Anti-VISTA constructs and uses thereof

Abstract

The present application provides anti-VISTA constructs (e.g., anti-VISTA antibodies) that bind to VISTA, nucleic acid molecules encoding the amino acid sequence of anti-VISTA, vectors comprising the nucleic acid molecules, host cells comprising the vectors, methods of preparing the anti-VISTA constructs, pharmaceutical compositions comprising the anti-VISTA constructs, and methods of using the anti-VISTA constructs or compositions. Exemplary anti-VISTA constructs include agonist antibodies capable of binding to and activating VISTA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 157,182, filed March 5, 2021, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] Technical Field The present disclosure relates to anti-VISTA constructs (such as anti-VISTA antibodies) and uses thereof. Submission of sequence listing as an ASCII text file

[0003] The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Format (CRF) of the Sequence Listing (Filename: 193852000440SEQLIST.TXT, Recorded Date: March 2, 2022, Size: 24,739 bytes). [Background technology]

[0004] Background of the Application VISTA (also known as programmed death-1 homolog, PD-1H, VSIR, Dies1, DD1α, Gi24) is a cell surface inhibitory molecule of the B7 / CD28 gene family expressed on T cells and myeloid cells. VISTA can function as an inhibitory ligand for antigen-presenting cells (APCs) and regulate T cell responses through an unknown receptor. In addition, VISTA can also function as an inhibitory receptor on T cells. For example, agonist VISTA monoclonal antibodies (mAbs) dramatically regulate antigen-specific CD4+ T cell responses and protect mice from graft-versus-host disease (GVHD) and experimental hepatitis. Mice deficient in VISTA on a C57BL / 6 background (B6 PD-1H KO) are more susceptible to autoimmune induction, such as experimental autoimmune encephalomyelitis and systemic lupus, when backcrossed with a lupus-susceptible strain. VISTA has been shown to be involved in peripheral immune tolerance and negatively regulate T cell activation. See, e.g., Sci Transl Med. 2019 Dec 11;11(522).

[0005] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. Summary of the Invention [Means for solving the problem]

[0006] BRIEF SUMMARY OF THE APPLICATION In one aspect, the application provides an anti-VISTA construct comprising an antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), the antibody portion competes for the binding epitope of VISTA with an antibody or antibody fragment comprising a second heavy chain variable region (VH-2) and a second light chain variable region (VL-2); a) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; b) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14; c) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; d) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30; or e) VH-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, and VL-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0007] In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR. In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR. In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR.In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR. In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR. In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 51, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44 or 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 or 57.In some embodiments, the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 43, ii) an HC-CDR2 comprising the amino acid sequence of any of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 45; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 or 53.

[0008] In another aspect, the present application provides an anti-VISTA construct comprising an antibody portion that specifically binds to VISTA, a) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 7, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 8; b) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 15, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 16; c) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 23, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 24; d) HC-CDR1, HC-CDR2 and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in a VH chain region having the sequence set forth in SEQ ID NO: 31, and LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in a VL chain region having the sequence set forth in SEQ ID NO: 32; or e) providing an anti-VISTA construct comprising HC-CDR1, HC-CDR2 and HC-CDR3, the amino acid sequences of which are CDR1, CDR2 and CDR3, respectively, within a VH chain region having the sequence set forth in SEQ ID NO: 39, and LC-CDR1, LC-CDR2 and LC-CDR3, the amino acid sequences of which are CDR1, CDR2 and CDR3, respectively, within a VL chain region having the sequence set forth in SEQ ID NO: 40.

[0009] In some embodiments according to any of the above anti-VISTA constructs, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31 and 39, or a variant having at least about 80% sequence identity; and / or the VL comprises an amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, 32 and 40, or a variant having at least about 80% sequence identity. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 7, or a variant having at least about 80% sequence identity; the VL comprises an amino acid sequence of SEQ ID NO: 8, or a variant having at least about 80% sequence identity. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 15, or a variant having at least about 80% sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 16, or a variant having at least about 80% sequence identity. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO:23, or a variant thereof having at least about 80% sequence identity; the VL comprises an amino acid sequence of SEQ ID NO:24, or a variant thereof having at least about 80% sequence identity. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO:31, or a variant thereof having at least about 80% sequence identity; the VL comprises an amino acid sequence of SEQ ID NO:32, or a variant thereof having at least about 80% sequence identity. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO:39, or a variant thereof having at least about 80% sequence identity; the VL comprises an amino acid sequence of SEQ ID NO:40, or a variant thereof having at least about 80% sequence identity.

[0010] In some embodiments according to any of the above anti-VISTA constructs, the antibody portion is an antibody or antigen-binding fragment thereof selected from the group consisting of a full length antibody, a bispecific antibody, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody. In some embodiments, the antibody portion is a full length antibody.

[0011] In some embodiments of any of the above anti-VISTA constructs, the antibody portion has an Fc fragment selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof. In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment. In some embodiments, the Fc fragment has an extended half-life compared to the corresponding wild-type Fc fragment.

[0012] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion of the anti-VISTA construct activates a downstream signaling pathway of VISTA.

[0013] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct is an agonistic antibody to VISTA, hi some embodiments, the antibody portion of the anti-VISTA construct activates or increases downstream signaling pathways of VISTA by at least about 20%.

[0014] In some embodiments according to any of the above anti-VISTA constructs, VISTA is human VISTA.

[0015] In another aspect, the present application provides a pharmaceutical composition comprising an anti-VISTA construct as described above and a pharma- ceutically acceptable carrier.

[0016] In another aspect, the application provides an isolated nucleic acid encoding the above-described anti-VISTA construct.

[0017] In another aspect, the present application provides a vector comprising the above-described isolated nucleic acid sequence.

[0018] In another aspect, the present application provides an isolated host cell comprising the above-described isolated nucleic acid sequence or vector.

[0019] In another aspect, the application provides an immunoconjugate comprising an anti-VISTA construct as described above, linked to a therapeutic agent or a label.

[0020] In another aspect, the present application provides a method for producing an anti-VISTA construct, the method comprising: a) culturing the above-mentioned isolated host cell under conditions effective to express the anti-VISTA construct; and b) obtaining the expressed anti-VISTA construct from the host cell.

[0021] In another aspect, the application provides a method of treating a disease or condition in an individual, comprising administering to the individual an effective amount of an anti-VISTA construct or pharmaceutical composition as described above. In some embodiments, the disease condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the anti-VISTA construct is administered intravenously or subcutaneously to the individual. In some embodiments, the anti-VISTA construct is administered at a dose of about 0.001 mg / kg to about 100 mg / kg. In some embodiments, the individual is a human.

[0022] In another aspect, the present application provides a kit comprising the anti-VISTA construct described above. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows anti-human VISTA antibody titers in the serum of three immunized VISTA knockout mice by ELISA.

[0024] [Diagram 2] FIG. 2 shows anti-mouse VISTA antibody titers in the serum of three immunized VISTA knockout mice by ELISA.

[0025] [Diagram 3] FIG. 3 shows the binding activity of various anti-VISTA antibodies to the human, mouse and cynomolgus monkey VISTA extracellular domains.

[0026] [Figure 4A] Figures 4A-4B show the binding activity of various anti-VISTA antibodies to Jurkat-hVISTA and Jurkat-mVISTA expressing cells using fluorescence-activated cell sorting (FACS). [Figure 4B] Figures 4A-4B show the binding activity of various anti-VISTA antibodies to Jurkat-hVISTA and Jurkat-mVISTA expressing cells using fluorescence-activated cell sorting (FACS).

[0027] [Figure 5A] 5A to 5B show activation of the VISTA downstream pathway by 9F9 at various concentrations in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells. [Figure 5B] 5A to 5B show activation of the VISTA downstream pathway by 9F9 at various concentrations in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0028] [Figure 6A] 6A to 6B show activation of the VISTA downstream pathway by 20E4 at various concentrations in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells. [Figure 6B] 6A to 6B show activation of the VISTA downstream pathway by 20E4 at various concentrations in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0029] [Figure 7] FIG. 7 compares various concentrations of different anti-VISTA antibodies in their ability to activate the VISTA downstream pathway in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0030] [Figure 8A] 8A to 8B show activation of the VISTA downstream pathway by 9F9 at various concentrations in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells. [Figure 8B] 8A to 8B show activation of the VISTA downstream pathway by 9F9 at various concentrations in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0031] [Figure 9A] 9A to 9B show activation of the VISTA downstream pathway by 20E4 at various concentrations in the presence of OKT3 in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells. [Figure 9B] 9A to 9B show activation of the VISTA downstream pathway by 20E4 at various concentrations in the presence of OKT3 in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0032] [Figure 10]FIG. 10 compares various concentrations of different anti-VISTA antibodies in their ability to activate the VISTA downstream pathway in the presence of OKT3 in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells.

[0033] [Figure 11A] Figures 11A-B show various recombinant mIgG1 anti-VISTA antibodies (9F9, 16A1, 17E7, 20E4, V4) that specifically bind to Jurkat-hVISTA and Jurkat-mVISTA expressing cell lines using fluorescence activated cell sorting (FACS). [Figure 11B] Figures 11A-B show various recombinant mIgG1 anti-VISTA antibodies (9F9, 16A1, 17E7, 20E4, V4) that specifically bind to Jurkat-hVISTA and Jurkat-mVISTA expressing cell lines using fluorescence activated cell sorting (FACS).

[0034] [Figure 12] FIG. 12 shows recombinant mIgG1 anti-VISTA (9F9) demonstrating activation in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cell lines using fluorescence activated cell sorting (FACS).

[0035] [Figure 13] FIG. 13 shows recombinant mIgG1 anti-VISTA (17E9) demonstrating activation in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cell lines using fluorescence activated cell sorting (FACS).

[0036] [Figure 14] FIG. 14 shows recombinant mIgG1 anti-VISTA (16A1) demonstrating activation in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cell lines using fluorescence activated cell sorting (FACS).

[0037] [Figure 15]FIG. 15 shows recombinant mIgG1 anti-VISTA (20E4) demonstrating activation in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cell lines using fluorescence activated cell sorting (FACS).

[0038] [Figure 16] FIG. 16 shows epitope binning assay of anti-VISTA antibodies by Octet competition.

[0039] [Figure 17A] Figures 17A-C show human, cynomolgus monkey and mouse VISTA antigen cross-binding activity of anti-VISTA mAbs by biolayer interferometry (BLI) assay. [Figure 17B] Figures 17A-C show human, cynomolgus monkey and mouse VISTA antigen cross-binding activity of anti-VISTA mAbs by biolayer interferometry (BLI) assay. [Figure 17C] Figures 17A-C show human, cynomolgus monkey and mouse VISTA antigen cross-binding activity of anti-VISTA mAbs by biolayer interferometry (BLI) assay.

[0040] [Figure 18A] Figures 18A-E show the binding activity of various anti-VISTA mAbs to human or cynomolgus VISTA. [Figure 18B] Figures 18A-E show the binding activity of various anti-VISTA mAbs to human or cynomolgus VISTA. [Figure 18C] Figures 18A-E show the binding activity of various anti-VISTA mAbs to human or cynomolgus VISTA. [Figure 18D] Figures 18A-E show the binding activity of various anti-VISTA mAbs to human or cynomolgus VISTA. [Figure 18E] Figures 18A-E show the binding activity of various anti-VISTA mAbs to human or cynomolgus VISTA.

[0041] [Figure 19] FIG. 19 shows the inhibitory effect of anti-VISTA mAbs on T cell proliferation.

[0042] [Figure 20] FIG. 20 outlines the experimental protocol for the mouse lupus treatment model.

[0043] [Figure 21] Figure 21A shows lymph node enlargement in mice treated with mIgG, MH5A, 9F9 and 20E4 at 12, 14 and 15 weeks of age, respectively, and Figure 21B shows a lymph node removed from the neck region of one mouse in the 20E4 group at 19 weeks.

[0044] [Figure 22] FIG. 22 shows serum levels of antinuclear immunoglobulin in mice treated with mIgG, MH5A, 9F9 and 20E4.

[0045] [Diagram 23] FIG. 23 shows serum levels of anti-dsDNA immunoglobulins in mice upon treatment with mIgG, MH5A, 9F9 and 20E4.

[0046] [Figure 24] FIG. 24 shows serum levels of IFNα in mice upon treatment with mIgG, MH5A, 9F9 and 20E4.

[0047] [Diagram 25] FIG. 25 shows urinary protein levels in 12 week old mice upon treatment with mIgG, MH5A, 9F9 and 20E4.

[0048] [Figure 26] FIG. 26 shows urinary protein levels in 15 week old mice upon treatment with mIgG, MH5A, 9F9 and 20E4.

[0049] [Figure 27] FIG. 27 shows changes in cutaneous lupus lesions in 17-week-old mice upon treatment with mIgG, MH5A, 9F9 and 20E4.

[0050] [Figure 28] FIG. 28 shows the weight change in mice injected with isotype control and treated with anti-VISTA antibodies 9F9 or 20E4.

[0051] [Figure 29] FIG. 29 shows the levels of human CD45+ cells in the blood of mice injected with isotype control and treated with anti-VISTA antibodies 9F9 or 20E4.

[0052] [Diagram 30] FIG. 30 shows skin denudation in mice injected with isotype control and treated with anti-VISTA antibodies 9F9 or 20E4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Detailed Description of the Application The present application provides novel anti-VISTA constructs that specifically bind to VISTA, methods of preparing the anti-VISTA constructs, and methods of using the constructs (e.g., methods of treating a disease or condition). Exemplary anti-VISTA constructs include agonist antibodies that can bind to and activate VISTA.

[0054] I. Definition The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0055] A full-length antibody contains two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains are each called "V H " and "V L ". The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs including HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are interposed between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain). Chimeric Fc regions (such as IgG2 / 4 mixtures) are also contemplated herein.

[0056] The term "antigen-binding fragment" as used herein refers to antibody fragments including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelid single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen that the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, an antigen-binding fragment can contain one or more CDRs from a particular human antibody grafted onto framework regions from a different human antibody from the fragment.

[0057] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a tightly non-covalently bound dimer of one heavy chain variable region domain and one light chain variable region domain. Folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but often with lower affinity than the entire binding site.

[0058] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is a VFv that is linked to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment that contains an antibody domain, which enables the scFv to form the desired structure for antigen binding. H Domain and V LIt further comprises a polypeptide linker between the domains. For a review of scFvs, see Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0059] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified by Kabat et al., J.Biol.Chem.252:6609-6616(1977);Kabat et al.,USDept.of Health and Human Services, "Sequences of proteins of immunological interest"(1991);Chothia et al.,J.Mol.Biol.196:901-917(1987);Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997);MacCallum et al.,J.Mol.Biol.262:732-745(1996);Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008);Lefranc MPet al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined by each of the above cited references are shown in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, e.g., Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015).The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and for possible inclusion in one or more claims herein. In some embodiments, the CDR sequences provided herein are based on the IMGT definition. For example, CDR sequences can be determined by the VBASE2 tool (http: / / www.vbase2.org / vbase2.php, see also Retter I, Althaus HH, Munch R, Muller W: VBASE2, an integrated V gene database. Nucleic Acids Res. 2005 Jan 1;33 (database issue):D671-4, incorporated herein by reference in its entirety). [Table 1]

[0060] The phrases "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody compilation of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of the FR or hypervariable region (HVR) of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbering sequence at the regions of homology.

[0061] Unless otherwise indicated herein, the numbering of residues in an immunoglobulin heavy chain is that of the EU index in Kabat et al., supra. "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0062] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0063] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of human immunoglobulin sequences. Humanized antibodies also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0064] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic animals, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) for human antibodies generated via human B-cell hybridoma technology.

[0065] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after the sequences are aligned, taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004.

[0066] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences multiplied by 100 divided by the number of positions compared. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the amino acid sequences TKLEIK and TALGIE share 50% homology. Generally, comparisons are made when the two sequences are aligned to obtain maximum homology.

[0067] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain is the C H 1. C H 2 and C H 3 domains (collectively C H ) and light chain CHL (or C L ) domain.

[0068] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0069] The "CH1 domain" (also called "C1" for "H1" domain) typically spans from about amino acid 118 to about amino acid 215 (EU numbering system).

[0070] The "hinge region" is generally defined as the region in IgG corresponding to Glu216-Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain S-S bonds in the same positions.

[0071] The "CH2 domain" (also called the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains in intact native IgG molecules. It has been speculated that the carbohydrates may provide a surrogate for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0072] The "CH3 domain" (also called the "C2" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 for IgG).

[0073] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from the amino acid residue at position Cys226 or the amino acid residue at Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. In some cases, the subsequent C-terminal glycine (residue 446 according to the EU numbering system) of the Fc region may also be removed. Thus, a composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population with no K447 residues removed, and an antibody population with a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0074] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, with FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Neonatal Fc receptors (FcRNs) are encompassed herein. Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0075] As used herein, the term "epitope" refers to the particular group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen.

[0076] As used herein, a first antibody or fragment thereof "competes" with a second antibody or fragment thereof for binding to a target antigen if the first antibody or fragment thereof inhibits target antigen binding of the second antibody or fragment thereof by at least about 50% (e.g., at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof, or vice versa. A high throughput process for "binning" antibodies based on cross-competition is described in PCT Publication No. WO 03 / 48731.

[0077] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding, between a target and an antibody or antibody portion that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to this target with higher affinity, avidity, more readily, and / or with longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10%. -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M, or ≤ 10 -12 Dissociation constant of M (K D). In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may include, but does not require, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-tests and peptide scans.

[0078] An "isolated" antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of association with all other components from its production environment.

[0079] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is not associated with all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide and antibody described herein is in a form other than the form or situation in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide and antibody described herein that naturally occurs in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0080] The term "control sequence" refers to a DNA sequence required for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0081] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0082] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0083] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0084] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0085] The term "immunoconjugate" includes reference to the covalent attachment of a therapeutic agent or detectable label to an antibody, such as an antibody portion described herein. The linkage can be direct or indirect, such as through a linker (such as a peptide linker).

[0086] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving or improving quality of life, increasing weight gain, and / or prolonging survival. The methods of the present application contemplate any one or more of these aspects of treatment.

[0087] The term "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, degree, or likelihood of that characteristic. "Reducing" or "inhibiting" is the reduction, reduction, or cessation of an activity, function, and / or amount compared to a reference. In certain embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 50% or more. In yet another embodiment, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0088] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. Reference can be obtained from healthy and / or non-disease samples. In some examples, reference can be obtained from untreated samples. In some examples, reference is obtained from non-disease or non-treated samples of individuals. In some examples, reference is obtained from one or more healthy individuals who are not individuals or patients.

[0089] As used herein, "delaying the onset of disease" means postponing, impeding, slowing, preventing, stabilizing, inhibiting and / or postponing the onset of disease. This delay can be of various lengths of time, depending on the disease being treated and / or the medical history of the individual. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0090] "Preventing" as used herein includes providing prophylaxis against the occurrence or recurrence of a disease in an individual who may have a predisposition to the disease, but has not yet been diagnosed with the disease.

[0091] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, an individual is a human.

[0092] An "effective amount" of an agent refers to an amount effective at the dosage and duration required to achieve the desired therapeutic or prophylactic result. The specific dosage may vary depending on one or more of the specific agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0093] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and does not contain additional components that are unacceptably toxic to the individual to whom the formulation is administered. Such formulations may be sterile.

[0094] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0095] A "sterile" preparation is sterile or essentially free of living microorganisms and their spores.

[0096] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0097] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time compared to the administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered within a time interval of about 60 minutes or less, e.g., about any of 30, 15, 10, 5, or 1 minutes or less.

[0098] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents, where administration of one or more agents is continued after discontinuing administration of the other agent or agents. For example, administration of the two or more therapeutic agents is administered at intervals of more than about 15 minutes, e.g., about 20, 30, 40, 50, or 60 minutes, one day, two days, three days, one week, two weeks, or one month or more.

[0099] As used herein, "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "in combination with" refers to the administration of one treatment modality before, during or after the administration of another treatment modality to an individual.

[0100] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for the use of such therapeutic product.

[0101] An "article of manufacture" is any article of manufacture (e.g., package or container) or kit that contains at least one reagent, such as a pharmaceutical agent for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0102] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of."

[0103] Reference herein to "about" a value or parameter includes (and accounts for) the variation that is directed to the value or parameter itself. For example, a statement of "about X" includes the statement "X."

[0104] As used herein, reference to a value or parameter "is not" generally means and describes a value or parameter "other than." For example, the method is not used to treat disease of type X means that the method is used to treat diseases other than type X.

[0105] Here, "about X to Y" is synonymous with "about X to about Y."

[0106] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0107] II. Anti-VISTA Constructs The present application provides anti-VISTA constructs comprising an anti-VISTA antibody portion that specifically binds to VISTA as described herein.

[0108] VISTA V-domain Ig inhibitor of T-cell activation (VISTA) (also called PD-1H, Gi24, Dies-1, or DD1α) is a more recently identified cell surface co-inhibitory molecule of the CD28 / B7 gene family. VISTA can function as an inhibitory ligand for antigen-presenting cells and regulate T-cell responses, and it has been reported that ablation of VISTA, either by gene knockout or antagonistic antibodies, can enhance T-cell immune responses against tumors in mouse models. VISTA may also play an important role in regulating inflammation and autoimmune diseases, as shown in mouse models of graft-versus-host disease (GVHD), acute hepatitis, encephalitis, lupus, asthma, and psoriasis. VISTA may also function as a co-inhibitory receptor on T cells. VISTA agonistic mAbs dramatically regulate antigen-specific CD4 T-cell responses and protect mice from GVHD, acute hepatitis, and asthma. See Files et al., J. Immunol. 187, 1537-1541 (2011); Files et al., J. Clin. Invest. 124, 1966-1975 (2014); and Liu et al., Cell. Mol. Immunol. 15, 838-845 (2018). Upregulated VISTA in prostate cancer patients was also shown to be associated with resistance to ipilimumab (CTLA-4 mAb). Furthermore, it has been shown that targeting VISTA may synergize with other non-redundant pathways, such as PD-1 blockade, to achieve optimal tumor clearance efficacy in experimental mouse models. See Liu et al., Proc. Natl. Acad. Sci. USA 112, 6682-6687 (2015). Thus, VISTA may be a key molecule in regulating immune responses and a potential target for immunotherapy.

[0109] The VISTA gene is located at 10q22.1. It is conserved in chimpanzees, cows, mice, rats, chickens, zebrafish and frogs. Human VISTA sequence can be found under NCBI reference number NM_022153. Human VISTA protein has 311 amino acids (NCBI reference number: NP_071436.1, SEQ ID NO: 59).

[0110] Anti-VISTA antibody part In some embodiments, the anti-VISTA construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0111] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; Lcomprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDR. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0112] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0113] In some embodiments, the antibody portion has the sequence shown in SEQ ID NO:7. H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:8. L The chain regions include LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

[0114] In some embodiments, V H includes a variant comprising an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; Lincludes variants comprising an amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0115] In some embodiments, the anti-VISTA construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0116] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDR. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0117] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0118] In some embodiments, the antibody portion has the sequence set forth in SEQ ID NO:15. H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 within the chain region, respectively, and V having the sequence shown in SEQ ID NO: 16 L The chain regions include LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

[0119] In some embodiments, V H includes a variant comprising an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L includes variants comprising an amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0120] In some embodiments, the anti-VISTA construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA,H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0121] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDR. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0122] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, Lcomprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0123] In some embodiments, the antibody portion has the sequence set forth in SEQ ID NO:23. H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 within the chain region, respectively, and V having the sequence shown in SEQ ID NO:24 L The chain regions include LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

[0124] In some embodiments, V H includes a variant comprising an amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L includes variants comprising an amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0125] In some embodiments, the anti-VISTA construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; L-2comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30.

[0126] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDR. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0127] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30.

[0128] In some embodiments, the antibody portion has the sequence set forth in SEQ ID NO:31. HHC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3 within the chain region, respectively, and V having the sequence shown in SEQ ID NO:32 L The chain regions include LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

[0129] In some embodiments, V H includes a variant comprising an amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L includes variants comprising an amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0130] In some embodiments, the anti-VISTA construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, L-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:36, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:38.

[0131] In some embodiments, V Hcomprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDR. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0132] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:38.

[0133] In some embodiments, the antibody portion has the sequence set forth in SEQ ID NO:39. H HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:40 L The chain regions include LC-CDR1, LC-CDR2 and LC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

[0134] In some embodiments, V H includes an amino acid sequence of SEQ ID NO: 39, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L includes variants comprising an amino acid sequence of SEQ ID NO:40, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0135] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47.

[0136] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47.

[0137] In some embodiments, V Hcomprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0138] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0139] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0140] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V Hcomprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0141] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0142] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0143] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, Lcomprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0144] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0145] In some embodiments, V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0146] In some embodiments, the anti-VISTA antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, Lcomprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0147] In some embodiments, the construct comprises an antibody portion, which may be a full length antibody, a diabody, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , V H H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody, or an antigen-binding fragment thereof.

[0148] In some embodiments, the anti-VISTA antibody portion is a full-length antibody.

[0149] In some embodiments, the anti-VISTA antibody portion is an scFv.

[0150] In some embodiments, the anti-VISTA antibody portion comprises an Fc fragment of an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the anti-VISTA antibody portion or full length antibody comprises an Fc fragment of an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof. In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment. In some embodiments, the Fc fragment has enhanced effector function compared to the corresponding wild-type Fc fragment.

[0151] In some embodiments, the antibody portion comprises a humanized antibody of any of the antibody portions described herein.

[0152] In some embodiments, the anti-VISTA antibody portion binds to both human VISTA and cynomolgus VISTA. In some embodiments, the anti-VISTA antibody portion binds to both human VISTA and mouse VISTA. In some embodiments, the anti-VISTA antibody portion binds to human VISTA, cynomolgus VISTA, and mouse VISTA. In some embodiments, the anti-VISTA antibody portion does not bind to cynomolgus VISTA and / or mouse VISTA.

[0153] In some embodiments, the antibody portion of the anti-VISTA construct activates downstream signaling pathways of VISTA, hi some embodiments, the anti-VISTA construct is an agonistic antibody of VISTA.

[0154] In some embodiments, the antibody portion of the anti-VISTA construct activates or increases a downstream signaling pathway of VISTA by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a reference construct (e.g., a corresponding construct that does not activate VISTA, e.g., a corresponding construct that includes a reference agonist anti-VISTA antibody such as 1E8).

[0155] In some embodiments, the anti-VISTA construct comprises or is an anti-VISTA fusion protein. In some embodiments, the anti-VISTA construct comprises an anti-VISTA antibody portion (e.g., an anti-VISTA scFv) and a second portion. In some embodiments, the second portion comprises a half-life extending moiety. In some embodiments, the half-life extending moiety is an albumin binding moiety (e.g., an albumin binding antibody portion). In some embodiments, the anti-VISTA antibody portion and the half-life extending moiety are linked via a linker (e.g., a peptide linker, e.g., a GS linker).

[0156] In some embodiments, the anti-VISTA construct comprises or is an anti-VISTA immunoconjugate comprising an anti-VISTA antibody moiety (e.g., any of the VISTA antibody moieties described herein) and a second agent. In some embodiments, the second agent is a therapeutic agent. In some embodiments, the second agent is a label.

[0157] In some embodiments, VISTA is human VISTA.

[0158] a) Antibody affinity The binding specificity of an antibody moiety can be determined experimentally by methods known in the art, including, but not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-test and peptide scan.

[0159] In some embodiments, the K of binding between the antibody moiety and VISTA D is about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10-10 M, about 10 -8 M~about 10 -10 M, or about 10 -7 M~about 10 -9 In some embodiments, the K of the bond between the antibody moiety and VISTA is D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, VISTA is human VISTA.

[0160] In some embodiments, the K of binding between the antibody moiety and VISTA on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is on is about 10 3 M -1 s -1~about 10 5 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is on is about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 In some embodiments, VISTA is human VISTA.

[0161] In some embodiments, the K of binding between the antibody moiety and VISTA off takes about 1 s -1 ~about 10 -6 s -1 , about 1s-1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 , or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is off is at least about 1s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 In some embodiments, VISTA is human VISTA.

[0162] In some embodiments, the binding affinity of an anti-VISTA antibody portion or anti-VISTA construct is higher (e.g., K ) than an existing anti-VISTA antibody (e.g., an anti-human VISTA antibody, e.g., 1E8). D value is smaller).

[0163] b) Chimeric or humanized antibodies In some embodiments, the anti-VISTA antibody portion is a chimeric antibody. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region from a mouse) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0164] In some embodiments, the anti-VISTA antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0165] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0166] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0167] It is understood that humanization of mouse-derived antibodies is a common and routinely used technique.Therefore, it is understood that any and all humanized formats of anti-VISTA antibodies disclosed in the sequence listing can be used in preclinical or clinical situations.When any humanized format of the referenced anti-VISTA antibody or its antigen-binding region is used in such preclinical or clinical situations, the humanized format is expected to have the same or similar biological activity and profile as the original non-humanized format.

[0168] c) Human antibodies In some embodiments, the anti-VISTA antibody portion is a human antibody (known as a human domain antibody or human DAb). Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are known in the art. See, e.g., US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0169] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is extrachromosomal, or is randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab®); U.S. Pat. No. 7,041,870 (describing KM MOUSE®), and U.S. Patent Application No. 2007 / 0061900 (describing VelociMouse®). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0170] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0171] Human antibodies (e.g., human DAbs) can also be made by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from an antibody library are described below.

[0172] d) Library-derived antibodies The anti-VISTA antibody portions described herein can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single domain antibody libraries have been described, see, e.g., U.S. Patent No. 7,371,849.

[0173] In certain phage display methods, V H and V LGene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either scFv fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without any immunization to provide a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0174] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0175] e) Substitutions, insertions, deletions and variants In some embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". More substantial changes are provided in Table 2 under the heading of "exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 2]

[0176] Amino acids can be grouped according to general side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0177] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0178] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0179] For example, modifications (e.g., substitutions) may be made to HVRs to improve antibody affinity. Such modifications may be made at HVR "hot spots," i.e., codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues encoded by the SDRs (a-CDRs), resulting in variant V H or V Lare tested for binding affinity. Affinity maturation by constructing and reselecting a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0180] In some embodiments, substitutions, insertions or deletions can occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such changes can be outside of HVR "hot spots" or CDRs.

[0181] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0182] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0183] f) Glycosylation variants In some embodiments, the anti-VISTA antibody portion is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0184] If the antibody moiety comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically have a C-terminal end with an Fc region. HThe oligosaccharides of the antibody moiety include branched, biantennary oligosaccharides that are generally linked by N-linkage to Asn297 of the 2 domain. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides of the antibody moiety may be performed to generate antibody variants with specific improved properties.

[0185] In some embodiments, the anti-VISTA antibody portion has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. For example, U.S. Patent Application Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778;WO2005 / 053742;WO2002 / 031140;Okazaki et al.J.Mol.Biol.336:1239-1249(2004);Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0186] In some embodiments, the anti-VISTA antibody portion has bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0187] g) Fc region variant In some embodiments, the anti-VISTA antibody portion comprises an Fc fragment.

[0188] The term "Fc region", "Fc domain", "Fc fragment" or "Fc" refers to the C-terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues in an IgG or Fc region follows the EU numbering system for antibodies, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0189] In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0190] In some embodiments, the Fc fragment has reduced effector function compared to a corresponding wild-type Fc fragment (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC).

[0191] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises a L234A mutation and / or a L235A mutation. In some embodiments, the IgG1 Fc fragment comprises a L235A mutation and / or a G237A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising S228P, F234A and / or L235A mutations. In some embodiments, the Fc fragment comprises a N297A mutation. In some embodiments, the Fc fragment comprises a N297G mutation.

[0192] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody moiety, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0193] In some embodiments, the Fc fragment has some, but not all, effector functions, making it a desirable candidate for applications where the half-life of the antibody portion in vivo is important, but where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0194] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc fragment comprises a N297A mutation. In some embodiments, the Fc fragment comprises a N297G mutation.

[0195] Certain antibody variants with improved or diminished binding to FcRs have been described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0196] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises a L234A mutation and / or a L235A mutation. In some embodiments, the IgG1 Fc fragment comprises a L235A mutation and / or a G237A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising S228P, F234A and / or L235A mutations.

[0197] In some embodiments, the antibody portion comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0198] In some embodiments, modifications are made to the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0199] In some embodiments, the Fc fragment has one or more mutations at Thr250, Met252, Ser254, The256, Thr307. Glu380, Met428, His433, and / or Asn434.

[0200] In some embodiments, the antibody partial variant comprises a variant Fc region that comprises one or more amino acid substitutions that alter half-life and / or change binding to neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to neonatal Fc receptor (FcRn), which is responsible for maternal IgG transfer to fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that alter binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the Fc region residues (e.g., substitution at Fc region residue 434) (U.S. Patent No. 7,371,826).

[0201] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0202] h) Cysteine ​​engineered antibody variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues are located at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues can be replaced with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibody moieties can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0203] i) Antibody derivative In some embodiments, the antibody moieties described herein may be further modified to include additional non-proteinaceous moieties that are known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for diagnosis under defined conditions, etc.

[0204] In some embodiments, the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. "Biological activity" or "biologically active", as used interchangeably herein, means to exhibit biological activity in the body to perform a specific function. For example, it may mean combination with a specific biomolecule, such as a protein, DNA, and then promoting or inhibiting the activity of such a biomolecule. In some embodiments, the biologically active protein or fragment thereof includes proteins and polypeptides administered to a patient as an active drug substance for the prevention or treatment of a disease or condition, as well as proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and polypeptides administered to a patient to prevent disease (such as vaccines).

[0205] III. Preparation method In some embodiments, methods are provided for preparing anti-VISTA constructs or antibody portions that specifically bind VISTA, and compositions such as polynucleotides, nucleic acid constructs, vectors, host cells, or culture media produced during the preparation of the anti-VISTA constructs or antibody portions. The anti-VISTA constructs or antibody portions or compositions described herein may be prepared by several processes as generally described below, and more specifically in the Examples.

[0206] Antibody expression and production The antibodies described herein can be prepared using any method known in the art, including those described below and in the Examples.

[0207] Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal, such as a hamster or llama, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). See also Example 1 for immunization in camels.

[0208] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBL") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0209] Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of hybridoma typically contains hypoxanthine, aminopterin and thymidine, which are substances that inhibit the growth of HGPRT-deficient cells (HAT medium).

[0210] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma lines, such as MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 cells (and their derivatives, such as X63-Ag8-653) (available from the American Type Culture Collection, Manassas, Va. USA), are preferred. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

[0211] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0212] The culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against desired antigens.Preferably, the binding affinity and specificity of monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA).Such techniques and assays are known in the art.For example, binding affinity can be determined by Scatchard analysis of Munson et al., Anal.Biochem.,107:220(1980).

[0213] After hybridoma cells that produce antibodies of desired specificity, affinity and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. A cell sorter may also be used. Additionally, hybridoma cells can be grown in vivo as tumors in mammals.

[0214] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0215] Monoclonal antibodies can also be produced as described above by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, HEK cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulin proteins, and synthesize the monoclonal antibody in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

[0216] In further embodiments, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications have described the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)) as strategies for constructing very large phage libraries. Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0217] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide is used in place of the constant domains of an antibody or in place of the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0218] The monoclonal antibodies described herein may be monovalent, and their preparation is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is generally truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residues may be replaced with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce their fragments, particularly Fab fragments, can be accomplished using routine techniques known in the art.

[0219] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds.Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0220] Nucleic acid molecules encoding antibody portions In some embodiments, a polynucleotide is provided that encodes any one of the anti-VISTA constructs or antibody portions described herein. In some embodiments, a polynucleotide is provided that is prepared using any one of the methods as described herein. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy or light chain of an antibody portion (e.g., an anti-VISTA antibody portion). In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of an antibody portion (e.g., an anti-VISTA antibody portion). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.

[0221] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides, either from one nucleic acid molecule or from two separate nucleic acid molecules, in some embodiments, for example when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0222] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody moiety (e.g., an anti-VISTA antibody moiety) comprises a nucleotide sequence that encodes a leader sequence located at the N-terminus of the heavy or light chain upon translation. As noted above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0223] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0224] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0225] nucleic acid construct In some embodiments, a nucleic acid construct is provided that includes any one of the polynucleotides described herein. In some embodiments, a nucleic acid construct is provided that is prepared using any of the methods described herein.

[0226] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide, hi some embodiments, the polynucleotide corresponds to a gene and the promoter is the wild-type promoter of the gene.

[0227] vector In some embodiments, vectors are provided that include any polynucleotide encoding the heavy and / or light chains of any one of the antibody moieties described herein (e.g., anti-VISTA antibody moieties) or nucleic acid constructs described herein. In some embodiments, vectors prepared using any of the methods described herein are provided. Also provided are vectors that include a polynucleotide encoding any of the anti-VISTA constructs, such as antibodies, scFvs, fusion proteins, or other forms of constructs described herein (e.g., anti-VISTA scFvs). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector includes a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, such as when the antibody is an scFv.

[0228] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into the host cell in similar amounts (such as similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of 5:1 to 1:5 of the first vector and the second vector are transfected into the host cell. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0229] In some embodiments, a vector is selected that is optimized for expression of a polypeptide in CHO or CHO-derived cells or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0230] host cell In some embodiments, a host cell is provided that comprises any of the polypeptides, nucleic acid constructs and / or vectors described herein. In some embodiments, a host cell is provided that is prepared using any of the methods described herein. In some embodiments, the host cell is capable of producing any of the antibody portions described herein under fermentation conditions.

[0231] In some embodiments, the antibody moieties described herein (e.g., anti-VISTA antibody moieties) may be expressed in prokaryotic cells, e.g., bacterial cells; or eukaryotic cells, e.g., fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO-S, CHO-GS, DG44; CHO cells, including Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); HEK cells, and NSO cells. In some embodiments, the antibody moieties described herein (e.g., anti-VISTA antibody moieties) may be expressed in yeast. See, e.g., U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to effect desired post-translational modifications to the heavy and / or light chains of the antibody moiety. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptides produced in 293 cells.

[0232] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into a desired host cell according to any suitable method.

[0233] The present application also provides a host cell comprising any of the polynucleotides or vectors described herein. In some embodiments, the present invention provides a host cell comprising an anti-VISTA antibody. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding antibodies, polypeptides or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtillis) and yeast (e.g., S. cerevisae, S. pombe; or K. lactis).

[0234] In some embodiments, the antibody portion is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0235] Culture medium In some embodiments, a culture medium is provided that comprises any of the antibody portions, polynucleotides, nucleic acid constructs, vectors, and / or host cells described herein. In some embodiments, a culture medium is provided that is prepared using any of the methods described herein.

[0236] In some embodiments, the medium contains hypoxanthine, aminopterin and / or thymidine (e.g., HAT medium). In some embodiments, the medium does not contain serum. In some embodiments, the medium contains serum. In some embodiments, the medium is D-MEM or RPMI-1640 medium.

[0237] In some embodiments, the culture medium is chemically defined, hi some embodiments, the culture medium is specifically derived from a particular cell line (e.g., CHO GS cells).

[0238] Purification of antibody moieties Anti-VISTA constructs may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and antibody constant regions. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind to the constant region and purify anti-VISTA constructs containing the Fc fragment. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for the purification of some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for the purification of some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for the purification of some polypeptides, such as antibodies. Many methods of purifying polypeptides are known in the art.

[0239] V. Treatment Method Also provided herein is a method of treating a disease or condition, or modulating an immune response in an individual (e.g., inhibiting proliferation of T cells), or modulating an immune response in an individual (e.g., inhibiting proliferation of T cells), comprising administering to an individual (e.g., a mammal such as a human) an anti-VISTA construct as described herein.

[0240] In some embodiments, methods of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual are provided, comprising administering to the individual an effective amount of an anti-VISTA construct as described herein. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0241] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual comprises administering to said individual a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, L-2In some embodiments, a method is provided comprising administering to an individual an effective amount of an anti-VISTA construct comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; L In some embodiments, the anti-VISTA antibody portion comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs. H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. H includes a variant comprising an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; Lcomprises an amino acid sequence of SEQ ID NO: 8, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0242] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual comprises administering to an individual a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L-2 In some embodiments, a method is provided comprising administering to an individual an effective amount of an anti-VISTA construct comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14. H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; LIn some embodiments, the anti-VISTA antibody portion comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs. H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14. H includes a variant comprising an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L comprises an amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0243] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual comprises administering to said individual a heavy chain variable region (V H) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, L-2 In some embodiments, a method is provided comprising administering to an individual an effective amount of an anti-VISTA construct comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22. H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; L In some embodiments, the anti-VISTA antibody portion comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs. H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22. Hincludes a variant comprising an amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L comprises an amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0244] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual comprises administering to an individual a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; L-2 In some embodiments, a method is provided comprising administering to an individual an effective amount of an anti-VISTA construct comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30. Hcomprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; L In some embodiments, the anti-VISTA antibody portion comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs. H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30. H includes a variant comprising an amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; Lcomprises an amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0245] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual comprises administering to an individual a heavy chain variable region (V H ) and the light chain variable region (V L ) for the binding epitope of VISTA, H-2 ) and a second light chain variable region (V L-2 ) and competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, L-2 In some embodiments, a method is provided comprising administering to an individual an effective amount of an anti-VISTA construct comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38. H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; LIn some embodiments, the anti-VISTA antibody portion comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs. H ) and the light chain variable region (V L ), a humanized antibody derived from an anti-VISTA antibody containing V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, L comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38. H includes an amino acid sequence of SEQ ID NO: 39, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L comprises an amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0246] In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 of the present application.

[0247] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual is provided, comprising administering to the individual an effective amount of an anti-VISTA construct comprising an antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 51, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47.

[0248] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual is provided, comprising administering to the individual an effective amount of an anti-VISTA construct comprising an antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44 or 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 or 57. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft versus host disease (GvHD) or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE).

[0249] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting T cell proliferation) in an individual is provided, comprising administering to the individual an effective amount of an anti-VISTA construct comprising an antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 43, ii) an HC-CDR2 comprising the amino acid sequence of any of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 45; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 or 53. In some embodiments, the disease or condition is associated with a dysregulated immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft versus host disease (GvHD) or a condition associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE).

[0250] In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a corresponding construct that does not activate VISTA (e.g., an isotype control). In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 5%, 10%, 15%, 20%, or 25% compared to a corresponding construct that includes a reference agonist anti-VISTA antibody (e.g., 1E8).

[0251] In some embodiments, methods of modulating a cell (e.g., an immune cell) are provided, the methods comprising contacting an immune cell with an anti-VISTA construct (such as any of the anti-VISTA constructs described herein). In some embodiments, the cell is a T cell (such as a CD4 and / or CD8 T cell). In some embodiments, the cell is a neutrophil. In some embodiments, the cell is a dendritic cell (e.g., a plasmacytoid dendritic cell). In some embodiments, the cell is a macrophage. In some embodiments, the contacting is performed in vitro.

[0252] In some embodiments, a method of genome editing a cell (e.g., an immune cell) is provided, comprising: a) a donor template comprising a nucleic acid sequence encoding any of the anti-VISTA constructs described herein; and b) introducing into the cell a DNA nuclease, or a nucleotide sequence encoding a DNA nuclease (e.g., a CRISPR-associated protein (Cas)). In some embodiments, the method further comprises administering the genome-edited cell to an individual having a disease or condition described herein.

[0253] In some embodiments, the subject is a mammal, such as a human.

[0254] In some embodiments, the individual has elevated serum levels of antinuclear antibodies (e.g., serum levels of antinuclear antibodies at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of anti-dsDNA antibodies (e.g., serum levels of anti-dsDNA antibodies at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of IFNα (e.g., serum levels of IFNα at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated urinary protein levels (e.g., urinary protein levels at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual).

[0255] Dosing and Administration Methods of Anti-VISTA Constructs The dosing regimen (such as the specific dosage and frequency) of an anti-VISTA construct used to treat a disease or disorder described herein administered to an individual may vary depending on the specific anti-VISTA construct, the mode of administration, and the type of disease or condition being treated. In some embodiments, an effective amount of an anti-VISTA construct is an amount effective to alleviate at least one symptom of a disease or condition. In some embodiments, an effective amount of an anti-VISTA construct is an amount sufficient to extend the overall survival of an individual. In some embodiments, an effective amount of an anti-VISTA construct is an amount sufficient to provide a clinical benefit in greater than about any of 50%, 60%, 70%, 80%, or 90% of a population of individuals treated with the anti-VISTA construct.

[0256] In some embodiments, an effective amount of an anti-VISTA construct is an amount that slows or inhibits progression of a disease or condition (e.g., at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an individual not receiving treatment. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an infectious disease.

[0257] In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of antinuclear antibodies by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of anti-dsDNA antibodies by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of IFNα by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces urinary protein levels by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, or 90% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct).

[0258] In some embodiments, an effective amount of an anti-VISTA construct is an amount that reduces the side effects (autoimmune responses) of a condition (e.g., transplant) by (e.g., at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%) compared to an individual not receiving treatment.

[0259] In some embodiments of any of the above aspects, the effective amount of the anti-VISTA construct is in the range of about 0.001 μg / kg to about 100 mg / kg of total body weight, e.g., about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg.

[0260] In some embodiments of any of the above aspects, the effective amount of the anti-VISTA construct for a human is a dose equivalent to 0.5 mg for a mouse.

[0261] In some embodiments of any of the above aspects, the anti-VISTA construct is administered weekly. In some embodiments of any of the above aspects, the anti-VISTA construct is administered every other week. In some embodiments, the anti-VISTA construct is administered weekly for at least about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 weeks.

[0262] The anti-VISTA constructs can be administered to an individual (such as a human) via a variety of routes including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the anti-VISTA construct is contained in a pharmaceutical composition while being administered to the individual. In some embodiments, sustained continuous release formulations of the composition can be used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered orally.

[0263] Combination therapy The application also provides methods of administering an anti-VISTA construct to an individual to treat a disease or condition, the method further comprising administering a second agent or treatment, in some embodiments, the second agent or treatment is a standard or commonly used agent or treatment for treating the disease or condition.

[0264] In some embodiments, the anti-VISTA construct is administered simultaneously with the second agent or treatment. In some embodiments, the anti-VISTA construct is administered with the second agent or treatment. In some embodiments, the anti-VISTA construct is administered sequentially with the second agent or treatment. In some embodiments, the anti-VISTA construct is administered prior to the second agent or treatment. In some embodiments, the anti-VISTA construct is administered after the second agent or treatment. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as the second agent or treatment. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form than the second agent or treatment. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as the second agent or treatment. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form than the second agent or treatment.

[0265] VI. Compositions, Kits and Articles of Manufacture Also provided herein are compositions (e.g., formulations) comprising any one of the anti-VISTA constructs or anti-VISTA antibody portions described herein, nucleic acids encoding the antibody portions, vectors comprising nucleic acids encoding the antibody portions, or host cells comprising the nucleic acids or vectors.

[0266] Suitable formulations of the anti-VISTA constructs described herein can be obtained by mixing the anti-VISTA constructs or anti-VISTA antibody portion having the desired purity with any pharma- ceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; The lyophilized formulations include proteins such as glycine, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Lyophilized formulations suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations using a suitable diluent, and the reconstituted formulations can be administered subcutaneously to the individual to be imaged, diagnosed, or treated herein.

[0267] Formulations to be used for in vivo administration must be sterile, which is readily accomplished for example, by filtration through sterile filtration membranes.

[0268] Kits are also provided that include any one of the anti-VISTA constructs or anti-VISTA antibody portions described herein, which may be useful for any of the methods of modulating cell compositions or treatments described herein.

[0269] In some embodiments, kits are provided that contain anti-VISTA constructs that specifically bind to VISTA.

[0270] In some embodiments, the kit further comprises a device that can deliver the anti-VISTA construct to an individual.One type of device for applications such as parenteral delivery is a syringe that is used to inject the composition into the subject's body.Inhalation devices can also be used for certain applications.

[0271] In some embodiments, the kit further comprises a therapeutic agent for treating a disease or condition, such as an infectious disease, an autoimmune disease, or a transplant.

[0272] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kits may provide additional components, such as buffers and interpretive information, as needed.

[0273] Thus, the application also provides an article of manufacture. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, and the like. Generally, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to image, diagnose, or treat a particular condition in an individual. The label or package insert further includes instructions for administering the composition to an individual and imaging the individual. The label may indicate instructions for reconstitution and / or use. The container holding the composition may be a multi-use vial that allows for repeated administration (e.g., from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions typically included in commercial packaging of diagnostic products, including information regarding indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such diagnostic products. Additionally, the article of manufacture may further comprise a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0274] The kit or article of manufacture may include a number of unit doses of the composition and instructions for use and may be packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy or compounding pharmacy.

[0275] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as limiting its scope in any way.

[0276] Exemplary embodiments Embodiment 1. An anti-VISTA construct comprising an antibody portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody portion competes for the binding epitope of VISTA with an antibody or antibody fragment comprising a second heavy chain variable region (VH-2) and a second light chain variable region (VL-2); a) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; b) VH-2 comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, and VL-2 comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14; c) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; d) VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30; or e) An anti-VISTA construct, wherein VH-2 comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, and VL-2 comprises LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0277] Embodiment 2. An anti-VISTA construct as described in embodiment 1, a) the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR; b) VH comprises i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; VL comprises i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR; c) VH comprises i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; VL comprises i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR; d) VH comprises i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; VL comprises i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR; e) VH comprises i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDR; VL comprises i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDR; f) VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 51, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; h) the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44 or 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45; the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 or 57; or An anti-VISTA construct, wherein i) the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 43, ii) an HC-CDR2 comprising the amino acid sequence of any of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 45; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 or 53.

[0278] Embodiment 3. An anti-VISTA construct as described in embodiment 2, wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0279] Embodiment 4. An anti-VISTA construct as described in embodiment 2, wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0280] Embodiment 5. An anti-VISTA construct as described in embodiment 3, wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

[0281] Embodiment 6. An anti-VISTA construct as described in embodiment 3, wherein the VH comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; and the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0282] Embodiment 7. An anti-VISTA construct comprising an antibody portion that specifically binds to VISTA, a) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 7, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 8; b) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 15, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 16; c) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VH chain region having the sequence shown in SEQ ID NO: 23, and LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in a VL chain region having the sequence shown in SEQ ID NO: 24; d) HC-CDR1, HC-CDR2 and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in a VH chain region having the sequence set forth in SEQ ID NO: 31, and LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in a VL chain region having the sequence set forth in SEQ ID NO: 32; or e) An anti-VISTA construct comprising HC-CDR1, HC-CDR2 and HC-CDR3, each of which comprises the amino acid sequences of CDR1, CDR2 and CDR3, within a VH chain region having the sequence set forth in SEQ ID NO: 39, and LC-CDR1, LC-CDR2 and LC-CDR3, each of which comprises the amino acid sequences of CDR1, CDR2 and CDR3, within a VL chain region having the sequence set forth in SEQ ID NO: 40.

[0283] Embodiment 8. An anti-VISTA construct described in any one of embodiments 1 to 7, wherein VH comprises a variant comprising an amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31 and 39, or an amino acid sequence having at least about 80% sequence identity; and / or VL comprises a variant comprising an amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, 32 and 40, or an amino acid sequence having at least about 80% sequence identity.

[0284] Embodiment 9. An anti-VISTA construct as described in embodiment 8, a) the VH comprises an amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% sequence identity; or b) the VH comprises an amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 80% sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% sequence identity; or c) the VH comprises an amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% sequence identity; or d) the VH comprises an amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% sequence identity; the VL comprises an amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% sequence identity; or e) An anti-VISTA construct, wherein VH comprises an amino acid sequence of SEQ ID NO: 39, or a variant thereof having an amino acid sequence with at least about 80% sequence identity; and VL comprises an amino acid sequence of SEQ ID NO: 40, or a variant thereof having an amino acid sequence with at least about 80% sequence identity.

[0285] Embodiment 10. An anti-VISTA construct described in any one of embodiments 1 to 9, wherein the antibody portion is an antibody or an antigen-binding fragment thereof selected from the group consisting of a full-length antibody, a bispecific antibody, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.

[0286] Embodiment 11 The anti-VISTA construct of embodiment 10, wherein the antibody portion is a full-length antibody.

[0287] Embodiment 12. An anti-VISTA construct described in any one of embodiments 1 to 11, wherein the antibody portion has an Fc fragment selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof.

[0288] Embodiment 13. An anti-VISTA construct according to embodiment 12, wherein the Fc fragment is selected from the group consisting of Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0289] Embodiment 14. An anti-VISTA construct according to embodiment 12 or embodiment 13, wherein the Fc fragment has a reduced effector function compared to the corresponding wild-type Fc fragment.

[0290] Embodiment 15. An anti-VISTA construct according to any one of embodiments 12 to 14, wherein the Fc fragment has a longer half-life compared to the corresponding wild-type Fc fragment.

[0291] Embodiment 16. An anti-VISTA construct described in any one of embodiments 1 to 15, wherein the antibody portion of the anti-VISTA construct activates a downstream signaling pathway of VISTA.

[0292] Embodiment 17. An anti-VISTA construct described in any one of embodiments 1 to 16, wherein the anti-VISTA construct is an agonistic antibody to VISTA.

[0293] Embodiment 18. An anti-VISTA construct according to any one of embodiments 16, wherein the antibody portion of the anti-VISTA construct activates or increases downstream signaling pathways of VISTA by at least about 20%.

[0294] Embodiment 19. An anti-VISTA construct according to any one of embodiments 1 to 18, wherein VISTA is human VISTA.

[0295] Embodiment 20. A pharmaceutical composition comprising an anti-VISTA construct according to any one of embodiments 1 to 19 and a pharma- ceutically acceptable carrier.

[0296] Embodiment 21. An isolated nucleic acid encoding an anti-VISTA construct according to any one of embodiments 1 to 20.

[0297] Embodiment 22. A vector comprising the isolated nucleic acid of embodiment 21.

[0298] Embodiment 23. An isolated host cell comprising the isolated nucleic acid of embodiment 21 or the vector of embodiment 22.

[0299] Embodiment 24. An immunoconjugate comprising an anti-VISTA construct according to any one of embodiments 1 to 19 linked to a therapeutic agent or a label.

[0300] Embodiment 25. A method of producing an anti-VISTA construct, comprising: a) culturing the isolated host cell of embodiment 23 under conditions effective to express the anti-VISTA construct; and b) obtaining the expressed anti-VISTA construct from a host cell.

[0301] Embodiment 26. A method for treating a disease or condition in an individual, comprising administering to the individual an effective amount of an anti-VISTA construct described in any one of embodiments 1 to 19, or a pharmaceutical composition described in embodiment 20.

[0302] Embodiment 27 The method of embodiment 26, wherein the symptomatic disease is associated with a dysregulated immune system.

[0303] Embodiment 28. The method of embodiment 26 or embodiment 27, wherein the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a condition associated with transplantation.

[0304] Embodiment 29. The method of embodiment 28, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorders, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

[0305] Embodiment 30 The method of any one of embodiments 26 to 29, wherein the anti-VISTA construct is administered to the individual intravenously or subcutaneously.

[0306] Embodiment 31 The method of any one of the embodiments, wherein the anti-VISTA construct is administered at a dose of about 0.001 mg / kg to about 100 mg / kg.

[0307] Embodiment 32. The method of any one of embodiments 22 to 31, wherein the individual is a human.

[0308] Embodiment 33. A kit comprising an anti-VISTA construct according to any one of embodiments 1 to 19. EXAMPLES

[0309] Working Example The following examples are intended to be purely illustrative of the present application and therefore should not be construed as limiting the present application in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0310] Example 1. Materials His-tagged human, mouse and cynomolgus VISTA extracellular domain proteins were generated in-house using standard protocols. hVISTA-mFc was purchased from Adipogen (cat. no. CHI-HF-211B7H5-C100). Alexa Fluor 647-labeled anti-human VISTA antibody (BD Biosciences, cat. no. 566670) and APC anti-mouse VISTA antibody (Biolegend, cat. no. 150205) were used to detect human and mouse VISTA-expressing Jurkat cells.

[0311] Jurkat A6 parental cells were transduced with pLenti7.3 / TOPO-mouse VISTA or human VISTA full-length sequence for binding assay by flow cytometry. Jurkat-nfkb-GFP reporter cells were transduced with lentivirus carrying CMV-human VISTA extracellular transmembrane along with CD3ζCAR-EF1-puro for reporter activation assay by flow cytometry. For each construct, VISTA-positive population of Jurkat cells was sorted twice. Cells of each construct are polyclonal.

[0312] Example 2. Immunization Three VISTA knockout BALB / c mice (female) were immunized four times according to the scheme outlined in Table I. Mice were immunized with both hVISTA (human VISTA-mFc) and mVISTA (mouse VISTA-his). The first three immunizations included subcutaneous and intraperitoneal injections. The final boost was an intraperitoneal injection only. Immune responses were analyzed by ELISA: serum samples collected on days 0, 42 and 74 were incubated with human VISTA ECD (2 μg / ml) or negative control antibody adsorbed to 96-well plates. Bound mouse IgG was detected by anti-mouse IgG horseradish peroxidase (Jackson ImmunoResearch, Cat. No. 615035214). The results are shown in Figures 1 and 2. [Table 5]

[0313] As shown in Figures 1 and 2, the use of hVISTA or mVISTA produced antibodies that bound to each antigen.

[0314] Example 3. Hybridoma Supernatant Production Spleens were harvested from mice. Hybridoma cells were prepared according to well-known procedures. Hybridoma cells were cultured in Hybridoma-SFM medium (Gibco) at 37°C for 7-10 days. Cells were pelleted by centrifugation and the supernatants were analyzed as described in the Examples below.

[0315] Example 4. Primary Screening (Binding to Human VISTA, Mouse VISTA, and Cynomolgus VISTA by ELISA) 50 μl of hybridoma supernatant was incubated with human VISTA ECD (2 μg / ml), mouse VISTA ECD (2 μg / mL), cynomolgus VISTA (2 μg / mL), or a negative mouse IgG1 control coated onto 96-well ELISA plates. Mouse anti-VISTA antibodies were detected by anti-mouse IgG-HRP (Jackson ImmunoResearch, catalog number 615035214). The results are shown in FIG. 3.

[0316] As shown in FIG. 3, 9E9, 15D11, 16A1, 17E9, and 20E4 hybridoma supernatants all bind effectively to human, cynomolgus and / or mouse VISTA extracellular domains, demonstrating cross-species reactivity (particularly between human and cynomolgus VISTA proteins).

[0317] Example 5. Secondary screening of hybridoma-derived antibodies that bind to VISTA by FACS assay Human and mouse VISTA-expressing Jurkat cells were cultured in RPMI1640 containing 10% FBS. Cells were plated in 96-well plates at 1x10 5Cells were seeded at 1000 x 1000 cells / well. The cells were then incubated with anti-VISTA hybridoma supernatant for 30 min at 4°C. 1E8 (Immunext, see WO 2017 / 181139) was used as a reference antibody. After washing with FACS buffer, the cells were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (H+L) (1 μg / ml) (Jackson ImmunoResearch, Cat. No. 61505214) for 30 min at 4°C. After washing twice with FACS buffer, the samples were run on a NovoCyte Flow Cytometer (Agilent). Data was analyzed using NovoExpress software.

[0318] Figures 4A-B show that all anti-VISTA hybridomas tested (9F9, 16A1, 17E9, and 20E4) show effective binding to the Jurkat-hVISTA expressing cell line and some binding affinity to the Jurkat-mVISTA expressing cell line.

[0319] Example 6. Reporter activation assay of hybridoma-derived antibodies In a 96-well plate, Jurkat-nfkb-GFP / human VISTA-hCD3z cells were plated at 1x10 in 200μl of RPMI1640 containing 10% FBS medium. 5 Cells were seeded at 1000 x 1000 cells / well. Hybridoma supernatants were added to the medium at increasing anti-VISTA concentrations (based on ELISA, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 10 and 30 μg / ml). GFP was measured by flow cytometry after 24 hours of incubation. The results are shown in Figures 5-7. To test the effect of OKT3 on cell activation, OKT3 (3 ng / ml) was added to the cells for 24 hours of incubation in the presence of increasing anti-VISTA concentrations from hybridoma-derived antibodies. The results are shown in Figures 8A-10.

[0320] Figures 5A-5B and 6A-6B show that upon incubation with various concentrations of 9F9 or 20E4 hybridoma supernatants, the VISTA downstream pathway was activated in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cells, as indicated by positive GFP staining. The degree of activation is concentration dependent. 16A1, 17E9, and 1E8 also show the ability to activate Jurkat-NFKb-GFP / hVISTA-hCD3z cells at certain concentrations. See Figure 7. Thus, 9F9, 20E4, 1E8, 16A1, and 17E9 all show agonistic effects on human VISTA, with 9F9 and 20E4 showing the highest agonistic effects among these antibodies.

[0321] Figures 8A-B, 9A-B and 10 show that OKT3 significantly increased the percentage of activated Jurkat-NFKb-GFP / hVISTA-hCD3z cells. The percentage of activated cells reached more than 60% for all antibodies tested except for 15D11. For cells incubated with 20E4 and 17E9, or 16A1, more than 80% of the cells were activated at the specific concentrations. These results indicate a strong agonistic effect of these antibodies.

[0322] Example 7. Sequence analysis Total RNA was isolated from hybridoma cell line cultures. The RNA was treated to remove aberrant transcripts and reverse transcribed using oligo(dT) primers. Samples of the resulting cDNA were amplified in separate PCRs using framework 1 and constant region primer pairs specific for either the heavy or light chain. Reaction products were separated on an agarose gel, size assessed, and recovered. Amplicons were cloned into pUC19 vector (Clontech). Ten colonies were selected and both plasmid DNA and PCR products were subjected to Sanger sequencing.

[0323] The DNA sequence data from all constructs was analyzed and the consensus sequences for the heavy and light chains were determined, see Tables 3 and 4 below.

[0324] [Table 3-1] [Table 3-2]

[0325] [Table 4-1] [Table 4-2]

[0326] Example 8. Anti-VISTA antibody expression and purification from hybridoma cells Hybridoma cells were cultured in Hybridoma-SFM medium (Gibco) at 37°C for 7-10 days. Cells were pelleted by centrifugation and the supernatant was collected for antibody purification using Protein G Sepharose (GE). The eluate from the column was buffer exchanged into 1XPBS (pH 7.4) by diafiltration with centrifugation. Purified antibodies were analyzed by SDS-PAGE gel electrophoresis to confirm purity and size. Antibody concentrations were measured using a spectrophotometer A 280 Purified antibodies were 0.2 μm filtered before storage.

[0327] Example 9. Recombinant expression and purification of anti-VISTA antibodies in Expi293 cells Anti-VISTA variable domains were cloned with mIgG1 constant domains and expressed in Expi293 cells. Briefly, mouse IgG1 variable domains were gene synthesized using human preferred codons from IDT. The gene fragment was then subcloned into a pcDNA3.4 vector containing a mouse antibody signal sequence and mIgG1 Fc fragment. Antibodies were produced by transient transfection into Expi293 cells using the ExpiFectamine293 transfection kit (Thermo Fisher Scientific). Five days after transfection, the supernatant from the transfected cells was collected and purified using Protein G Sepharose (GE). Bound antibodies were eluted using 0.1M glycine buffer (pH 2.7) and dialyzed overnight against 1XPBS (pH 7.4). Purified antibodies were analyzed by reducing and non-reducing SDS-PAGE to confirm purity and size. Recombinant protein concentrations were measured using a spectrophotometer A 280 It was decided by.

[0328] Example 10. Binding of recombinant anti-VISTA antibodies to the cell surface of human and mouse VISTA expressing Jurkat cells as determined by fluorescence-activated cell sorting (FACS) assay Human and mouse expression Jurkat cells were cultured in RPMI1640 containing 10% FBS. Cells were plated in 96-well plates at 1x10 5 Cells were seeded at 1000 x 1000 cells / well. The cells were then incubated with recombinant anti-VISTA antibodies 9F9, 16A1, 17E9, 20E4 or V4 (Hummingbird) (10 μg / ml) for 30 min at 4°C. After washing with FACS buffer, the cells were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (H+L) (1 μg / ml) (Jackson ImmunoResearch, Cat. No. 615605214) for 30 min at 4°C. The cells were washed twice with FACS buffer and the samples were run on a NovoCyte Flow Cytometer (Agilent). The data was analyzed using NovoExpress software. The results are shown in Figures 11A-B.

[0329] Figures 11A-B demonstrate that the recombinant mIgG1 anti-VISTA antibodies (9F9, 16A1, 17E9, and 20E4) all show effective binding to Jurkat-hVISTA expressing cell lines. Additionally, 9F9 also shows effective binding to Jurkat-mVISTA, demonstrating cross-species reactivity.

[0330] Example 11. Reporter activation assay of recombinant anti-VISTA antibodies In a 96-well plate, Jurkat-nfkb-GFP / human VISTA-hCD3z cells were plated at 1x10 in 200 μl of RPMI 1640 containing 10% FBS medium. 5 Cells were seeded at 1000 x 1000 cells / well. Recombinant anti-VISTA antibodies were added to the medium at increasing VISTA concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 10, 30 μg / ml) or (0.05, 0.5, 5 and 50 μg / ml). GFP was measured by flow cytometry after 24 hours of incubation. To test the effect of OKT3 on cell activation, OKT3 (3 ng / ml) was added to the cells in the presence of increasing anti-VISTA concentrations and GFP was measured after 24 hours of incubation.

[0331] Figures 12-15 show that recombinant mIgG1 anti-VISTA antibodies (9F9, 16A1, 17E9, and 20E4) induce effective activation in Jurkat-NFKb-GFP / hVISTA-hCD3z expressing cell lines, with 15%-50% of cells being activated at a concentration of 5 μg / mL.

[0332] Example 12. Epitope binning assay of anti-VISTA antibodies by Octet competition The bins of anti-VISTA antibody epitopes were determined using an Octet QKe (ForteBio). Human VISTA recombinant protein (Sino Biological Inc., Cat. No. 13485-H08H) was biotinylated using EZ-LINK NHS-PEG4 Biotin (Thermo Fisher Scientific). Biotinylated VISTA protein was captured (300 s at 5 μg / ml) using a streptavidin biosensor chip (ForteBio). After stabilizing a baseline measurement for 60 s in 1x kinetic buffer (Fortebio), a primary anti-VISTA antibody (10 μg / ml) was allowed to associate with the captured protein for 300 s. A series of secondary anti-VISTA antibodies (10 μg / ml) were then allowed to associate with the antigen and primary antibody complex for an additional 300 s. Signals were recorded for each binding event and data analysis was performed with ForteBio Data Analysis HT 11.1 software.

[0333] Epitope binning assays of lead anti-VISTA antibodies showed three group binding epitopes in this Octet analysis. Figure 16 shows that 16A1, 17E9 and 20E4 compete for the same epitope on VISTA, while 1E8, 9F9 and 15D11 bind to different epitopes. The binding epitopes from all these antibodies bind are different from the binding epitopes of the benchmark control antibodies Onvatilimab and IE8 (Immunext).

[0334] Example 13. Demonstration of human, cynomolgus monkey and mouse VISTA antigen cross-binding activity of anti-VISTA mAbs using Biolayer Interferometry (BLI) assay Human, cynomolgus and mouse VISTA antigen cross-binding activity of anti-VISTA antibodies was measured by biolayer interferometry using an Octet QKe (ForteBio). Human VISTA recombinant protein (Sino Biological Inc., Cat. No. 13482-H08H), mouse VISTA (Sino Biological Inc., Cat. No. 51550-M08H) or cynomolgus VISTA protein (in-house) was biotinylated using EZ-LINK NHS-PEG4 Biotin (Thermo Fisher Scientific). Biotinylated VISTA protein was loaded (5 μg / ml for 300 s) using a streptavidin biosensor (ForteBio). After stabilizing baseline measurements for 60 s in 1X kinetic buffer (ForteBio), 5 μg / ml of anti-VISTA antibody was allowed to associate with the captured protein for 300 s. The sensor was then dissociated for 600 s in 1X kinetic buffer. Data analysis was performed with ForteBio Data Analysis HT 11.1 software.

[0335] Figures 17A-C show that all antibodies tested show effective binding to human, cynomolgus monkey and mouse VISTA. In comparison, the benchmark control antibody ombatilimab does not cross-react with mouse VISTA.

[0336] Example 14. Binding affinity of anti-VISTA antibodies to human and cynomolgus VISTA determined by biolayer interferometry (BLI) assay The binding affinity of anti-VISTA antibodies to human and cynomolgus monkey was determined by biolayer interferometry using an Octet QKe (ForteBio). Human VISTA recombinant protein (Sino Biological Inc., Cat. No. 13482-H08H) or cynomolgus monkey VISTA protein (produced in-house) was biotinylated using EZ-LINK NHS-PEG4 Biotin (Thermo Fisher Scientific). Biotinylated VISTA protein was loaded (5 μg / ml for 300 s) using a streptavidin biosensor (ForteBio). After stabilizing baseline measurements for 60 s in 1X kinetic buffer (ForteBio), anti-VISTA antibodies at serial dilutions (50, 25, 12.5, 6.25 and 3.125 μg / ml) were allowed to associate with the captured protein for 300 s. The sensor was then dissociated for 600 s in 1X kinetic buffer. Data analysis was performed with ForteBio Data Analysis HT 11.1 software.

[0337] Figures 18A-E show the binding affinity of all anti-VISTA antibodies to human and cynomolgus VISTA.

[0338] Example 15. Analysis of the ability of anti-VISTA antibodies to inhibit T cell proliferation The ability of anti-VISTA antibodies to inhibit T cell proliferation was analyzed in an in vitro assay. Anti-CD3 (OKT3) and VISTA-Ig at a concentration ratio of 1:1 (2.5 μg / ml anti-CD3:2.5 μg / ml) were coated onto 96-well plates overnight at 37°C. Human PBMCs were purified from freshly collected buffy coats and labeled with CFSE (Invitrogen, Cat. No. C34554). The 96-well plate wells were then washed three times with 1XPBS buffer and 200,000 CFSE-labeled PBMC cells were added to each well in the presence of anti-VISTA antibodies (50 μg / ml) or mouse IgG control (50 μg / ml) using CTS OpTmizer T cell proliferation SFM (Invitrogen, Cat. No. A1048501). After 5 days of treatment, cells were harvested, labeled with APC-conjugated anti-human CD3 antibody (Biolegend Cat. No. 300311) and run on a NovoCyte Flow Cytometer (Agilent). Data was analyzed using NovoExpress software. Results are shown in FIG. 19.

[0339] Benchmark control antibody 1E8 (Immunext) was used as a positive control, and mouse IgG isotype was used as a negative control. As shown, approximately 50%-70% of cells incubated with OKT3 only, OKT3+VISTA-Ig, or OKT3+VISTA-Ig+mIgG proliferated as shown by CFSE staining. In contrast, the presence of anti-VISTA antibodies (1E8, 9F9, 15D11, 16A1, 17E9, and 20E4) significantly suppressed T cell proliferation. Of these, 9F9, 15D11, 16A1, and 20E4 show better inhibitory effects than 1E8.

[0340] Example 16. Animal studies Four-week-old female MRL-lpr mice were used for the lupus treatment model. Different groups of mice received weekly treatments of 200 μg of mIgG1 (control) or anti-VISTA constructs (MH5A, 9F9, or 20E4) for weeks 5–12. Most measurements were taken after week 12, with some measurements of serum antibody levels taken before week 12. A graphic summary of the protocol used for the lupus treatment model is shown in Figure 20. The following experiments A–F were designed using the main clinical indicators of lupus in investigating the efficacy of the anti-VISTA constructs tested herein.

[0341] A. Anti-VISTA Constructs in the Treatment of Lymph Node Enlargement MRL-lpr mice express a spontaneously mutated Fas lpr This autoimmune mouse model develops skin lesions and is a common denominator used as a model for lupus erythematosus. Beginning at 10 weeks of age, enlarged lymph nodes can be felt by gentle pressure around the neck area. Enlarged lymph nodes may be an early sign reflecting a lupus condition, and skin lesions form as the animal ages.

[0342] Four groups of mice treated with mIgG, MH5A, 9F9 or 20E4 were examined for the size of their lymph nodes at 12, 14 and 15 weeks of age, respectively. The results are shown in FIG. 21A, with the data bars for each week from left to right being the mIgG, MH5A, 9F9 and 20E4 treatment groups. The size of lymph nodes was classified into five values: 0 (not palpable), 1 (mung bean size), 2 (pea size), 3 (peanut size), and 4 (walnut size). FIG. 21A shows that MH5A and 9F9 significantly reduced lymph node size in mice, and 20E4 slightly reduced lymph node size, compared to the control group (mIgG1 treatment). FIG. 21B shows exemplary sizes of lymph nodes removed from the neck region of mice used in the experiment.

[0343] B. Anti-VISTA Constructs in Reducing Serum Levels of Antinuclear Immunoglobulin MRL-lpr mice develop systemic autoimmune symptoms, including the spontaneous development of autoantibodies against cell nuclei (antinuclear antibodies, ANA). The ANA test is used in humans to diagnose lupus; a positive ANA test indicates that the immune system has launched a misdirected attack against a person's own tissues.

[0344] Serum ANA levels in mice treated with mIgG1, MH5A, 9F9, or 20E4 were measured at weeks 5, 6, 9, 12, and 15, respectively. The results are shown in Figure 22. The data bars for each week from left to right in Figure 22 are the mIgG, MH5A, 9F9, and 20E4 treatment groups. MH5A and 9F9 significantly reduced serum ANA levels compared to mIgG1, and 20E4 mildly reduced serum ANA levels in mice by week 15.

[0345] C. Anti-VISTA Constructs in Reducing Serum Levels of Anti-dsDNA Immunoglobulins In the clinic, high levels of anti-dsDNA antibodies in the blood are strongly associated with lupus and often increase significantly during or just before a flare-up. A positive anti-dsDNA antibody test, associated with other clinical signs and symptoms related to lupus, is used to diagnose lupus.

[0346] Serum levels of anti-dsDNA antibodies in mice treated with mIgG1, MH5A, 9F9, or 20E4 were measured at weeks 9, 12, and 15, respectively. The results are shown in Figure 23. All anti-VISTA constructs tested (MH5A, 9F9, and 20E4) were able to reduce anti-dsDNA antibodies in mouse serum at week 12, and levels remained low 3 weeks after treatment at week 15.

[0347] D. Anti-VISTA Constructs in Reducing Serum Levels of IFNα An example of a key cytokine involved in the etiology and pathogenesis of lupus is interferon alpha. IFNα is a key protein in immune regulation. IFNα is a pleiotropic cytokine that can affect multiple cell types involved in lupus. Elevated serum levels of IFNα and altered expression of several genes in the interferon pathway are associated with risk of lupus, suggesting a role for this pathway in pathogenesis.

[0348] Serum levels of IFNα in mice treated with mIgG1, MH5A, 9F9, or 20E4 were measured at weeks 12 and 15. Figure 24 clearly shows that all three anti-VISTA constructs significantly reduced serum levels of IFNα at week 12, which remained significantly lower at week 15, compared to mIgG1 (control).

[0349] E. Anti-VISTA Constructs in Reducing Urinary Protein Levels The MRL-lpr mice used herein spontaneously develop lupus nephritis. Urinary protein levels can reflect kidney disease pathogenesis. In addition to blood tests, urine tests, including urine protein levels, are used in clinics to diagnose and monitor the effects of lupus on the kidney.

[0350] Urinary protein levels were measured at weeks 12 and 15 for mice treated with mIgG1, MH5A, 9F9 or 20E4. Each group had 6 mice. The pie charts in Figures 25 and 26 demonstrate the distribution of mice in different urinary protein level categories in each treatment group. The percentage values ​​below the charts indicate the percentage of mice in each treatment group with protein levels of ≥ 100 mg / dL or ≥ 300 mg / dL. All treatment groups with anti-VISTA constructs had fewer mice in the 2+ group (100-300 mg / dL protein) and 3+ group (300-1000 mg / dL protein) compared to the mIgG group at week 12. MH5A and 9F9 showed a long-term effect on maintaining reduced urinary protein levels at week 15.

[0351] F. Anti-VISTA Constructs in Reducing Cutaneous Lupus Lesions The protective effect of anti-VISTA constructs against cutaneous lupus lesions in mice was examined at week 17. As shown in Figure 27, mice treated with MH5A and 9F9 had reduced skin lesions compared to mice treated with mIgG1.

[0352] G. Conclusion MH5A, 9F9 and 20E4 were tested in MRL-lpr mice for their therapeutic effects against lupus, including systemic lupus erythematosus (SLE). Lymph node enlargement, autoantibody and cytokine levels in serum, urinary protein levels, and skin appearance were used to evaluate the severity of disease in each group after treatment. 9F9 and MH5A showed promising preclinical efficacy in the MRL-lpr mouse model, significantly reducing lymph node enlargement, serum antinuclear and anti-dsDNA autoantibody levels, serum IFNα levels, and urinary protein levels in MRL-lpr mice. 20E4 also showed a protective effect in reducing serum anti-dsDNA and IFNα levels compared to mIgG1 (control).

[0353] Example 17. Efficacy study of anti-VISTA antibodies in a graft-versus-host (GvHD) mouse model The aim of this study was to evaluate the efficacy of anti-VISTA antibodies 9F9 and 20E4 in a mouse model of graft-versus-host disease (GvHD).

[0354] The efficacy of the study was evaluated by measuring body weight and by visual evaluation of the skin denudation of the mice. The overall activity of the mice was evaluated throughout the study. The level of human CD45+ cell engraftment, which indicates the level of GvHD development in the blood, was also measured. See, for example, Ali et al., PLoS One. 2012; 7(8): e44219.

[0355] NOD-scid IL2rg null (NSG) mice were used in this study. All mice were injected with 10 million human PBMCs by intravenous (IV) injection on day 0 and divided into three groups. Group 1 received mouse IgG isotype control (0.5 mg / mouse) treatment on day 0. Groups 2 and 3 were treated with 9F9 (0.5 mg / mouse) or 20E4 (0.5 mg / mouse) by intraperitoneal (ip) injection, respectively. Body weights were collected weekly. A second dose of test article was given in 2 weeks. Blood samples were taken at weeks 2 and 5 for flow cytometry analysis. Mice were sacrificed on day 37.

[0356] Measurements collected as raw data were analyzed using GraphPad software Prism. P<0.05 was considered statistically significant.

[0357] Graft-versus-host disease leads to weight loss as animals become lethargic, and activity decreases as the disease progresses. Figure 28 shows that treatment of mice with anti-VISTA antibodies 9F9 and 20E4 prevents weight loss during the study period. Weight changes were statistically different in the 9F9 and 20E4 treated groups compared to the isotype control group. A decrease in activity was observed in three out of four mice treated with isotype control. No change in activity was observed for either the 9F9 or 20E4 antibody treated groups.

[0358] Mice suffering from GvHD appear with skin denudation over time as the disease progresses. On day 33, mice were photographed to capture the level of skin denudation observed in the different groups. Figure 30 shows skin denudation in mice injected with isotype control and mice treated with anti-VISTA antibodies 9F9 or 20E4.

[0359] Serum was collected on the 14th and 37th days after PBMC transfer. Cells were collected and stained for human and mouse CD45 for flow cytometry analysis. Figure 29 shows the percentage of human CD45+ cells in different groups. Each symbol represents an individual mouse in a group. Compared to control, there was a statistically significant reduction in human CD45+ cells in mice treated with either 9F9 or 20E4 antibody. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

Claims

1. Heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) , wherein the antibody portion comprises a second heavy chain variable region (V Ｈ－２ ) and a second light chain variable region (V Ｌ－２ ) competes for the binding epitope of VISTA with an antibody or antibody fragment comprising a) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; b) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; c) Said V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:22; d) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27; Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30; or e) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35; Ｌ－２ an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:36, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

38.

2. 2. The anti-VISTA construct of claim 1, a) the V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs; b) the V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDR; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs; c) Said V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs; d) the V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:30, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs; e) the V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the HC-CDRs; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising 5, 4, 3, 2, or 1 amino acid substitution in the LC-CDRs; f) Said V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 51, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; g) the V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44 or 52, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45; Ｌ comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 or 57; or h) the V Ｈ comprising i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 43, ii) an HC-CDR2 comprising the amino acid sequence of any of SEQ ID NO: 58, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 45; Ｌ an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:48, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:49, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:50 or 53.

3. The V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; Ｌ The anti-VISTA construct of claim 2, comprising i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

4. The V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11; Ｌ The anti-VISTA construct of claim 2, comprising i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

14.

5. The V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; Ｌ The anti-VISTA construct of claim 2, comprising i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:21, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

22.

6. The V Ｈ comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:27; Ｌ The anti-VISTA construct of claim 2, comprising i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

30.

7. 1. An anti-VISTA construct comprising an antibody portion that specifically binds to VISTA, a) V having the sequence shown in SEQ ID NO:7 Ｈ HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:

8. Ｌ LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the chain regions, respectively; b) V having the sequence shown in SEQ ID NO: 15 Ｈ HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:

16. Ｌ LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the chain regions, respectively; c) V having the sequence shown in SEQ ID NO:23 Ｈ HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:

24. Ｌ LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 within the chain regions, respectively; d) V having the sequence shown in SEQ ID NO:31 Ｈ HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:

32. Ｌ LC-CDR1, LC-CDR2 and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain regions; or e) V having the sequence shown in SEQ ID NO:39 Ｈ HC-CDR1, HC-CDR2 and HC-CDR3, which contain the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within the chain region, and V having the sequence shown in SEQ ID NO:

40. Ｌ An anti-VISTA construct comprising LC-CDR1, LC-CDR2 and LC-CDR3, which comprise within their chain regions the amino acid sequences of CDR1, CDR2 and CDR3, respectively.

8. The V Ｈ comprises an amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31 and 39, or a variant thereof having at least about 80% sequence identity; and / or Ｌ The anti-VISTA construct of any one of claims 1 to 7, wherein said construct comprises an amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, 32 and 40, or a variant comprising an amino acid sequence having at least about 80% sequence identity.

9. 9. The anti-VISTA construct of claim 8, a) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; Ｌ comprises the amino acid sequence of SEQ ID NO:8, or a variant comprising an amino acid sequence having at least about 80% sequence identity; b) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 15, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; Ｌ comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; c) Said V Ｈ comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; Ｌ comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% sequence identity; d) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 31, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; Ｌ comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% sequence identity; or e) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 39, or a variant thereof having an amino acid sequence having at least about 80% sequence identity; Ｌ An anti-VISTA construct comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having an amino acid sequence having at least about 80% sequence identity.

10. The antibody portion may be a full-length antibody, a bispecific antibody, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) ２ 10. The anti-VISTA construct of any one of claims 1 to 9, which is an antibody or antigen-binding fragment thereof selected from the group consisting of: an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.

11. The anti-VISTA construct of claim 10 , wherein the antibody portion is a full-length antibody.

12. The anti-VISTA construct of any one of claims 1 to 11, wherein the antibody portion has an Fc fragment selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof.

13. The anti-VISTA construct of claim 12, wherein the Fc fragment is selected from the group consisting of Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

14. The anti-VISTA construct of claim 12 or claim 13, wherein the Fc fragment has a reduced effector function compared to the corresponding wild-type Fc fragment.

15. The anti-VISTA construct of any one of claims 12 to 14, wherein the Fc fragment has a longer half-life compared to the corresponding wild-type Fc fragment.

16. The anti-VISTA construct of any one of claims 1 to 15, wherein the antibody portion of the anti-VISTA construct activates a downstream signaling pathway of VISTA.

17. The anti-VISTA construct of any one of claims 1 to 16, wherein the anti-VISTA construct is an agonistic antibody of VISTA.

18. The anti-VISTA construct of claim 16, wherein the antibody portion of the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 20%.

19. The anti-VISTA construct of any one of claims 1 to 18, wherein said VISTA is human VISTA.

20. A pharmaceutical composition comprising an anti-VISTA construct according to any one of claims 1 to 19 and a pharma- ceutically acceptable carrier.

21. An isolated nucleic acid encoding an anti-VISTA construct according to any one of claims 1 to 19.

22. A vector comprising the isolated nucleic acid of claim 21.

23. 23. An isolated host cell comprising the isolated nucleic acid of claim 21 or the vector of claim 22.

24. 20. An immunoconjugate comprising the anti-VISTA construct of any one of claims 1 to 19 linked to a therapeutic agent or a label.

25. 1. A method of producing an anti-VISTA construct, comprising: a) culturing the isolated host cell of claim 23 under conditions effective to express the anti-VISTA construct; and b) obtaining the expressed anti-VISTA construct from said host cell.

26. A composition comprising an anti-VISTA construct according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20, for treating a disease or condition in an individual.

27. 27. The composition of claim 26, wherein the disease or condition is associated with a dysregulated immune system.

28. 28. The composition of claim 26 or claim 27, wherein the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related condition.

29. 29. The composition of claim 28, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune bowel disorder, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE).

30. A composition described in any one of claims 26 to 29, characterized in that it is administered to the individual intravenously or subcutaneously.

31. The composition according to any one of claims 26 to 30, wherein the anti-VISTA construct is administered at a dose of about 0.001 mg / kg to about 100 mg / kg.

32. The composition of any one of claims 26 to 31, wherein the individual is a human.

33. A kit comprising an anti-VISTA construct according to any one of claims 1 to 19.

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