K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 331 P1-069063 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAAATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGACGATGAATGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 332 Pl-069063 VL№K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG

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GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGCCCTGGGACCAAAGTGG ATATC AAA 333 P1-069065 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATGAAGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGACGACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 334 P1-069065 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 335 P1-069067 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAAATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGACGATGAATGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 336 P1-069067 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA

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337 P1-069069 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAAATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGACGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 338 P1-069069 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 339 P1-069071 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATGAAGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGAAGGCTGGA TTGACGCTTTTGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 340 Pl-069071 VL№K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 341 P1-069073 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATGAAGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAAATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA

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CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 342 Pl-069073 USDNK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 343 P1-069075 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGGACAGTGCTGAAATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 344 P1-069075 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 345 P1-069077 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAAGATGAAGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGACGAAATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 346 P1-069077 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC

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CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 347 P1-068761 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 348 Pl-068761 VL№K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 349 PI-068767 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 350 Pl-068767 SKDNK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 351 P1-068773 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG

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GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGATAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCCAAACTCCCTGTATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAA GGGACAATGG TCACCGTCTCTTCA 352 Pl-068773 UDNK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 353 P1-068765 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCGATGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 354 P1-068765 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 355 P1-061029 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA

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356 Pl-061029 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 357 Pl-068757 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 358 P1-068757 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 359 P1-068771 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTCATGAGATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 360 Pl-068771 VLflHK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA

- 175 046477

CCATTCACTTTCGGGCCCTGGGACCAAAGTGGATATC AAA 361 P1-068775 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 362 PI-068775 UKDNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 363 PI-068769 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGATCACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 364 PI-068769 VLAHK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 365 Pl-068759 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC

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AGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 366 Pl-068759 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 367 P1-068763 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 368 Pl-068763 VL№K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 369 Pl-061015 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTAGCTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCAATTATATGGTATGATGGAAGTAATAAATACT ATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTG TATCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA CTGTGCGAGAGATAGTGGTTTTTACTCCTCGTACTA CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA 370 Pl-061015 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG

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GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 371 P1-068748 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTCACCATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGCTGGAGTGGG TGGCAATTATATGGTATGATGGAAGTAATGACGACT ATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTA TCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA CTGTGCGAGAGATAGTGGTTTTTACGACTCGTACTA CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA 372 P1-068748 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 373 P1-068744 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGAGTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACATATATGGTATGATGGAAGTAATAAATAC GAGGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTG TATCTGCAAA TGAACAGCCTGAGAGCCGAGGACACGGCTGTGTAT TACTGTGCGAGAGATAGTGGTTTTTACGAATCGTAC TACTTTGACGAGTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA 374 P1-068744 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 375 P1-068736 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT

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CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGAGTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGCTGGAGTGGG TGGCAATTGATTGGTATGATGGAAGTAATAAAGAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACG GCTGTGTATT ACTGTGCGAGAGATAGTGGTTTTTACGACTCGTACT ACTTTGACGACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 376 P1-068736 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 377 PI-068752 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTAGCTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCAGAGATATGGTATGATGGAAGTAATAAAGAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCT GTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT ACTGTGCGAGAGATAGTGGTTTTTACGACTCGTACT ACTTTGACGAGTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 378 PI-068752 VL^HK GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 379 PI-068740 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGACTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGCTGGAGTGGG TGGCAATTATATGGTATGATGGAAGTGATAAAGAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCTG TATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT ACTGTGCGAGAGATAGTGGTTTTTACGACTCGTACT

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ACTTTGACGACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 380 Pl-068740 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 381 P1-068742 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGACTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGCTGGAGTGGG TGGCAATTATATGGTATGATGGAAGTGATAAAGAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCTG TATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT ACTGTGCGAGAGATAGTGGTTTTTACGAAGATTACT ACTTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 382 P1-068742 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 383 P1-068746 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTAGCTATGCCATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCAATTATATGGTATGATGGAAGTAATCACCACT ATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTG TATCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA CTGTGCGAGAGATAGTGGTTTTTACGACTCGTACTA CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA 384 P1-068746 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT

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TTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 385 Pl-068750 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGACTATGACATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGCTGGAGTGGG TGGCAGAGATATGGGATGATGGAAGTAATAAATAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCTGTA TCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT ACTGTGCGAGAGATGAGGAATTTTACTCCTCGTACT ACTTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 386 Pl-068750 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 387 P1-06873 8 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGAGTATGCCCATCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCAATTATATGGGATGATGGAAGTAATCACTACT ATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTA TCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTA CTGTGCGAGAGATAGTGGTTTTTACGAAGATTACTA CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA 388 P1-06873 8 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 389 Pl-068754 VH DNA CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC GTCTGGATTCACCTTCAGTGACTATGACATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG

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TGGCAGAGATATGGGATGATGGAAGTAATAAATAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT ACTGTGCGAGAGATAGTGGTTTTTCACTCCGATTACT ACTTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA 390 P1-068754 VL DNA GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCC CAGCCAGGTTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGGAGCCTGA AGAT TTTGCAACTTATTACTGCCAACAGTATAATAGTTAC CCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 391 P1-070864 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 392 P1-070864 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 393 P1-070866 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 394 P1-070866 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT

- 182 046477

TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAG CTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 395 P1-070868 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAGATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 396 P1-070868 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 397 P1-070870 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 398 Pl-070870 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC

- 183 046477

AAA 399 P1-070872 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 400 P1-070872 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 401 P1-070874 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 402 P1-070874 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 403 P1-070876 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT

- 184 046477

GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 404 P1-070876 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 405 P1-070878 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 406 P1-070878 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 407 P1-070880 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 408 P1-070880 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC

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TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 409 Ρ1-070882 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAGATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 410 Ρ1-070882 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 411 PI-070884 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 412 PI-070884 VL^HK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 413 Pl-070886 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC

- 186 046477

CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATA GCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 414 Pl-070886 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 415 P1-070888 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 416 P1-070888 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 417 P1-070890 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAGATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG

- 187 046477

TCACCGTCTCTTCA 418 P1-070890 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 419 PI-070892 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 420 P I-070892 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 421 Pl-070894 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 422 PI-070894 VL^HK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT

- 188 046477

TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 423 P1-070896 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 424 PI-070896 VLAHK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 425 Pl-070898 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTGAGATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 426 PI-070898 VLAHK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 427 P1-070900 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC

- 189 046477

TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 428 P1-070900 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 429 P1-070902 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 430 P1-070902 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 431 P1-070904 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 432 P1-070904 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG

- 190 046477

GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGCCCTGGGACCAAAGTGG ATATC AAA 433 P1-070906 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 434 P1-070906 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 435 Pl-070908 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 436 PI-070908 VL^HK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA

- 191 046477

437 P1-070910 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 438 P1-070910 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 439 Pl-070912 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 440 Pl-070912 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 441 Pl-070914 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT

- 192 046477

ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 442 Pl-070914 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 443 P1-070916 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 444 P I-070916 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 445 Pl-070918 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 446 Pl-070918 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC

- 193 046477

CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 447 P1-070920 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGTCTGGGGCCAAGGGACAATG GTCACCGTCTCTTCA 448 P1-070920 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 449 P1-070922 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 450 P1-070922 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 451 P1-070924 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG

- 194 046477

GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGACTGGGGCCAA GGGACAAT GGTCACCGTCTCTTCA 452 P1-070924 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 453 P1-070926 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 454 P1-070926 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 455 P1-070928 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTTTTGATGACTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA

- 195 046477

456 Pl-070928 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 457 P1-07093 0 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTG TATCTGCAAAT GAACAGTCTGAGAACTGAGGACACGGCCTTGTATT ACTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGG ATTGACGCTGAAGATGTCTGGGGCCAAGGGACAAT GGTCACCGTCTCTTCA 458 Pl-070930 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 459 P1-070932 VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 460 Pl-070932 VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA

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CCATTCACTTTCGGGCCCTGGGACCAAAGTGGATATC AAA 461 IgG1.3 heavy chain constant region DNA GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGC CCTCCAGCAGCTTGGGCACCCAGACCTA CATCTGCA ACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAAGCCGAAG GGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC ATCCCGGGAGGAGATGACC AAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGT GGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGC ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG CCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAAC CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTC ACACAT GCCCACCGTGCCCAGCACCTGAAGCCGAA GGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCC AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAG

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AACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTTAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 462 Approximate DNA light chain constant region CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCG CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAA CACAA AGTCTACGCCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTTAG 463 Pl-068761 VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 464 Pl-068761 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 465 Pl-068761 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSEEIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 466 Pl-068761 K16RT84A DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAGAGATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCC CTGTATCTGCAAAT GAACAGTCTGAGAgCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCCATGGCTGGAT TGACGCTGAAGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 467 Pl-061029 VHK16R Т84А EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S 468 Pl-061029 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S 469 Р1-061029 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S

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470 Pl-061029 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 471 Pl-068767 VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 472 Pl-068767 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 473 P1-068767 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 474 Pl-068767 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAgCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 475 Pl-070868 (Pl068761_E55A) VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 476 Pl-070868 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 477 P1-070868 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDEAMHW VRQAPGKGLEWVSGINWNSAEIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSHGWIDA ED VWGQGTMVT VS S 478 Pl-070868 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG

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TCTCAGGTATTGATTGGAACAGTGAAAACATAGGC TATGCGGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT GAACAGTCTGAGAgCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 479 Pl-70906 (Pl068767_D52N) VH K16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 480 Pl-70906 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 481 Pl-70906 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSENIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 482 Pl-70906 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGAAAACATAGGC TATGCGGACTCTTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACTCCCT GTATCTGCAAAT GAACAGTCTGAGAgCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAAGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 483 Pl-70908 (Pl068767_E55A) VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 484 Pl-70908 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 485 Pl-70908 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGIDWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 486 Pl-70908 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTGATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAGCTGAGGACACGGCCTTGTATTAC

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TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTGAAGATGACTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 487 Pl-70916 (Pl068767_D52N_E55A) VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 488 Pl-70916 VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 489 Pl-70916 VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLEDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 490 Pl-70916 VHK16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGAGGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTGAAGATGACTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 491 Pl-72000 (Pl061029_F100fE_V102D) VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 492 Pl-72000 (Pl061029_F100fE_V102D) VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 493 Pl-72000 (Pl061029_F100fE_V102D) HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 494 Pl-72000 (Pl061029_F100fE_V102D) LC EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

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NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 495 Pl-72000 (Pl061029_F100fE_V102D) VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 496 Pl-72000 (Pl061029_F100fE_V102D) scdna GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 497 Pl-72000 (Pl061029_F100fE_V102D) NS DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTGTATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC

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TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACG CCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCCCCGGGTTGA 498 Pl-72000 (Pl061029_F100fE_V102D) LC DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTTAG 499 Pl-72000 (Pl061029_F100fE_V102D) VHK16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 500 Pl-72000 (Pl061029_F100fE_V102D) VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 501 Pl-72000 (Pl061029_F100fE_V102D) VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVT VS S 502 Pl-72000 (Pl061029_F100fE_V102D) VH K16R T84A DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG

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AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTGAGGATGACTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 503 Pl-72002 (Pl061029_F100fE) VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 504 Pl-72002 (Pl061029_F100fE) VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 505 Pl-72002 (Pl061029_F100fE) HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLG TQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 506 Pl-72002 (Pl061029_F100fE) LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSK AD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 507 Pl-72002 (Pl061029 F100fE)VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 508 Pl-72002 (Pl061029_F100fE) VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC

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AAA 509 Pl-72002 (Pl061029 F100ffi)HC DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCG AACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAG CCGAAGGGGCCCCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACG CCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCCCCGGGTTGA 510 Pl-72002 (Pl061029_F100fE) LC DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC

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CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCAT CAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTTAG 511 Pl-72002 (Pl061029_F100fE) VH K16RT84A EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 512 Pl-72002 (Pl061029_F100fE) VH K16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 513 Pl-72002 (Pl061029_F100fE) VH T84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA EDDWGQGTMVTVSS 514 Pl-72002 (Pl061029_F100fE) VH K16RT84A DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTGAGGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 515 Pl-72004 (Pl061029_V102D) VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVT VS S 516 Pl-72004 (Pl-061029_ V102D) VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 517 Pl-72004 (Pl-061029_ V102D)HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD

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SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 518 Pl-72004 (Pl-061029_ V102D)LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPP SDEQLK SGT AS VVCLLNNF YPREAK VQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK H KVYACEVTHQGLSSPVTKSFNRGEC 519 Pl-72004 (Pl-061029_ V102D) VHflHK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 520 Pl-72004 (Pl-061029_ V102D) VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 521 Pl-72004 (Pl-061029_ V102D) NS DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGACTGGGGCCAAGGGACAATGG TCACCGTCTCTTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAA CCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCC GAAGGGGCCCCGTCAGTCTTCCTCTT

- 207 046477

CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCAAACCACTACACGCAGAAGAGCCTCTCCCT GTCCCCGGGTTGA 522 Pl-72004 (Pl-061029_ V102D) LC DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTTAG 523 Pl-72004 (Pl-061029_ V102D) VHK16RT84A EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVT VS S 524 Pl-72004 (Pl-061029_ V102D) VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVT VS S 525 Pl-72004 (Pl-061029_ V102D) VHT84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDYAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FDDWGQGTMVT VS S 526 Pl-72004 (Pl-061029 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT

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V102D) VHK16RT84A DNA ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATTATGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTGTATCTGCAAATG AACAGTCTGAGAgCTGAGGACA CGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTTTTGATGACTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 527 Pl-72006 (Pl061029_Y32E) VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSS 528 Pl-72006 (Pl-061029_ Y32E)VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 529 Pl-72006 (Pl-061029_ Y32E)HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGT QTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 530 Pl-72006 (Pl-061029_ Y32E)LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQ SGNSQES VTEQD SKD ST YSLS STLTLSKAD YEK HKVYACEVTHQGLSSPVTKSFNRGEC 531 Pl-72006 (Pl-061029_ Y32E) VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 532 Pl-72006 (Pl-061029_ Y32E) VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG

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GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 533 Pl-72006 (Pl-061029_ Y32E) NS DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCAGCGTCGACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCC CGAACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGA AGCCGAAGGGGCCCCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCAC GCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCCCCGGGTTGA 534 Pl-72006 (Pl-061029_ Y32E) LC DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC

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TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC CCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGA GAGTGTTAG 535 Pl-72006 (Pl-061029_ Y32E) VHK16RT84A EVQLVESGGGLVQPGRSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S 536 Pl-72006 (Pl-061029_ Y32E)VHK16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S 537 Pl-72006 (Pl-061029_ Y32E) VH T84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA FD VWGQGTMVT VS S 538 Pl-72006 (P1-061029Y32E) VHK16RT84A DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTTTTGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 539 Pl-72008 (Pl061029_Y32E_F100fE) VH EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 540 Pl-72008 (Pl-061029_ Y32E_F100fE) VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 541 Pl-72008 (Pl-061029_ Y32E_F100fE) HC EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNS ANIGYAD S VKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA EDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

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GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 542 Pl-72008 (Pl-061029_ Y32E_F100fE) LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVY ACEVTHQGLSSPVTKSFNRGEC 543 Pl-72008 (Pl-061029_ Y32E_F100fE) VH DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCA 544 Pl-72008 (Pl-061029_ Y32E_F100fE) VL DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAA 545 Pl-72008 (Pl-061029_ Y32E_F100fE) NS DNA GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAAGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCTG TATCTGCAAATG AACAGTCTGAGAACTGAGGACACGGCCTTGTATTA CTGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGAT TGACGCTGAGGATGTCTGGGGCCAAGGGACAATGG TCACCGTCTCTTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCG AACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTTCCCGGCT

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GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCATGATCTCCC g AGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATA GCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCAAACCACTACACGCAGAAGAGCCTCTCCCT GTCCCCGGGTTGA 546 Pl-72008 (Pl-061029_ Y32E_F100ffi) LC DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGACTGGAGCCTGA AGAT TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATC AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTTAG 547 Pl-72008 (Pl-061029_ Y32E_F100ffi) VH K16R T84A EVQLVESGGGLVQPGRSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 548 Pl-72008 (Pl-061029_ Y32E_F100ffi) VH K16R EVQLVESGGGLVQPGRSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRTEDTALYYCAKVPGYSGGWIDA

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ED VWGQGTMVT VS S 549 Pl-72008 (Pl-061029_ Y32E_F100ffi) VH T84A EVQLVESGGGLVQPGKSLRLSCAASGFTLDDEAMHW VRQAPGKGLEWVSGINWNSANIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKVPGYSGGWIDA ED VWGQGTMVT VS S 550 Pl-72008 (Pl-061029_ Y32E_F100ffi) VH K16R T84ADNK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGCAgGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCCTTGATGATGAGGCCATGCACTG GGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGG TCTCAGGTATTAATTGGAACAGTGCTAACATAGGCT ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCAAGAACTCCCT GTATCTGCAAATG AACAGTCTGAGAgCTGAGGACACGGCCTTGTATTAC TGTGCAAAAGTTCCTGGGTATAGCGGTGGCTGGATT GACGCTGAGGATGTCTGGGGCCAAGGGACAATGGT CACCGTCTCTTCA 551 VISTA.4 VH 41F11 With signal sequence that is emphasized MEFGLSWVFLVAIIKGVOCQVOLVESGGGLVKPGG SLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISN SGSPIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED T AVYYC ARDLPGW YFDLWGRGTL VT VS S 552 VISTA.4 VK1 VL 41F11 With a signal sequence that is underlined MEAPAQLLFLLLWLPDTTGEIVLTQSPATLSLSPGE RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSASGSGTDFTLTISSLEPEDFAVYYCQQRN NWPRTFGQGTKVEIK 553 VISTA.4 VK2 VL 41F11 With a signal sequence that is underlined MDMRVLAQLLGLLLLCFPGARCDIOMTOSPSSLSAS VGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YNSYPRTFGQGTKVEIK 554 VISTA.4 VK3 VL 41F11 With a signal sequence that is underlined METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGE RATLSCRASQ S VS S S YL AW YQQKPGQ APRLLIYGAS S RATGIPDRFSGSGGTDFTLTISRLEPEDFAVYYCQQY GS SPWTFGQGTKVEIK 555 VISTA.4 VH DNA 41F11 With a signal sequence that is underlined ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTG CTATTATAAAAGGTGTCCAGTGTCAGGTGCAGTT GGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAG GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA CCTTCAGTGACTATTACATGAGCTGGATCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATT AGTAATAGTGGTAGTCCCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGGGACAACGC CAAGAACTCACTGTATCTGCAAATG AACAGCCTGA GAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA GATCTCCCGGGCTGGTACTTCGATCTCTGGGGCCGT GGCACCCTGGTCACTGTCTCCTCA 556 VISTA.4 VK1 VL DNA With signal ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGC TACTCTGGCTCCCAGATACCACCGGAGAAATTGT

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sequence that is emphasized GTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCCTGCAGGGCCAGTC AGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAG AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTT CAGTGCCAGTGGGTCTGGGACAGACTTCACTCTCAC CATCAGCAGCCTAGCCTGAAGATTTT GCAGTTTA TTACTGTCAGCAGCGTAACAACTGGCCTCGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAA 557 VISTA.4.A64G VK1 VL With signal sequence which is underlined MEAPAOLLFLLLLWLPDTTGEIVLTOSPATLSLSPGE RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRN NWPRTFGQGTKVEIK 558 VISTA.4. A64G LC With signal sequence that is emphasized MEAPAOLLFLLLLWLPDTTGEIVLTOSPATLSLSPGE RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRN NWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSF NRGEC 559 VISTA. 5 VH AVQLQESGPGLVRPSQSLSLTCTVTDYSITSDYAWNW IRQFPGSKLEWLGFIGYSGNTNYNPSLESRISITRHTSK NQFFLHLNSMTTEDTATYYCARSLYGGSHWYFDVW GAGTTVTVSS 560 VISTA. 5 VK1, VL DIVLTQSPASLAVSLGQRATISCRGSESVEYYGTILMQ WYQQKPGQPPKLLIYGASNVESGVPARFSGSGSGTDF SLNH4PVEEDDIAMYFCQQSRKVPWTFGGGTKLEIK 561 VIST A. 5 VH DNA GCTGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGT GAGACCTTCTCAGTCTCTGTCCCTCACCTGCACTGT CACTGACTACTCAATCACCAGTGATTATGCCTGGAA CTGGATCCGGCAGTTTCCAGGAAGCAAACTGGAGT GGCTGGGCTTCATAGGCTACAGTGGTAACACTAACT ACAACCCATCTCTCGAAAGTCGAATCTCTATCACTC GACACACATCCAAGAACCAGTTCTTC CTGCACTTGA ATTCTATGACTACTGAGGACACAGCCACATATTACT GTGCAAGATCCCTCTACGGTGGTAGTCACTGGTACT TCGATGTCTGGGGCGCAGGGACCACGGTCACCGTC TSCTSA 562 VISTA. 5 VK1, VL DNA GACATTGTGCTCACCCAATCTCCAGCTTCTTTGGCT GTGTCTCTAGGGCAGAGCCACCATCTCCTGCAG AGGCAGTGAAAGTGTTGAATATTATGGCACAATTT AATGCAGTGGTACCAACAGAAACCAGGACAGCCAC CCAAACTCCTCATCTATGGTGCATCCAACGTAGAAT CTGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTG GGACAGACTTCAGCCTCAACATC CATCCTGTGGAG GAGGATGATATTGCAATGTATTTCTGTCAGCAAAGT AGGAAGGTTCCGTGGACGTTCGGTGGAGGCACCAA GCTGGAAATCAAA

Claims (26)

1. An antibody that binds to the V domain of human T cell suppressor Ig activation (hVISTA) under acidic conditions, where the antibody contains the complementarity determining region of the heavy chain variable region (VH) 1 (CDR1), CDR2 and CDR3 a) P1-061029_F100fE_V102D, where VH CDR1 P1-061029_F100fE_V102D contains residues 26-35 SEQ ID NO: 491, VH CDR2 P1-061029_F100fE_V102D contains residues 50-66 of SEQ ID NO: 491, and VH CDR3 P1-061029_F100fE_V102D contains residues 99-112 SEQ ID NO: 491; b) P1-061029_F100fE, where VH CDR1 P1-061029_F100fE contains residues 26-35 SEQ ID NO: 503, VH CDR2 P1-061029_F100fE contains residues 50-66 of SEQ ID NO: 503, and VH CDR3 P1-061029_F100fE contains residues 99-112 SEQ ID NO: 503; c) P1-061029_V102D, where VH CDR1 P1-061029_V102D contains residues 26-35 SEQ ID NO: 515, VH CDR2 P1-061029_V102D contains residues 50-66 of SEQ ID NO: 515, and VH CDR3 P1-061029_V102D contains residues 99-112 SEQ ID NO: 515; d) P1-061029_Y32E, where VH CDR1 P1-061029_Y32E contains residues 26-35 SEQ ID NO: 527, - 215 046477 VH CDR2 P1-061029_Y32E contains residues 50-66 of SEQ ID NO: 527, and VH CDR3 P1-061029_Y32E contains residues 99-112 of SEQ ID 527; or e) P1-061029_Y32E_F100fE, where VH CDR1 P1-061029_Y32E_F100fE contains residues 26-35 SEQ ID NO: 539, VH CDR2 P1-061029_Y32E_F100fE contains residues 50-66 of SEQ ID NO: 539, and VH CDR3 P1-061029_Y32E_F100fE contains residues 99-112 of SEQ ID NO: 539, and where the antibody contains a light chain variable region (VL) containing CDR1, CDR2 and CDR3 VL from VL P1-061029, where CDR1 VL P1-061029 contains residues 24-35 SEQ ID NO: 68, CDR2 VL P1-061029 contains residues 51-57 of SEQ ID NO: 68, and CDR3 VL P1-061029 contains residues 90-98 of SEQ ID NO: 68. 2. 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 P1-061029_F100fE_V102D (SEQ ID NO: 491), P1- 061029_F100fE (SEQ ID NO: 503), P1-061029_V102D (SEQ ID NO: 515), P1-061029_Y32E (SEQ ID NO: 527) or P1-061029_Y32E_F100fE (SEQ ID NO: 539). 3. An antibody that binds to hVISTA under acidic conditions, wherein the antibody contains VH P1061029 (SEQ ID NO: 67), P1-068757 (SEQ ID NO: 71), P1-068759 (SEQ ID NO:87), P1-068761 (SEQ ID NO: 51), P1-068763 (SEQ ID NO:91), P1-068765 (SEQ ID NO:63), P1-068767 (SEQ ID NO: 55), P1-068769 (SEQ ID NO:83), P1-068771 (SEQ ID NO:75), P1-068773 (SEQ ID NO: 59), P1-068775 (SEQ ID NO:79), P1-069059 (SEQ ID NO: 11), P1-069061 (SEQ ID NO: 15), P1-069063 (SEQ ID NO: 19), P1-069065 (SEQ ID NO: 23), P1-069067 (SEQ ID NO: 27), P1-069069 (SEQ ID NO:31), P1-069071 (SEQ ID NO:35), P1-069073 (SEQ ID NO: 39), P1-069075 (SEQ ID NO:43), P1-069077 (SEQ ID NO:47), P1-068761_E55A (SEQ ID NO: 193), P1-068761_H100G (SEQ ID NO: 201), P1-068761_E56N (SEQ ID NO: 197), P1-068761_E55A_E56N (SEQ ID NO: 213), P1-068761_E30D (SEQ ID NO: 185), P1-068761_E30D_E55A (SEQ ID NO: 237), P1-068761_E56N_H100G (SEQ ID NO: 257), P1-068761_E30D_H100G (SEQ ID NO: 245), P 1 -068761_E30D_E56N (SEQ ID NO: 241), P1-068761_E100fF (SEQ ID NO: 205), P1-068761_E55A_E100fF (SEQ ID NO: 253), P1-068761_H100G_E100fF (SEQ ID NO: 271), P1-06 8761_E30D_E100fF (SEQ ID NO : 249), P1-068761_E56N_E100fF (SEQ ID NO: 261), P1-068761_E32Y (SEQ ID NO:189), P1-068761_E32Y_E55A (SEQ ID NO: 221), P1-068761_E32Y_E56N (SEQ ID NO: 225), P1 - 068761_E30D_E32Y (SEQ ID NO: 209), P1-068761_E32Y_H100G (SEQ ID NO: 229), P1-068761_E32Y_E100fF (SEQ ID NO: 233), P1-068767_D52N_D102V (SEQ ID NO: 317), P1 -068767_D52N (SEQ ID NO: 269), P1-068767_D52N_E55A (SEQ ID NO: 289), P1-068767_E55A_D102V (SEQ ID NO: 321), P1-068767_D102V (SEQ ID NO: 281), P1-068767_E55A (SEQ ID NO: 273), P1-06876 7_E30D_D52N (SEQ ID NO:285) 7 ), P1-068767_E55A_E100fF (SEQ ID NO: 293), P1-068767_D52N_E100fF (SEQ ID NO: 313), P1-068767_E100fF (SEQ ID NO: 277), P1-068767_E30D_E100fF (SEQ ID NO: 305), P1-061029_F100fE_V102D ( SEQ ID NO: 491), P1-061029_F100fE (SEQ ID NO: 503), P1-061029_V102D (SEQ ID NO: 515), P1-061029_Y32E (SEQ ID NO: 527), or P1-061029_Y32E_F100fE (SEQ ID NO: 539), which is modified by one or both of the K16R and T84A substitutions, and where the antibody contains CDR1 VL, CDR2 and CDR3 VL P1-061029, where CDR1 VL P1-061029 contains residues 24-35 of SEQ ID NO: 68, CDR2 VL P1-061029 contains residues 51-57 of SEQ ID NO: 68, CDR3 VL P1-061029 contains residues 90-98 of SEQ ID NO: 68. 4. An antibody according to any one of claims 1 to 3, wherein the antibody comprises a VL containing an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical to the corresponding amino acid sequence of SEQ ID NO: 68. 5. The antibody of claim 4, wherein the antibody comprises a VL containing the amino acid sequence of SEQ ID NO: 68. 6. Antibody according to any one of claims 1-5, where the antibody has one or more properties: a) binds to hVISTA under acidic conditions with an affinity that is higher than that under neutral or physiological conditions; b) binds to hVISTA under acidic conditions with a K _D that is at least 10 times lower than its K _D under neutral or physiological conditions; c) binds to hVISTA under acidic conditions with a koff that is at least 5 times lower than its _koff under neutral or physiological conditions; d) inhibits the binding of hVISTA to human T cells under conditions having a lower pH, - 216 046477 than pH 7.0; e) inhibits the binding of hVISTA to huPSGL-1 and/or competes with huPSGL-1 for binding to hVISTA under conditions having a pH less than pH 7.0; f) inhibits the binding of hVISTA to heparan sulfate proteoglycans; g) inhibits the binding of hVISTA to heparan sulfate proteoglycans under conditions having a pH less than pH 7.0; h) stimulates T cell activation, as evidenced by increased T cell proliferation; increased production of IFN-γ by T cells; and/or stimulation of T cell receptor-mediated NF-kB signaling; i) reduces VISTA-mediated cell-cell adhesion; j) has a mean retention time (MRT) of at least 100, 200, 300, 400 or 500 hours in cynomolgus monkeys; k) binds to VISTA-positive cells in the peripheral blood of the subject to which it is administered with lower affinity than to VISTA-positive cells under acidic conditions; l) depletes VISTA-positive cells in the peripheral blood of the subject to which it is administered to a lower level than VISTA-positive cells under acidic conditions; m) binds at or near a histidine-rich region of hVISTA, such as the histidine-rich β-sheet extension; And n) binds in or near the histidine-rich region of hVISTA, such as the histidine-rich β-sheet extension, under conditions having a pH of 6.0-6.5. 7. The antibody according to any one of claims 1 to 6, wherein the antibody binds with higher affinity to unmodified hVIST than to hVISTA containing mutations in 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 and R127. 8. The antibody according to any one of claims 1 to 7, wherein the antibody binds with higher affinity to unmodified hVISTAs than to hVISTAs containing mutations at 2, 3, 4, 5 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. 9. The antibody of claim 7 or 8, wherein the antibody does not bind to hVISTA containing mutations at one or more of the following residues: H68, F97, L115, V117, I119, H121, H122, S124, E125, R127. 10. The antibody according to any one of claims 1 to 9, wherein the antibody binds to hVISTA under acidic conditions, wherein the antibody inhibits the interaction between hVISTA and: (a) T cells and/or (b) PSGL-1, and where the antibody binds to hVISTA on one or more hVISTA residues selected from one of: (i) V34, T35, Y37, K38, T39, Y41, S52, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125 or R127; (ii) V34, T35, Y37, T39, Y41, S52, R54, F62, L65, H66, H68, L115, V117, I119, R120, H121, H122, S124 or E125; or (iii) Y37, T39, R54, F62, H66, L115 or V117, as determined by yeast surface display, and where the numbering corresponds to the numbering of mature hVISTA (SEQ ID NO: 2). 11. The antibody according to any one of claims 1 to 10, wherein the antibody binds to hVISTA under acidic conditions, wherein the antibody inhibits the interaction between hVISTA and: (a) T cells and/or (b) PSGL-1, and wherein the antibody binds to hVISTA on one or more hVISTA residues selected from: Y37, T39, R54, F62, H66, V117, I119 or S124, as determined by yeast surface display, and where the numbering corresponds to the numbering of mature hVISTA (SEQ ID NO: 2). 12. Antibody according to any one of claims 1-11, where the antibody: a) binds to the FG loop of hVISTA; b) binds to the histidine-rich β-sheet extension of hVISTA, as determined by crystallography; c) contacts H121, H122 and H123 (histidine triad) of mature hVISTA (SEQ ID NO: 2) at a distance of 4.0 angstroms (A) or less as determined by crystallography; d) contacts hVISTA through CDR1 VH and CDR3 VH with higher affinity than through CDR2 VH and/or through CDR VL; e) wherein amino acid residues 110 and 112 of the antibody form hydrogen bonds with H121 and H122 of hVISTA, respectively, and as determined by crystallography; f) wherein the amino acid residue of the antibody forms a hydrogen bond with H123 hVISTA, as determined by crystallography; g) contacts hVISTA through at least one or more glutamic acid, aspartic acid or histidine residues that are located in CDR1, CDR2 or CDR3 of VH, as determined by crystallography; and/or h) binds to hVISTA under acidic conditions with a KD (or ko _ff ) that is at least 10-fold, 100-fold, or 1000-fold lower than its KD and/or _{koff of} binding to hVISTA under neutral or physiological conditions - 217 046477 checal pH. 13. Antibody according to any one of claims 1-12, where the antibody: inhibits the interaction between hVISTA and: (a) T cells and/or (b) PSGL-1; increases T cell activation by increasing T cell proliferation; increased production of IFN-γ by T cells; and/or stimulation of T cell receptor-mediated NF-kB signaling; contacts hVISTA at one or more hVISTA residues selected from Y37, T39, R54, F62, H66, V117, I119 or S124, as determined by yeast surface display; and where the numbering corresponds to the numbering of mature hVISTA (SEQ ID NO: 2); binds to the histidine-rich β-sheet extension of hVISTA as determined by crystallography; contacts H121, H122 and/or H123 of mature hVISTA (SEQ ID NO: 2) at a distance of 4.0 angstroms (A) or less) as determined by crystallography; contacts Area 1:57LGPVDKGHDVTF68; Area 2: ₈₆ RRPIRNLTFQDL ₉₇ ; And Region 3: i ₄₈ VVEIRHHHSEHRVHGAMEi ₆₅ hVISTA having SEQ ID NO: 1, as determined by MS-HDX; competes for binding of hVISTA (bidirectional competition) with one or more antibodies containing VH and VL containing the amino acid sequences of SEQ ID NO: 67 and 68, respectively, SEQ ID NO: 51 and 52, respectively, or SEQ ID NO: 55 and 56 , respectively; contacts hVISTA through at least one or more glutamic acid, aspartic acid or histidine residues that are located in CDR1, CDR2 or CDR3 of VH, as determined by crystallography; and/or has a mean retention time (MRT) of at least 100, 200, 300, 400, 500, 600 or 700 hours in cynomolgus monkeys. 14. The antibody according to any one of claims 1 to 12, wherein the antibody binds to hVISTA under acidic conditions with a KD (and/or koff) that is at least 10 times, 100 times or 1000 times lower than its KD or koff of binding with hVISTA at neutral or physiological pH, and where the antibody: inhibits the interaction between hVISTA and: (a) T cells and/or (b) PSGL-1; increases T cell activation by increasing T cell proliferation; increased production of IFN-γ by T cells; and/or stimulation of T cell receptor-mediated NF-kB signaling; contacts hVISTA through one or more residues selected from Y37, T39, R54, F62, H66, V117, I119 or S124, as determined by yeast surface display; and where the numbering corresponds to the numbering of mature hVISTA (SEQ ID NO: 2); binds to the histidine-rich β-sheet extension of hVISTA as determined by crystallography; contacts H121, H122 and/or H123 of mature hVISTA (SEQ ID NO: 2) at a distance of 4.0 angstroms (A) or less, as determined by crystallography; contacts Area 1:57LGPVDKGHDVTF68; Area 2:86RRPIRNLTFQDL97; And Region 3: i ₄₈ VVEIRHHHSEHRVHGAMEi ₆₅ hVISTA having SEQ ID NO: 1, as determined by MS-HDX; contacts hVISTA through at least one or more glutamic acid, aspartic acid or histidine residues that are located in CDR1, CDR2 or CDR3 of VH, as determined by crystallography; and/or has a mean retention time (MRT) of at least 100, 200, 300, 400, 500, 600 or 700 hours in cynomolgus monkeys. 15. The antibody according to any one of claims 1 to 14, wherein the antibody has an isoelectric point (pI) between 6.5 and 6.8, as measured by icIEF. 16. An antibody according to claims 1 to 15, which is a human IgG1, IgG2 or IgG4 antibody. 17. The antibody of claim 16, which is a human IgG4 antibody with S228P. 18. An antibody-drug conjugate (ADC) that binds to the V domain of human T cell suppressor Ig activation (hVISTA) under acidic conditions, where the conjugate contains an antibody that contains the complementarity determining region of the heavy chain variable region (VH) 1 ( CDR1), CDR2 and CDR3 a) P1-061029_F100fE_V102D, where VH CDR1 P1-061029_F100fE_V102D contains residues 26-35 SEQ ID NO: 491, VH CDR2 P1-061029_F100fE_V102D contains residues 50-66 of SEQ ID NO: 491, and VH CDR3 P1-061029_F100fE_V102D contains residues 99-112 SEQ ID NO: 491; b) P1-061029_F100fE, where - 218 046477 VH CDR1 P1-061029_F100fE contains residues 26-35 SEQ ID NO: 503, VH CDR2 P1-061029_F100fE contains residues 50-66 of SEQ ID NO: 503, and VH CDR3 P1-061029_F100fE contains residues 99-112 SEQ ID NO: 503; c) P1-061029_V102D, where VH CDR1 P1-061029_V102D contains residues 26-35 SEQ ID NO: 515, VH CDR2 P1-061029_V102D contains residues 50-66 of SEQ ID NO: 515, and VH CDR3 P1-061029_V102D contains residues 99-112 SEQ ID NO: 515; d) P1-061029_Y32E, where VH CDR1 P1-061029_Y32E contains residues 26-35 of SEQ ID NO:527, VH CDR2 P1-061029_Y32E contains residues 50-66 of SEQ ID NO:527, and VH CDR3 P1-061029_Y32E contains residues 99-112 of SEQ ID 527; or e) P1-O61O29_Y32E_F1OO1E, where VH CDR1 P1-061029_Y32E_F100fE contains residues 26-35 of SEQ ID NO:539, VH CDR2 P1-061029_Y32E_F100fE contains residues 50-66 of SEQ ID NO:539, and VH CDR3 P1-061029_Y32E_F100fE contains residues 99-112 SEQ ID NO:539; and wherein the antibody comprises a light chain variable region (VL) comprising VL CDR1, CDR2 and CDR3 of VL P1-061029, wherein CDR1 VL P1-061029 contains residues 24-35 SEQ ID NO: 68, CDR2 VL P1-061029 contains residues 51-57 of SEQ ID NO: 68, and CDR3 VL P1-061029 contains residues 90-98 of SEQ ID NO: 68. 19. Nucleic acid encoding an antibody according to any one of claims 1 to 17. 20. Nucleic acid encoding the heavy chain and light chain of an antibody according to any one of claims 1 to 17. 21. An antibody expression composition according to any one of claims 1 to 17, comprising a nucleic acid encoding an antibody heavy chain and a nucleic acid encoding an antibody light chain. 22. A cell for expressing an antibody according to any one of claims 1 to 17, including an isolated nucleic acid according to claim 19 or 20 or a composition according to claim 21. 23. A method for producing an antibody, comprising culturing the cell according to claim 22 under conditions under which the antibody is expressed. 24. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 17 or an antibody-drug conjugate according to claim 18 and a pharmaceutically acceptable carrier. 25. A method of treating a cancer patient, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 17, an antibody-drug conjugate according to claim 18, or a pharmaceutical composition according to claim 24. 26. A method of treating an infectious disease in a patient, comprising administering to the patient a therapeutically effective amount of an antibody according to any one of claims 1 to 17, an antibody-drug conjugate according to claim 18, or a pharmaceutical composition according to claim 24.