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 the anti-VISTA, vectors comprising the nucleic acid molecules, host cells containing the vectors, methods of making the anti-VISTA constructs, pharmaceutical compositions containing the anti-VISTA constructs, and methods of using the anti-VISTA constructs or compositions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 404,470, filed on September 7, 2022, the content of which is incorporated herein by reference in its entirety for all purposes.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (193852000740SEQLIST.xml; size: 60,285 bytes; creation date: September 6, 2023) is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to anti - VISTA constructs (such as anti - VISTA antibodies) and their uses. Background Art

[0006] 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 act as an inhibitory ligand on antigen - presenting cells (APCs) and regulate T - cell responses through an unknown receptor. In addition, VISTA can also act as an inhibitory receptor on T cells. For example, agonistic VISTA monoclonal antibodies (mAbs) significantly 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 autoimmunity induction (such as experimental autoimmune encephalomyelitis and systemic lupus) when backcrossed with lupus - prone strains. Studies have shown that VISTA is involved in peripheral immune tolerance and negatively regulates T - cell activation. See, e.g., Sci Transl Med. December 11, 2019; 11(522).

[0007] 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

[0008] This application provides, in one aspect, an anti - VISTA construct, the anti - VISTA construct comprising an anti - VISTA antibody portion, the anti - VISTA antibody portion comprising a heavy - chain variable region (V H ) and a light - chain variable region (V L ), wherein: a) V Hcomprising an HC-CDR1 containing the amino acid sequence of SEQ ID NO:45, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:46, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:47, and V L comprising an LC-CDR1 containing the amino acid sequence of SEQ ID NO:48, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:49, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:50, wherein V H comprising the amino acid sequence of any one of SEQ ID NO:53 - 57, and wherein V L comprising the amino acid sequence of any one of SEQ ID NO:58 - 62; or b) V H comprising an HC-CDR1 containing the amino acid sequence of any one of SEQ ID NO:1 - 3, an HC-CDR2 containing the amino acid sequence of any one of SEQ ID NO:4 - 6, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprising an LC-CDR1 containing the amino acid sequence of any one of SEQ ID NO:8 - 10, an LC-CDR2 containing the amino acid sequence of any one of SEQ ID NO:11 - 13, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, wherein V H comprising the amino acid sequence of any one of SEQ ID NO:16 - 30, and wherein V L comprising the amino acid sequence of any one of SEQ ID NO:32 - 44.

[0009] In some embodiments, V H comprising the amino acid sequence of any one of SEQ ID NO:53 - 57, and wherein V L comprising the amino acid sequence of any one of SEQ ID NO:58 - 62. In some embodiments, V H comprising the amino acid sequence of SEQ ID NO:53, and V L comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, V H comprising the amino acid sequence of SEQID NO:55, and V L comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, V H comprising the amino acid sequence of SEQ ID NO:56, and V L comprising the amino acid sequence of SEQ ID NO:61.

[0010] In some embodiments, V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO:8, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0011] In some embodiments, V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO:9, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0012] In some embodiments, V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO:10, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0013] In some embodiments, V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO:2, an HC-CDR2 containing the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO:8, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0014] In some embodiments, VH comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:2, HC-CDR2 containing the amino acid sequence of SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:9, LC-CDR2 containing the amino acid sequence of SEQ ID NO:12, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0015] In some embodiments, V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:2, HC-CDR2 containing the amino acid sequence of SEQ ID NO:5, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:10, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0016] In some embodiments, V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:3, HC-CDR2 containing the amino acid sequence of SEQ ID NO:6, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:8, LC-CDR2 containing the amino acid sequence of SEQ ID NO:11, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0017] In some embodiments, V H comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO:3, HC-CDR2 containing the amino acid sequence of SEQ ID NO:6, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:7, and V L comprises LC-CDR1 containing the amino acid sequence of SEQ ID NO:9, LC-CDR2 containing the amino acid sequence of SEQ ID NO:12, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0018] In some embodiments, V Hcomprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:10, 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.

[0019] In some embodiments of any of the anti-VISTA constructs described above, V H comprises an amino acid sequence of any one of SEQ ID NOs: 16-30, and wherein V L comprises an amino acid sequence of any one of SEQ ID NOs: 32-44. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:30, and V L comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:16, and V L comprises the amino acid sequence of SEQ IDNO:32. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:16, and V L comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:16, and V L comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:16, and V L comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:17, and V L comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:17, and V L comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, V H comprises the amino acid sequence of SEQID NO:17, and V L comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:17, and V L comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:18, and V L comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:18, and V L comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:18, and V L comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:18, and V L comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:19, and V L comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:19, and V L comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:19, and V L comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:19, and V L comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:20, and V L comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:21, and V L comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:22, and V L comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, V Hcomprises the amino acid sequence of SEQ ID NO: 23, and V L comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 24, and V L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 25, and V L comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 25, and V L comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 25, and V L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 26, and V L comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 26, and V L comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 26, and V L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 29, and V L comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 29, and V L comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, V H comprises the amino acid sequence of SEQ ID NO: 30, and V L comprises the amino acid sequence of SEQ ID NO: 43.

[0020] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion is an antibody or an antigen-binding fragment thereof selected from the group consisting of: full-length antibody, bispecific antibody, single-chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, and tetrabody. In some embodiments, the antibody portion is a full-length antibody.

[0021] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion has an Fc fragment selected from the group consisting of the following Fc fragment forms: 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.

[0022] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion of the anti-VISTA construct activates the downstream signaling pathway of VISTA.

[0023] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct is an agonistic antibody of VISTA.

[0024] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion of the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 20%.

[0025] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct is an antagonistic antibody of VISTA.

[0026] In some embodiments of any of the anti-VISTA constructs described above, VISTA is human VISTA.

[0027] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct inhibits PBMC and / or T cell activation by at least 20%.

[0028] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct reduces the production of IL-17A by at least 30%.

[0029] In another aspect, the present application provides a pharmaceutical composition comprising any of the anti-VISTA constructs described above and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present application provides an isolated nucleic acid encoding any of the anti-VISTA constructs described above.

[0031] In another aspect, the present application provides a vector comprising any of the isolated nucleic acids described above.

[0032] In another aspect, the present application provides an isolated host cell comprising any of the isolated nucleic acids and / or vectors described above.

[0033] In another aspect, the present application provides an immunoconjugate comprising any of the anti-VISTA constructs described above linked to a therapeutic agent or a label.

[0034] In another aspect, the present application provides a method for producing an anti-VISTA construct, the method comprising: a) culturing any of the isolated host cells described above under conditions effective for expressing the anti-VISTA construct; and b) obtaining the expressed anti-VISTA construct from the host cell.

[0035] In another aspect, the present application provides a method for treating a disease or disorder in an individual, the method comprising administering to the individual an effective amount of any one of the anti-VISTA constructs described above, or any one of the pharmaceutical compositions described above. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is associated with activated T cells. In some embodiments, the activated T cells are CD3+CD25+ T cells and / or CD45+ T cells. In some embodiments, the disease or disorder is associated with VISTA-positive cells. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a disorder associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, 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. In some embodiments, the anti-VISTA construct is administered together with a second agent. In some embodiments, the second agent is a VISTA agonist. In some embodiments, the second agent is a VISTA ligand. In some embodiments, the second agent is a polypeptide. In some embodiments, the VISTA ligand comprises VSIG3 or a fragment thereof. In some embodiments, VSIG3 or a fragment thereof is fused to an Fc domain.

[0036] In another aspect, the present application provides a kit comprising any one of the anti-VISTA constructs described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 SDS-PAGE gels showing exemplary anti-VISTA antibody VH / VL combinations. The gels were run under non-reducing conditions.

[0038] Figures 2A - 2E Binding activities of 9E9 and 20E4 parental and humanized anti-VISTA antibodies against Jurkat cells expressing human VISTA, detected using fluorescence-activated cell sorting (FACS).

[0039] Figures 3A - 3F Binding activities of 9E9 and 20E4 parental and humanized anti-VISTA antibodies against Jurkat cells expressing murine VISTA, detected using fluorescence-activated cell sorting (FACS).

[0040] Figures 4A - 4BShows the binding of humanized 9F9 anti-VISTA antibody to human VISTA detected by Octet.

[0041] Figure 4C Shows the binding of humanized 20E4 anti-VISTA antibody to human VISTA detected by Octet.

[0042] Figures 5A - 5B Shows the binding of humanized 9F9 anti-VISTA antibody to mouse VISTA detected by Octet.

[0043] Figure 5C Shows the binding of humanized 20E4 anti-VISTA antibody to mouse VISTA detected by Octet.

[0044] Figures 6A - 6C Depicts the activation of the VISTA signaling pathway by different concentrations of humanized 9F9 anti-VISTA antibody in cells expressing Jurkat-NFKb-GFP / hVISTA-hCD3z (human VISTA fused with hCD3z, hCD3z expressed on the cell surface) in the presence of anti-CD3 antibody (OKT3).

[0045] Figure 6D Depicts the activation of the downstream VISTA pathway by different concentrations of humanized 20E4 anti-VISTA antibody in cells expressing Jurkat-NFKb-GFP / hVISTA-hCD3z in the presence of anti-CD3 antibody (OKT3). Human VISTA is fused with hCD3z; activation of the VISTA pathway stimulates cell FITC signal.

[0046] Figure 7 Describes a summary of the experimental protocol in a murine graft-versus-host treatment model.

[0047] Figure 8 Shows the change in body weight over time of graft-versus-host mice treated with isotype control compared to mice treated with 9F9 or 20E4 anti-VISTA antibody.

[0048] Figure 9 Shows the degree of skin exfoliation of graft-versus-host mice injected with isotype control compared to mice treated with 9F9 or 20E4 anti-VISTA antibody.

[0049] Figure 10 Shows the level of human CD45+ cells in the blood of graft-versus-host mice treated with isotype control compared to mice treated with 9F9 or 20E4 anti-VISTA antibody. Samples were collected on day 14 and day 37.

[0050] Figure 11 Shows the forward and side scatter of CD45+ cells stained in the blood of mice injected with isotype control and mice treated with humanized 9E9 or 20E4 anti-VISTA antibodies. Samples were collected on day 14 and day 37.

[0051] Figure 12 Describes a summary of the experimental protocol in a murine graft-versus-host treatment model.

[0052] Figure 13 Shows the degree of skin exfoliation in graft-versus-host mouse model mice injected with isotype control and 20E4 VH3 / VL3 anti-VISTA antibody.

[0053] Figure 14A Shows the percentage of human CD45+ cells in different treatment groups on day 13.

[0054] Figure 14B Shows the percentage of human CD45+ cells in different treatment groups on day 22.

[0055] Figure 15A Shows the inhibition of 9F9 anti-VISTA antibody on PBMC and T cell activation.

[0056] Figure 15B Shows the inhibition of 20E4 anti-VISTA antibody on PBMC and T cell activation. * indicates p<0.05; ** indicates p<0.01.

[0057] Figure 16 Shows the efficacy of 9F9 and 20E4 hIgG1 and hIgG2 antibodies in an imiquimod (IMQ)-induced psoriatic-like BALB / c-hVISTA mouse model. * indicates p<0.05. Detailed Description

[0058] This application provides novel anti-VISTA constructs that specifically bind to VISTA, methods for preparing the anti-VISTA constructs, and methods of using the constructs (e.g., methods of treating diseases or disorders). Exemplary anti-VISTA constructs include agonistic antibodies that are capable of binding and activating VISTA.

[0059] I. Definitions

[0060] The term "antibody" is used in its broadest sense and encompasses various 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, provided that they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0061] Full-length antibodies contain 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 may be referred to as "V H " and "V L ", respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are interspersed between flanking segments called 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 do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Chimeric Fc regions (such as IgG2 / 4 mixtures) are also contemplated herein.

[0062] As used herein, the term "antigen-binding fragment" 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 Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of antibodies 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 the complete antibody structure. Antigen-binding fragments are capable of binding to the same antigen to which the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, the antigen-binding fragment may contain one or more CDRs from a particular human antibody that are grafted into the framework region from one or more different human antibodies.

[0063] "Fv" is the smallest antibody fragment containing the complete antigen recognition and binding site. This fragment consists of a dimer of a heavy-chain variable domain and a light-chain variable domain in a tight, non-covalent association. From the folding of these two domains arise six hypervariable loops (three loops from each of the heavy and light chains), which contribute the amino acid residues for antigen binding and confer antibody antigen-binding specificity. However, although the affinity is usually lower than that of the whole binding site, even a single variable domain (or half of the Fv containing only three CDRs specific for the antigen) is capable of recognizing and binding the antigen.

[0064] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment containing the V H and V L antibody domains joined into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V H domain and the V L domain, which enables the scFv to assume the structure required for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994).

[0065] As used herein, the term "CDR" or "complementary determining region" is intended to refer to non-contiguous antigen-combining sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described in: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. 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 M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, application of any definition to refer to the CDRs of an antibody or its grafted antibody or variant 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 references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, 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 content of the references cited in this paragraph is incorporated herein by reference in its entirety for use in this application and may be included 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, Münch R, Müller W: VBASE2, an integrative V gene database. Nucleic Acids Res. January 1, 2005; 33 (Database issue): D671-4, which is incorporated herein by reference in its entirety).

[0066] Table 1: CDR Definitions

[0067]

[0068] 1 Residue numbering follows the nomenclature of Kabat et al., ibid.

[0069] 2 Residue numbering follows the nomenclature of Chothia et al., ibid.

[0070] 3 Residue numbering follows the nomenclature of MacCallum et al., ibid.

[0071] 4 Residue numbering follows the nomenclature of Lefranc et al., ibid.

[0072] 5 Residue numbering follows the nomenclature of Honegger and Plückthun, ibid.

[0073] The expression "residue numbering in the variable domain as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system for the heavy chain variable domain or the light chain variable domain used in antibody editing in Kabat et al. (ibid.). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion into the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence.

[0074] Unless otherwise specified herein, the numbering of residues in immunoglobulin heavy chains is as per the EU index in Kabat et al. (supra). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0075] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as defined herein.

[0076] A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains the minimal sequence derived from the non-human antibody. To a large extent, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the hypervariable regions (HVRs) of the recipient are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, the framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Additionally, a humanized antibody may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are the FRs of the human immunoglobulin sequence. A humanized antibody will also optionally contain at least a portion of the immunoglobulin constant region (Fc) (usually the constant region of a human immunoglobulin). For more 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).

[0077] "Human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody prepared using any of the techniques for preparing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. A variety of techniques known in the art, including phage display libraries, can be used to generate human antibodies. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also useful for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 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 an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation, but whose endogenous loci have been disabled, e.g., an immunized xenomouse (for the XENOMOUSE TM technology, see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584). For human antibodies generated by human B cell hybridoma technology, also see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0078] The "percent amino acid sequence identity (%)" or "homology" for the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues identical with the amino acid residues in the polypeptide being compared after aligning the sequences and considering any conservative substitutions as part of the sequence identity. The alignment for purposes of determining the percent amino acid sequence identity can be achieved in various ways within the capabilities of those of ordinary skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, the amino acid sequence identity values % are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0079] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When the positions in both of the two sequences being compared are occupied by the same base or amino acid monomer subunit, e.g., if the position in each 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 divided by the number of positions being compared times 100. For example, if 6 of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the amino acid sequences TKLEIK and TALGIE are 50% homologous. In general, comparison is made when the two sequences are aligned to give maximal homology.

[0080] The term "constant domain" refers to the part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of the immunoglobulin (the variable domain) that contains the antigen-binding site. The constant domain contains the C H 1, C H 2 and C H 3 domains (collectively C H ) and the CHL (or C L ) domain of the light chain.

[0081] The "light chain" of an antibody (immunoglobulin) from any mammalian species can be assigned to one of two distinct types (referred to as kappa ("κ") and lambda ("λ")) based on the amino acid sequence of its constant domain.

[0082] The "CH1 domain" (also referred to as "C1" of the "H1" domain) typically extends from approximately amino acid 118 to approximately amino acid 215 (EU numbering system).

[0083] The "hinge region" is typically defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161 - 206 (1985)). The 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 bond in the same positions.

[0084] The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique in that it does not pair tightly with another domain. Instead, two N - linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrate provides an alternative to domain - domain pairing and helps to stabilize the CH2 domain. Burton, Molec Immunol. 22:161 - 206 (1985).

[0085] The "CH3 domain" (also referred to as the "C2" domain) contains the extension of the residues C - terminal to the CH2 domain in the Fc region (i.e., from approximately amino acid residue 341 of the antibody sequence to the C - terminus (usually at amino acid residue 446 or 447 of IgG)).

[0086] The term "Fc region" or "fragment crystallizable region" as used 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 the immunoglobulin heavy - chain Fc region may vary, the human IgG heavy - chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from position Pro230 to its carboxyl terminus. The C - terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. In some cases, the subsequent C - terminal glycine of the Fc region (residue 446 according to the EU numbering system) can also be removed. Thus, a composition of intact antibodies can contain a population of antibodies in which all K447 residues have been removed, a population of antibodies in which no K447 residues have been removed, and a population of antibodies having a mixture of antibodies with or without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0087] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Additionally, preferred FcRs are receptors that bind IgG antibodies (gamma receptors), and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. 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 receptor (FcRN) is covered herein. The term "FcR" as used herein also covers other FcRs, including FcRs yet to be identified.

[0088] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0089] As used herein, a first antibody or fragment thereof "competes" with a second antibody or fragment thereof for binding to a target antigen when the first antibody or fragment thereof inhibits binding of the second antibody or fragment thereof to the target antigen by at least about 50% (such as any of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of the first antibody or fragment thereof at equimolar concentration, or vice versa. A high-throughput method for "binning" antibodies based on antibody-based cross-competition is described in PCT Publication No. WO 03 / 48731.

[0090] As used herein, the terms "specifically bind," "specifically recognize," and "is specific for" refer to a measurable and reproducible interaction, such as the binding between a target and an antibody or antibody portion, which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which can be an epitope) is an antibody or antibody portion that binds to this target with greater affinity, avidity, more readily, and / or for a longer duration than its binding to other targets. In some embodiments, the extent to which the antibody binds to an irrelevant 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 a target has a dissociation constant (K -5 M, ≤ 10 -6 M, ≤ 10 -7 M, ≤ 10 -8 M, ≤ 10 - 9 M, ≤ 10 -10 M, ≤ 10 -11 M, or ≤ 10 -12 M). 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 can 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 blotting, ELISA-, RIA-, ECL-, IRMA-, EIA, BIACORE D -testing, and peptide scanning. TM -testing, and peptide scanning.

[0091] An "isolated" antibody (or construct) is an antibody (e.g., natural or recombinant) that has been identified, separated, and / or recovered from the components of its production environment. Preferably, the isolated polypeptide is not associated with all of the other components from its production environment.

[0092] An "isolated" nucleic acid molecule encoding a construct, antibody or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is associated in its natural environment of production. Preferably, the isolated nucleic acid is not associated with all of the components associated with the natural environment of production. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from that in which it is found in nature or in its natural environment. Thus, an isolated nucleic acid molecule is different from the nucleic acids encoding the polypeptides and antibodies described herein that occur naturally in a cell. Isolated nucleic acids include nucleic acid molecules 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.

[0093] The term "control sequence" means a DNA sequence that is necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes, for example, include a promoter, an optional operator sequence, and a ribosome-binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0094] 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 to facilitate 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. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide linkers or adaptors are used in accordance with conventional practice.

[0095] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors in the form of self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are herein referred to as "expression vectors".

[0096] The terms "transfection" or "transformation" or "transduction" as used herein refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell into which exogenous nucleic acid has been transfected, transformed, or transduced. The cell includes the primary subject cell and its progeny.

[0097] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include “transformants” and “transformed cells” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The nucleic acid content of the progeny may not be identical to that of the parental cell and may contain mutations. This includes mutant progeny that have been selected or chosen and that have the same function or biological activity as the originally transformed cell.

[0098] The term “immunoconjugate” includes reference to the covalent attachment of a therapeutic agent or detectable label to an antibody, such as an antibody moiety as described herein. The attachment can be direct or indirect through a linker, such as a peptide linker.

[0099] As used herein, “treatment” or “treating” is a course of action that is used to obtain a beneficial or desired result, including a clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by a disease, reduction of the extent of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of the spread of the disease (e.g., metastasis), prevention or delay of the recurrence of the disease, delay or slowing of the progression of the disease, improvement of the disease state, provision of a remission (partial or complete) of the disease, reduction of the dose of one or more other medications required to treat the disease, delay of the progression of the disease, improvement of the quality of life, increase in body weight gain, and / or extension of survival. The methods of this application contemplate any one or more of these aspects of treatment.

[0100] The term “inhibition” or “inhibit” refers to a decrease or cessation of any phenotypic characteristic, or to a decrease or cessation in the incidence, degree, or likelihood of that characteristic. “Decrease” or “inhibit” means a reduction, decrease, or suppression of activity, function, and / or amount as compared to a reference. In certain embodiments, “decrease” or “inhibit” means being able to effect an overall decrease of 20% or greater. In another embodiment, “decrease” or “inhibit” means being able to effect an overall decrease of 50% or greater. In yet another embodiment, “decrease” or “inhibit” means being able to effect an overall decrease of 75%, 85%, 90%, 95% or greater.

[0101] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy and / or non-diseased sample. In some instances, the reference can be obtained from an untreated sample. In some instances, the reference is obtained from a non-diseased or untreated sample of an individual. In some instances, the reference is obtained from one or more healthy individuals who are not the individual or patient.

[0102] As used herein, "delaying the development of a disease" means postponing, hindering, slowing, arresting, stabilizing, suppressing, and / or retarding the development of a disease. This delay can have different time lengths, depending on the disease history and / or the individual receiving treatment. As will be apparent to those skilled in the art, a sufficient or significant delay can actually encompass prevention, since the individual does not develop the disease.

[0103] "Prevention" as used herein includes providing prophylaxis against the occurrence or recurrence of a disease in an individual who may be predisposed to the disease but has not been diagnosed with the disease.

[0104] The terms "subject", "individual", or "patient" are used interchangeably herein to refer to a mammal, including but not limited to a human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0105] An "effective amount" of an agent is an amount that is effective to achieve the desired therapeutic or prophylactic result at the necessary dosage for the necessary period of time. The specific dosage can vary depending on one or more of the following: the particular agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying it.

[0106] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that permits the biological activity of one or more active ingredients to be effective and that does not contain additional components that are unacceptably toxic to the individual to whom the formulation will be administered. Such formulations can be sterile.

[0107] A "pharmaceutically acceptable carrier" refers to conventional non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, formulation aids, or carriers in the art that are used in conjunction with a therapeutic agent to constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed.

[0108] A "sterile" formulation is sterile or substantially free of viable microorganisms and their spores.

[0109] "Administering in combination" with one or more additional therapeutic agents includes administering simultaneously (in parallel) and sequentially or in series in any order.

[0110] The term "parallel" as used herein refers 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 shorter time period relative to the administration of another therapeutic agent. For example, the two or more therapeutic agents are administered at intervals of no more than about 60 minutes, such as at intervals of no more than about 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.

[0111] The term "sequentially" as used herein refers to the administration of two or more therapeutic agents, where the administration of one or more agents continues after the cessation of the administration of one or more other agents. For example, the two or more therapeutic agents are administered at intervals of more than about 15 minutes, such as at intervals of about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, or longer.

[0112] As used herein, "in combination with" means the administration of another therapeutic modality in addition to one therapeutic modality. Thus, "in combination with" means that one therapeutic modality is administered to an individual before, during, or after the administration of another therapeutic modality.

[0113] The term "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic product and that contain information regarding indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings for the use of such therapeutic product.

[0114] An "article" is any article (e.g., a package or container) or kit that contains at least one reagent, such as an agent for treating a disease or disorder, or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0115] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0116] The mention of "about" a value or parameter herein includes (and describes) variations that relate to that value or parameter itself. For example, the description of "about X" includes the description of "X".

[0117] As used herein, the mention of "not" a value or parameter generally means and describes "different from" a value or parameter. For example, a method not being used to treat a disease of type X means that the method is used to treat a disease of a type different from X.

[0118] The term "about X - Y" as used herein has the same meaning as "about X to about Y".

[0119] Unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms "a", "an", "or", and "the" include plural referents.

[0120] II. Anti-VISTA Constructs

[0121] This application provides anti-VISTA constructs that comprise an anti-VISTA antibody portion that specifically binds to VISTA as described herein.

[0122] In some embodiments, there are provided anti-VISTA constructs that comprise an anti-VISTA antibody portion that comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein: VH comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 47, and VL comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 50, wherein VH comprises the amino acid sequence of any one of SEQ ID NOs: 53-57, or a variant having an amino acid sequence with at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein VL comprises the amino acid sequence of any one of SEQ ID NOs: 58-62, or a variant having an amino acid sequence with at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0123] In some embodiments, there are provided anti-VISTA constructs that comprise an anti-VISTA antibody portion that comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein: VH comprises an HC-CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1-3, an HC-CDR2 having the amino acid sequence of any one of SEQ ID NOs: 4-6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 7, and V LAn LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 8-10, an LC-CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 11-13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, wherein V H comprises an amino acid sequence of any one of SEQ ID NOs: 16-30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises an amino acid sequence of any one of SEQ ID NOs: 32-44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0124] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0125] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0126] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0127] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 59, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0128] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0129] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0130] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:62, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0131] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 54, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0132] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 54, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 59, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0133] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 54, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0134] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 54, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0135] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 54, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 62, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0136] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0137] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:59, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0138] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0139] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 62, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0140] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0141] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 59, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0142] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0143] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:62, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0144] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:57, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0145] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 59, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0146] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0147] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0148] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, an HC-CDR2 having the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:8, an LC-CDR2 having the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0149] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, an HC-CDR2 having the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0150] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, an HC-CDR2 having the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:10, an LC-CDR2 having the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0151] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:2, an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:8, an LC-CDR2 having the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0152] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:2, an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0153] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:2, an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:10, an LC-CDR2 having the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0154] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:3, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:8, an LC-CDR2 having the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0155] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:3, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14.

[0156] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:3, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and V LComprising an LC-CDR1 containing the amino acid sequence of SEQ ID NO:10, an LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0157] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0158] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0159] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0160] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0161] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0162] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0163] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0164] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0165] In some embodiments, provided are anti-VISTA constructs that comprise an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0166] In some embodiments, provided are anti-VISTA constructs that comprise an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0167] In some embodiments, provided are anti-VISTA constructs that comprise an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0168] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0169] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0170] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V Lcomprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0171] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0172] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0173] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0174] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0175] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0176] In some embodiments, provided is an anti-VISTA construct that comprises an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0177] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0178] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0179] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0180] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0181] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0182] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0183] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0184] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0185] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0186] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0187] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0188] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0189] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0190] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0191] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0192] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0193] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0194] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0195] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0196] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0197] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0198] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0199] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0200] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0201] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0202] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0203] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0204] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0205] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0206] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0207] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0208] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0209] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0210] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0211] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0212] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V Lcomprising the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0213] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0214] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0215] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0216] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0217] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0218] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0219] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0220] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0221] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0222] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0223] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0224] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0225] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0226] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0227] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0228] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0229] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0230] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0231] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0232] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0233] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0234] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0235] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0236] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0237] In some embodiments, provided are anti-VISTA constructs, the anti-VISTA constructs comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0238] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0239] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0240] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0241] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0242] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0243] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0244] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0245] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0246] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0247] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0248] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0249] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0250] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0251] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0252] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0253] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0254] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0255] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0256] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0257] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0258] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0259] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0260] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0261] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0262] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0263] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0264] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0265] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0266] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0267] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0268] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0269] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0270] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0271] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0272] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0273] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:24, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0274] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0275] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0276] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0277] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0278] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0279] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0280] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0281] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0282] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V Lcomprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0283] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0284] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0285] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0286] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:25, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0287] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0288] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0289] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0290] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0291] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0292] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0293] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0294] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0295] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0296] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0297] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0298] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0299] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 26, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0300] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0301] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0302] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0303] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0304] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0305] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0306] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0307] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0308] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0309] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0310] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprising the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0311] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0312] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 27, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0313] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO:28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0314] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0315] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO:28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0316] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0317] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0318] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0319] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0320] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0321] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0322] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0323] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0324] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0325] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 28, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0326] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H Comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0327] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0328] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0329] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0330] In some embodiments, provided is an anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0331] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0332] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0333] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0334] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0335] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0336] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0337] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0338] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V LComprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0339] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 32, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0340] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 33, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0341] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V Hcomprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 34, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0342] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 35, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0343] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0344] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0345] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 38, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0346] In some embodiments, provided is an anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 39, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0347] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0348] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 41, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0349] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 42, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0350] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 43, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0351] In some embodiments, an anti-VISTA construct is provided, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO: 44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0352] In some embodiments, compared to a reference anti-VISTA construct, the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the reference anti-VISTA construct is 1E8. In some embodiments, the reference anti-VISTA construct is a parental antibody (e.g., 9F9 or 20E4 disclosed herein).

[0353] In some embodiments, the construct comprises or is an antibody or an antigen-binding fragment thereof selected from the group consisting of: full-length antibodies, bispecific antibodies, single-chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , VH H Fc fusions, scFv-Fc fusions, scFv-Fv fusions, diabodies, triabodies, and tetra-bodies.

[0354] In some embodiments, the anti-VISTA antibody moiety is a full-length antibody.

[0355] In some embodiments, the anti-VISTA antibody moiety described above 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 moiety or full-length antibody described above 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. In some embodiments, the Fc fragment has an extended half-life compared to the corresponding wild-type Fc fragment.

[0356] In some embodiments, the anti-VISTA antibody moiety of the anti-VISTA construct activates the downstream signaling pathway of VISTA. In some embodiments, the anti-VISTA construct is an agonistic antibody of VISTA. In some embodiments, the antibody moiety of the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 20%. In some embodiments, compared to a reference construct, the antibody moiety of the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% (e.g., a corresponding construct that does not activate VISTA, e.g., a corresponding construct comprising a reference agonistic anti-VISTA antibody (such as a parental VISTA antibody)). In some embodiments, the anti-VISTA construct is an antagonistic antibody of VISTA.

[0357] In some embodiments, the anti-VISTA antibody portion binds to both human VISTA and murine VISTA. In some embodiments, the anti-VISTA antibody portion binds to human VISTA. In some embodiments, the anti-VISTA antibody portion binds to murine VISTA. In some embodiments, the anti-VISTA antibody portion does not bind to murine VISTA.

[0358] 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 portion. In some embodiments, the half-life extending portion is an albumin binding portion (e.g., an albumin binding antibody portion). In some embodiments, the anti-VISTA antibody portion and the half-life extending portion are linked by a linker (such as a peptide linker, such as a GS linker).

[0359] In some embodiments, the anti-VISTA construct comprises or is an anti-VISTA immunoconjugate that comprises an anti-VISTA antibody portion (such as any of the VISTA antibody portions 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.

[0360] VISTA

[0361] T cell activation V-domain Ig suppressor (VISTA) (also known as PD-1H, Gi24, Dies-1 or DD1α) is a recently discovered cell surface co-inhibitory molecule of the CD28 / B7 gene family. It has been reported that VISTA can act as an inhibitory ligand of antigen-presenting cells and regulate T cell responses, and elimination of VISTA by gene knockout or antagonistic antibodies can enhance T cell immune responses against tumors in mouse models. VISTA can also play a key role in the regulation of inflammatory and autoimmune diseases, as demonstrated in mouse models of graft-versus-host disease (GVHD), acute hepatitis, encephalitis, lupus, asthma and psoriasis. VISTA can also act as a co-inhibitory receptor on T cells. VISTA agonist mAb significantly regulates antigen-specific CD4 T cell responses and protects 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). Studies have also shown that upregulation of VISTA in patients with prostate cancer is associated with tolerance to ipilimumab (CTLA-4 mAb). In addition, studies have shown that targeting VISTA can 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. U.S.A. 112, 6682–6687 (2015). Thus, VISTA can be an important molecule for regulating immune responses and a potential target for immunotherapy.

[0362] The VISTA gene is located on 10q22.1 and is conserved in chimpanzee, cow, mouse, rat, chicken, zebrafish and frog. The human VISTA sequence can be found in NCBI reference number NM_022153. The human VISTA protein has 311 amino acids (NCBI reference number: NP_071436.1, SEQ ID NO:59).

[0363] a) Antibody affinity

[0364] The binding specificity of the antibody moiety can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE TM testing and peptide scanning.

[0365] In some embodiments, the K of the binding between the antibody moiety and VISTA Dis about 10 -7 M to about 10 -12 M, about 10 - 7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M or about 10 -7 M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and VISTA D is greater than 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.

[0366] In some embodiments, the K of the binding between the antibody portion and VISTA on is about 10 3 M -1 s -1 to about 10 8 M -1 s -1 、about 10 3 M -1 s -1 to about 10 4 M-1 s -1 about 10 4 M -1 s -1 to about 10 5 M -1 s -1 about 10 5 M -1 s -1 to about 10 6 M -1 s -1 about 10 6 M -1 s -1 to about 10 7 M -1 s -1 or about 10 7 M -1 s -1 to about 10 8 M -1 s -1 。 In some embodiments, the K of the binding between the antibody portion and VISTA on is about 10 3 M -1 s -1 to about 10 5 M -1 s -1 about 10 4 M -1 s -1 to about 10 6 M -1 s -1 about 10 5 M -1 s -1 to about 10 7 M -1 s -1 about 10 6 M -1 s -1 to about 10 8 M -1 s -1 about 10 4 M -1 s -1 to about 10 7 M -1 s -1 or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 。 In some embodiments, the K of the binding between the antibody portion and VISTAon not more than 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 or any one of 10 s. In some embodiments, VISTA is human VISTA.

[0367] In some embodiments, the K of the binding between the antibody portion and VISTA off is about 1 s -1 to about 10 -6 s -1 、 about 1 s -1 to about 10 -2 s -1 、 about 10 -2 s -1 to about 10 -3 s -1 、 about 10 -3 s -1 to about 10 -4 s -1 、 about 10 -4 s -1 to about 10 -5 s -1 、 about 10 -5 s -1 to about 10 -6 s -1 、 about 1 s -1 to about 10 -5 s -1 、 about 10 -2 s -1 to about 10 -6 s -1 、 about 10 -3 s -1 to about 10 -6 s -1 、 about 10 -4 s -1 to about 10 -6 s -1 、 about 10 -2 s -1 to about 10 -5 s-1 or about 10 -3 s -1 to about 10 -5 s -1 。In some embodiments, the K of the binding between the antibody portion and VISTA off is at least about 1 s -1 、10 -2 s -1 、10 -3 s -1 、10 -4 s -1 、10 -5 s -1 or 10 -6 s -1 or any one of 10 s. In some embodiments, VISTA is human VISTA.

[0368] In some embodiments, the binding affinity of the anti-VISTA antibody portion or anti-VISTA construct is high (e.g., having a smaller K D value) than existing anti-VISTA antibodies (e.g., anti-human VISTA antibodies, e.g., 1E8).

[0369] b) Chimeric antibody or humanized antibody

[0370] 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 derived 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 parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0371] In some embodiments, the anti-VISTA antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental 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 human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or enhance antibody specificity or affinity.

[0372] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, 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 "surface 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 the "guided selection" approach to FR shuffling).

[0373] Human framework regions useful 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 the consensus sequence of human antibodies of a particular subgroup 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 (somatic mutation) 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 FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0374] It should be understood that humanization of mouse-derived antibodies is a common and routinely used technique. Thus, it should be understood that any and all humanized forms of anti-VISTA antibodies disclosed in the Sequence Listing can be used in preclinical or clinical settings. In any case where a humanized form of any of the anti-VISTA antibodies or their antigen-binding regions is used in such preclinical or clinical settings, it is expected that the humanized form will have the same or similar biological activity and profile as the original non-humanized form.

[0375] c) Human antibodies

[0376] In some embodiments, the anti-VISTA antibody portion is a human antibody (referred to 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, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1 and WO2004049794.

[0377] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigenic stimulation. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes the K-M technology and U.S. Patent Application Publication No. US 2007 / 0061900, which describes technology). The human variable regions from intact antibodies generated from such animals can be further modified, for example, by combining with different human constant regions.

[0378] Human antibodies (e.g., human DAbs) can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., 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 by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional 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).

[0379] Human antibodies (e.g., human DAbs) can also be generated by isolating variable domain sequences of Fv clones selected from a phage display library derived from humans. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0380] d) Antibodies from libraries

[0381] The anti-VISTA antibody moieties described herein can be isolated by screening a combinatorial library of antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., 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 et al., 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., 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, for example, U.S. Patent No. 7,371,849.

[0382] In certain phage display methods, V H and V LThe repertoire of genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, and can then be used to screen antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv fragments or as Fab fragments. A library from an immunogen provides high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a variety of non-self antigens as well as self antigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a naive library can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions and effecting 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. Patent No. 5,750,373 and U.S. Patent Publications No. 2005 / 0079574, No. 2005 / 0119455, No. 2005 / 0266000, No. 2007 / 0117126, No. 2007 / 0160598, No. 2007 / 0237764, No. 2007 / 0292936, and No. 2009 / 0002360.

[0383] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0384] e) Substitutions, insertions, deletions, and variants

[0385] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutations include HVRs (or CDRs) and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody and the product screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0386] Table 2. Amino Acid Substitutions

[0387]

[0388]

[0389] Amino acids can be grouped according to the nature of their common side chains: (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.

[0390] Non-conservative substitutions would entail replacing a member of one of these categories with a member of another category.

[0391] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). In general, the resulting variant selected for further study will have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parental antibody and / or will have certain biological properties of the parental antibody that are substantially retained. Exemplary substitution variants are affinity matured antibodies, which can be readily generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and it is screened for a particular biological activity (e.g., binding affinity).

[0392] Changes (e.g., substitutions) can be made to the HVRs, for example, to improve antibody affinity. Such changes can be made to HVR “hot spots,” i.e., residues encoded by codons that are mutated at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDR (a-CDR), where the resulting variant V H or V LTesting binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described, e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O’Brien et al., eds., 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 having 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, e.g., using alanine-scan mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0393] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made to the HVRs. Such changes can be in HVR “hot spots” or outside of the CDRs.

[0394] A method that can be used to identify residues or regions in an antibody that can be targets for mutagenesis is called “alanine-scan mutagenesis,” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a group of residues or target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they possess the desired properties.

[0395] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0396] f) Glycosylation variants

[0397] In some embodiments, the anti-VISTA antibody portion is altered to increase or decrease the degree of construct glycosylation. Adding glycosylation sites to or removing glycosylation sites from the antibody can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0398] In the case where the antibody portion comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached via an N-linkage to Asn297 in the C H 2 domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the biantennary oligosaccharide structure “backbone”. In some embodiments, modifications can be made to the oligosaccharide in the antibody portion to create antibody variants with certain improved properties.

[0399] In some embodiments, the anti-VISTA antibody portion has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications / disclosures related to "defucosylated" or "fucose-lacking" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 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 afucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cell line (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines such as the α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, 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).

[0400] In some embodiments, the anti-VISTA antibody moiety has a bisected oligosaccharide, e.g., where the biantennary oligosaccharide attached to the antibody Fc region is bisected by GlcNAc. Such antibody variants can 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 can have improved CDC function. This antibody variant is described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0401] g) Fc region variants

[0402] In some embodiments, the anti-VISTA antibody moiety comprises an Fc fragment.

[0403] The terms "Fc region", "Fc domain", "Fc fragment", or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In some embodiments, the 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 specifically stated herein, the numbering of amino acid residues in IgG or the Fc region is according to the EU numbering system for antibodies, also known as 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.

[0404] In some embodiments, the Fc fragment is 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 from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0405] In some embodiments, the Fc fragment has reduced effector function (such as reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of effector function as measured by the level of antibody-dependent cell cytotoxicity (ADCC)) compared to the corresponding wild-type Fc fragment.

[0406] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment contains the L234A mutation and / or the L235A mutation. In some embodiments, the IgG1 Fc fragment contains the L235A mutation and / or the 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 containing the S228P, F234A, and / or L235A mutations. In some embodiments, the Fc fragment contains the N297A mutation. In some embodiments, the Fc fragment contains the N297G mutation.

[0407] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody portion, 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 amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0408] In some embodiments, the Fc fragment has some but not all effector functions, which makes the Fc fragment a desirable candidate for applications where the in vivo half-life of the antibody portion is important, while 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 / elimination of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains the ability to bind FcRn. The major cells mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs 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 for assessing the ADCC activity of a molecule of interest are described in U.S. Patent 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 can be employed (see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model such as the animal model 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 does not bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO2005 / 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, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0409] Antibodies with reduced effector function include those antibodies having substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc fragment contains an N297A mutation. In some embodiments, the Fc fragment contains an N297G mutation.

[0410] Certain antibody variants with improved or attenuated binding to FcRs have been described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0411] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises an L234A mutation and / or an L235A mutation. In some embodiments, the IgG1 Fc fragment comprises an 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 an S228P, F234A, and / or L235A mutation.

[0412] In some embodiments, the antibody portion comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of the residues) in the Fc region.

[0413] In some embodiments, changes are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178 - 4184 (2000).

[0414] In some embodiments, the Fc fragment has one or more mutations at Thr250, Met252, Ser254, The256, Thr307, Glu380, Met428, His433, and / or Asn 434.

[0415] In some embodiments, the antibody portion variant comprises a variant Fc region that comprises one or more amino acid substitutions that alter the half-life and / or alter the binding to the neonatal Fc receptor (FcRn). Antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the 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.). Those antibodies comprise an Fc region having one or more substitutions therein that alter the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues in the Fc region residues (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0416] See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relate to other examples of Fc region variants.

[0417] h) Cysteine-engineered antibody variants

[0418] In some embodiments, it may be desirable to create cysteine-engineered antibody moieties, e.g., “thioMAbs”, in which one or more residues of the antibody are replaced with cysteine residues. In a particular embodiment, the residues to be replaced are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to create an immunoconjugate, 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 as described, for example, in U.S. Patent No. 7,521,541.

[0419] i) Antibody derivatives

[0420] In some embodiments, the antibody moieties described herein can be further modified to include additional non-protein moieties known in the art and readily available. Moieties suitable for antibody derivatization 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 / propanediol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide co-polymer, 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 can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if 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 specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for diagnostic purposes under defined conditions, etc.

[0421] In some embodiments, the antibody portion can be further modified to include one or more bioactive proteins, polypeptides, or fragments thereof. "Bioactive" or "biologically active" are used interchangeably herein and mean exhibiting biological activity that performs a specific function in the body. For example, it can mean combining with a specific biomolecule such as a protein, DNA, etc., and then promoting or inhibiting the activity of such biomolecule. In some embodiments, the bioactive protein or fragment thereof includes proteins and polypeptides administered to a patient as an active pharmaceutical substance to prevent or treat a disease or disorder, as well as proteins and polypeptides used for diagnostic purposes, such as enzymes for diagnostic tests or in vitro assays, and proteins and polypeptides administered to a patient to prevent a disease, such as a vaccine.

[0422] III. Preparation methods

[0423] In some embodiments, a method of preparing an anti-VISTA construct or antibody portion that specifically binds to VISTA is provided, as well as a composition produced during the preparation of the anti-VISTA construct or antibody portion, such as a polynucleotide, nucleic acid construct, vector, host cell, or culture medium. The anti-VISTA construct or antibody portion or composition described herein can be prepared by a variety of methods generally described below and more specifically described in the examples.

[0424] Antibody expression and production

[0425] The antibodies described herein can be prepared using any known method in the art, including the methods described below and in the examples.

[0426] Monoclonal antibodies

[0427] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is 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 property of the antibody as not being a mixture of discrete antibodies. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or 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 a llama, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind the protein used for immunization. Alternatively, the 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 the immunization of the camel in Example 1).

[0428] The immunizing agent will generally comprise the antigenic protein or a fusion variant thereof. Generally, if cells of human origin are desired, peripheral blood lymphocytes ("PBL") are used, or if cells of non-human mammalian origin are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0429] The immortalized cell line is generally a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Generally, a rat or mouse myeloma cell line is used. The hybridoma cells thus prepared are inoculated and grown in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma will generally contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of cells lacking HGPRT.

[0430] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to media such as HAT medium. Among them, preferred are murine myeloma lines (such as those derived from MOPC-21 and MPC-11 mouse tumors, which are available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA) and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) (available from the American Type Culture Collection, Manassas, Va., USA). Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are also described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0431] Determine the production of monoclonal antibodies against the antigen in the medium in which the hybridoma cells are growing. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0432] The presence of monoclonal antibodies against the desired antigen in the medium in which the hybridoma cells are cultured can be determined. Preferably, the binding affinity and specificity of the monoclonal antibody 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, the binding affinity can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0433] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and the clones can be grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. A cell sorter can also be used. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

[0434] The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites or serum by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0435] Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567 and as described above. The DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as Escherichia coli (E. coli) cells, simian COS cells, HEK cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins, so as to 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 Plückthun, Immunol. Revs. 130:151-188 (1992).

[0436] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). The isolation of murine and human antibodies using phage libraries is described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), respectively. Subsequent publications describe high-affinity (nM range) human antibody production by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are suitable alternatives to the traditional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0437] DNA can also be modified, for example, by replacing homologous murine sequences with the coding sequences for human heavy and light chain constant domains (U.S. Patent 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 sequences of non-immunoglobulin polypeptides to immunoglobulin coding sequences. Typically, the constant domains of an antibody are replaced with such non-immunoglobulin polypeptides, or the variable domains of one antigen-binding site of an antibody are replaced with them, to create a chimeric bivalent antibody containing one antigen-combining site specific for one antigen and another antigen-combining site specific for a different antigen.

[0438] The monoclonal antibodies described herein can 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 typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues can be replaced with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also applicable for the preparation of monovalent antibodies. Digestion of an antibody to produce its fragments, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

[0439] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking 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.

[0440] A nucleic acid molecule encoding an antibody portion

[0441] In some embodiments, polynucleotides encoding any of the anti-VISTA constructs or antibody portions described herein are provided. In some embodiments, polynucleotides prepared using any of the methods described herein are provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody portion (e.g., an anti-VISTA antibody portion). In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding a heavy chain of an antibody portion (e.g., an anti-VISTA antibody portion) and a polynucleotide encoding 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 encoding a heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0442] In some such embodiments, the heavy and light chains are expressed from one nucleic acid molecule or as two separate polypeptides from two separate nucleic acid molecules. In some embodiments, such as when the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising the heavy and light chains linked together.

[0443] In some embodiments, the polynucleotide encoding the heavy or light chain of the antibody portion (e.g., the anti-VISTA antibody portion) comprises a nucleotide sequence encoding a leader sequence that, when translated, is located at the N-terminus of the heavy or light chain. As discussed above, the leader sequence can be the native heavy or light chain leader sequence or can be another heterologous leader sequence.

[0444] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0445] The nucleic acid molecule can be constructed using conventional recombinant DNA techniques in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0446] Nucleic acid construct

[0447] In some embodiments, a nucleic acid construct comprising any one of the polynucleotides described herein is provided. In some embodiments, a nucleic acid construct prepared by any of the methods described herein is provided.

[0448] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide. In some embodiments, the polynucleotide corresponds to a gene, and the promoter is the wild-type promoter of the gene.

[0449] Vector

[0450] In some embodiments, a vector is provided that comprises any polynucleotide encoding a heavy chain and / or a light chain of an antibody portion described herein (e.g., an anti-VISTA antibody portion) or any of the nucleic acid constructs described herein. In some embodiments, a vector prepared by any of the methods described herein is provided. Also provided is a vector that comprises a polynucleotide encoding any anti-VISTA construct, such as an antibody, scFv, fusion protein, or other forms of the constructs described herein (e.g., an anti-VISTA scFv). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and the light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and the light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

[0451] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain and a second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into a host cell in similar amounts, such as similar molar amounts or similar mass amounts. In some embodiments, the first vector and the second vector are transfected into a host cell at a molar ratio or mass ratio of 5:1 to 1:5. In some embodiments, the mass ratio of the vector encoding the heavy chain to the vector encoding the light chain used is between 1:1 and 1:5. In some embodiments, the mass ratio of the vector encoding the heavy chain to the vector encoding the light chain used is 1:2.

[0452] In some embodiments, a vector optimized for expressing a polypeptide in CHO or CHO-derived cells or NSO cells is selected. Such exemplary vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0453] Host cell

[0454] In some embodiments, a host cell is provided that comprises any polypeptide, nucleic acid construct, and / or vector described herein. In some embodiments, a host cell prepared by any of the methods described herein is provided. In some embodiments, the host cell is capable of producing any antibody portion described herein under fermentation conditions.

[0455] In some embodiments, the antibody portions described herein (e.g., anti-VISTA antibody portions) can be expressed in prokaryotic cells such as bacterial cells or in eukaryotic cells such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells useful for expressing polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-GS, DG44, Lec13 CHO cells, and FUT8 CHO cells; Crucell; HEK cells, and NSO cells. In some embodiments, the antibody portions described herein (e.g., anti-VISTA antibody portions) can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications to the heavy and / or light chains of the antibody portion. For example, in some embodiments, polypeptides produced by CHO cells have a higher level of sialylation compared to the same polypeptides produced in 293 cells.

[0456] One or more nucleic acids can be introduced into the desired host cell 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, 3 3rd Edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cell according to any suitable method.

[0457] This application also provides host cells comprising any of the polynucleotides or vectors described herein. In some embodiments, the invention provides host cells comprising anti-VISTA antibodies. Any host cell capable of overexpressing heterologous DNA can be used to isolate the gene encoding the antibody, polypeptide, or protein 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. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes such as Escherichia coli (E. coli) or Bacillus subtilis (B. subtilis) and yeast such as Saccharomyces cerevisiae (S. cerevisae), Schizosaccharomyces pombe (S. pombe); or Kluyveromyces lactis (K. lactis).

[0458] 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).

[0459] culture medium

[0460] 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 by any of the methods described herein.

[0461] In some embodiments, the culture medium comprises hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the culture medium does not contain serum. In some embodiments, the culture medium contains serum. In some embodiments, the culture medium is D-MEM or RPMI-1640 medium.

[0462] In some embodiments, the culture medium is chemically defined. In some embodiments, the culture medium is specifically derived for a particular cell line (e.g., CHO GS cells).

[0463] Purification of the antibody portion

[0464] The anti-VISTA construct can be purified by any suitable method. Such methods include, but are not limited to, using an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include the ROR1 ECD and ligands that bind to the antibody constant region. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column can be used to bind the constant region and purify an anti-VISTA construct that contains an Fc fragment. Hydrophobic interaction chromatography, such as a butyl or phenyl column, may also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides such as antibodies. Many methods for purifying polypeptides are known in the art.

[0465] V. Methods of treatment

[0466] The present disclosure also provides methods for treating a disease or disorder in an individual or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells) or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of T cells, such as CD3+CD25+ T cells or CD45+ T cells). The methods include administering to the individual (e.g., a mammal, such as a human) an anti-VISTA construct described herein.

[0467] In some embodiments, provided is a method for treating a disease or disorder in an individual or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method including administering to the individual an effective amount of an anti-VISTA construct described herein. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, an inflammation, an infection, graft-versus-host disease (GvHD), or a transplant-related disorder. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0468] In some embodiments, provided is a method for treating a disease or disorder in an individual or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method including administering to the individual an effective amount of an anti-VISTA construct (such as any of the anti-VISTA constructs disclosed herein).

[0469] In some embodiments, provided is a method for treating a disease or disorder in an individual or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method including administering to the individual an effective amount of an anti-VISTA construct that includes an antibody portion that includes a heavy chain variable region (VH) and a light chain variable region (V L ), where VH includes the amino acid sequence of SEQ ID NO:30, or a variant that includes an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and where V LComprises the amino acid sequence of SEQ ID NO:44, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related disorder. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0470] In some embodiments, provided is a method of treating a disease or disorder in an individual or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method 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 (V L ), wherein VH comprises the amino acid sequence of SEQ ID NO:53, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L Comprises the amino acid sequence of SEQ ID NO:58, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related disorder. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0471] In some embodiments, provided is a method of treating a disease or disorder in an individual or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method 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 (V L), wherein VH comprises the amino acid sequence of SEQ ID NO:55, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:60, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related disorder. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0472] In some embodiments, provided is a method of treating a disease or disorder in an individual or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), the method 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 (V L ), wherein VH comprises the amino acid sequence of SEQ ID NO:56, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein V L comprises the amino acid sequence of SEQ ID NO:61, or a variant comprising an amino acid sequence having at least about 80% (such as any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a transplant-related disorder. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0473] In some embodiments, compared to a corresponding construct that does not activate VISTA (e.g., an isotype control), an anti-VISTA construct for modulating the immune response of an individual or modulating immune cells (e.g., T cells) inhibits the proliferation of T cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, compared to a corresponding construct comprising a reference agonistic anti-VISTA antibody (e.g., 1E8), an anti-VISTA construct for modulating the immune response of an individual or modulating immune cells (e.g., T cells) inhibits the proliferation of T cells by at least about 5%, 10%, 15%, 20%, or 25%. In some embodiments, the cell is a VISTA-positive cell. In some embodiments, the cell is a CD3+CD25+ cell. In some embodiments, the cell is a CD45+ T cell.

[0474] In some embodiments, provided is a method of modulating a cell (e.g., an immune cell), the method comprising contacting the 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 CD3+CD25+ cell. In some embodiments, the cell is a CD45+ 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 cell is a VISTA-positive cell. In some embodiments, the contacting occurs in vitro.

[0475] In some embodiments, provided is a method of genome editing a cell (e.g., an immune cell), the method comprising introducing into the cell: a) a donor template comprising a nucleic acid sequence encoding any of the anti-VISTA constructs described herein, and b) a DNA nuclease (e.g., a CRISPR-associated protein (Cas)) or a nucleotide sequence encoding a DNA nuclease. In some embodiments, the method further comprises administering the genome-edited cell to an individual suffering from a disease or disorder described herein.

[0476] In some embodiments, the subject is a mammal (such as a human).

[0477] In some embodiments, the individual has an elevated level of serum anti-nuclear antibodies (e.g., the serum anti-nuclear antibody level is at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than that of a healthy individual). In some embodiments, the individual has an elevated level of serum anti-dsDNA antibodies (e.g., the serum anti-dsDNA antibody level is at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than that of a healthy individual). In some embodiments, the individual has an elevated level of serum IFNα (e.g., the serum IFNα level is at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than that of a healthy individual). In some embodiments, the individual has an elevated level of urinary protein (e.g., the urinary protein level is at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than that of a healthy individual).

[0478] Methods of Administration and Dosage of Anti-VISTA Constructs

[0479] The dosing regimen (such as the specific dose and frequency) of an anti-VISTA construct administered to an individual for treating a disease or disorder as described herein can vary depending on the particular anti-VISTA construct, the mode of administration, and the type of disease or disorder being treated. In some embodiments, an effective amount of the anti-VISTA construct is an amount that is effective to alleviate at least one symptom of the disease or disorder. In some embodiments, an effective amount of the anti-VISTA construct is an amount sufficient to extend the overall survival of an individual (such as a human). In some embodiments, an effective amount of the anti-VISTA construct is an amount sufficient to produce a clinical beneficial effect of more than about 50%, 60%, 70%, 80%, or 90% in a population of individuals treated with the anti-VISTA construct.

[0480] In some embodiments, an effective amount of the anti-VISTA construct is an amount that slows or inhibits the progression of the disease or disorder (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an untreated individual. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an infection. In some embodiments, the disease or disorder is psoriasis.

[0481] In some embodiments, an effective amount of the anti-VISTA construct reduces the serum anti-nuclear antibody level by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% compared to a reference individual (e.g., an individual having the same disease or disorder but not treated with the anti-VISTA construct). In some embodiments, an effective amount of the anti-VISTA construct reduces the serum anti-dsDNA antibody level by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% compared to a reference individual (e.g., an individual having the same disease or disorder but not treated with the anti-VISTA construct). In some embodiments, an effective amount of the anti-VISTA construct reduces the serum IFNα level by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% compared to a reference individual (e.g., an individual having the same disease or disorder but not treated with the anti-VISTA construct). In some embodiments, an effective amount of the anti-VISTA construct reduces the serum IL-17A level by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% compared to a reference individual (e.g., an individual having the same disease or disorder but not treated with the anti-VISTA construct). In some embodiments, an effective amount of the anti-VISTA construct reduces the urinary protein level by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80% or 90% compared to a reference individual (e.g., an individual having the same disease or disorder but not treated with the anti-VISTA construct).

[0482] In some embodiments, an effective amount of the anti-VISTA construct is an amount that reduces the side effects (autoimmune responses) of a disorder (e.g., transplantation) by at least about 5%, 10%, 15%, 20%, 30%, 40% or 50% compared to an untreated individual.

[0483] 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 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.

[0484] In some embodiments of any of the above aspects, an effective amount of the anti-VISTA construct for humans is a dose equivalent to 0.5 mg for mice.

[0485] In some embodiments of any of the above aspects, the anti-VISTA construct is administered once a week. In some embodiments of any of the above aspects, the anti-VISTA construct is administered once every two weeks. In some embodiments, the anti-VISTA construct is administered once a week for at least about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, or about 20 weeks.

[0486] The anti-VISTA construct can be administered to an individual (such as a human) by various routes, including, for example, intravenous, intraarterial, intraperitoneal, intralung, oral, inhalation, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the anti-VISTA construct is included in a pharmaceutical composition when administered to an individual. In some embodiments, a sustained release formulation 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.

[0487] Combination therapy

[0488] The present application also provides a method of administering an anti-VISTA construct to an individual for treating a disease or disorder, wherein the method further comprises administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or disorder. In some embodiments, the disease or disorder is psoriasis.

[0489] In some embodiments, the anti-VISTA construct is administered concomitantly with a second agent or therapy. In some embodiments, the anti-VISTA construct is administered in parallel with a second agent or therapy. In some embodiments, the anti-VISTA construct is administered sequentially with a second agent or therapy. In some embodiments, the anti-VISTA construct is administered prior to the second agent or therapy. In some embodiments, the anti-VISTA construct is administered after the second agent or therapy. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form from the second agent or therapy. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form from the second agent or therapy. In some embodiments, the second agent or therapy is an agonist of VISTA. In some embodiments, the second agent or therapy is a VISTA ligand.

[0490] In some embodiments, the second agent or therapy is a polypeptide. In some embodiments, the polypeptide comprises VSIG3 or a fragment thereof. VSIG3 (V-Set and immunoglobulin domain-containing protein 3) is a ligand of VISTA. It is known that VSIG3 can inhibit T cells through inhibitory functions (see, e.g., Xie et al., Front. Immunol., 12:625808 (2021)). In some embodiments, the polypeptide is a VSIG3-Fc fusion. In some embodiments, the polypeptide is an antibody or a fragment thereof.

[0491] In some embodiments, the second agent or therapy is a small molecule (e.g., a small molecule inhibitor).

[0492] VI. Use of Anti-VISTA Constructs

[0493] Also provided herein is the use of an anti-VISTA construct as disclosed herein for the manufacture of a medicament for treating a disease or disorder in an individual or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of T cells). The use includes administering to an individual (e.g., a mammal, such as a human) a medicament comprising the anti-VISTA construct described herein.

[0494] In some embodiments, provided is the use of an anti-VISTA construct as disclosed herein for manufacturing a medicament for treating a disease or disorder in an individual or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cells), said use comprising administering to the individual an effective amount of the anti-VISTA construct described herein. In some embodiments, the disease or disorder is associated with an immune system disorder. In some embodiments, the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a disorder associated with transplantation. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE). In some embodiments, the disease or disorder is psoriasis.

[0495] In some embodiments, provided is the use of an anti-VISTA construct as disclosed herein for manufacturing a medicament for treating a disease or disorder in an individual or modulating the immune response of an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, such as CD3+CD25+ T cells or CD45+ T cel...

Claims

1. An anti-VISTA construct, the anti-VISTA construct comprising an anti-VISTA antibody portion, the anti-VISTA antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (V L ). Wherein: a) said V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO: 45, an HC-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO: 47, and said V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO: 48, an LC-CDR2 containing the amino acid sequence of SEQ ID NO: 49, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO: 50, wherein said V H comprises the amino acid sequence of any one of SEQ ID NOs: 53 - 57, and wherein said V L comprises the amino acid sequence of any one of SEQ ID NOs: 58 - 62; or b) said V H comprises an HC-CDR1 having an amino acid sequence of any one of SEQ ID NOs: 1-3, an HC-CDR2 having an amino acid sequence of any one of SEQ ID NOs: 4-6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 having an amino acid sequence of any one of SEQ ID NOs: 8-10, an LC-CDR2 having an amino acid sequence of any one of SEQ ID NOs: 11-13, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 14, wherein said V H comprises an amino acid sequence of any one of SEQ ID NOs: 16-30, and wherein said V L comprises an amino acid sequence of any one of SEQ ID NOs: 32-44.

2. The anti-VISTA construct according to claim 1, wherein the V H comprises the amino acid sequence of any one of SEQ ID NO: 53-57, and wherein the V L comprises the amino acid sequence of any one of SEQ ID NO: 58-62.

3. The anti-VISTA construct according to claim 2, Wherein: 1) The V H comprises the amino acid sequence of SEQ ID NO:53, and wherein the V L comprises the amino acid sequence of SEQ ID NO:58; 2) said V H comprises the amino acid sequence of SEQ ID NO:55, and wherein said V L comprises the amino acid sequence of SEQ ID NO:60; or 3) said V H comprises the amino acid sequence of SEQ ID NO:56, and wherein said V L comprises the amino acid sequence of SEQ ID NO:

61.

4. The anti-VISTA construct according to claim 1, Wherein: a) said V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 4, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 14; b) said V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, an HC-CDR2 having the amino acid sequence of SEQ ID NO:4, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and said V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14; or c) said V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 containing the amino acid sequence of SEQ ID NO: 4, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14; d) said V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 containing the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14; e) said V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:2, an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and said V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14; f) said V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14; g) said V H comprises an HC-CDR1 containing the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 containing the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 containing the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 containing the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 containing the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14; h) said V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO:3, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7, and said V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:9, an LC-CDR2 having the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:14; or i) said V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 7, and said V L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:

14.

5. The anti-VISTA construct according to claim 1 or claim 4, wherein the V H comprises an amino acid sequence selected from any one of SEQ ID NOs: 16-30, and wherein the V L comprises an amino acid sequence selected from any one of SEQ ID NOs: 32-44.

6. The anti-VISTA construct according to claim 5, Wherein: 1) said V H comprises the amino acid sequence of SEQ ID NO: 30, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 44; 2) said V H comprises the amino acid sequence of SEQ ID NO: 16, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 32; 3) said V H comprises the amino acid sequence of SEQ ID NO: 16, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 33; 4) said V H comprises the amino acid sequence of SEQ ID NO: 16, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 34; 5) said V H comprises the amino acid sequence of SEQ ID NO:16, and wherein said V L comprises the amino acid sequence of SEQ ID NO:35; 6) said V H comprises the amino acid sequence of SEQ ID NO:17, and wherein said V L comprises the amino acid sequence of SEQ ID NO:32; 7) said V H comprises the amino acid sequence of SEQ ID NO: 17, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 33; 8) said V H comprises the amino acid sequence of SEQ ID NO:17, and wherein said V L comprises the amino acid sequence of SEQ ID NO:34; 9) said V H comprises the amino acid sequence of SEQ ID NO:17, and wherein said V L comprises the amino acid sequence of SEQ ID NO:35; 10) said V H comprises the amino acid sequence of SEQ ID NO: 18, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 32; 11) said V H comprises the amino acid sequence of SEQ ID NO:18, and wherein said V L comprises the amino acid sequence of SEQ ID NO:33; 12) The V H comprises the amino acid sequence of SEQ ID NO:18, and wherein said V L comprises the amino acid sequence of SEQ ID NO:34; 13) The V H comprises the amino acid sequence of SEQ ID NO:18, and wherein said V L comprises the amino acid sequence of SEQ ID NO:35; 14) said V H comprises the amino acid sequence of SEQ ID NO:19, and wherein said V L comprises the amino acid sequence of SEQ ID NO:32; 15) The V H comprises the amino acid sequence of SEQ ID NO:19, and wherein the V L comprises the amino acid sequence of SEQ ID NO:33; 16) said V H comprises the amino acid sequence of SEQ ID NO:19, and wherein said V L comprises the amino acid sequence of SEQ ID NO:34; 17) said V H comprises the amino acid sequence of SEQ ID NO: 19, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 35; 18) said V H comprises the amino acid sequence of SEQ ID NO: 20, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 36; 19) said V H comprises the amino acid sequence of SEQ ID NO: 21, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 37; 20) said V H comprises the amino acid sequence of SEQ ID NO: 22, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 38; 21) said V H comprises the amino acid sequence of SEQ ID NO: 23, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 39; 22) said V H comprises the amino acid sequence of SEQ ID NO:24, and wherein said V L comprises the amino acid sequence of SEQ ID NO:40; 23) said V H comprises the amino acid sequence of SEQ ID NO: 25, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 38; 24) said V H comprises the amino acid sequence of SEQ ID NO:25, and wherein said V L comprises the amino acid sequence of SEQ ID NO:39; 25) said V H comprises the amino acid sequence of SEQ ID NO: 25, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 40; 26) said V H comprises the amino acid sequence of SEQ ID NO: 26, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 38; 27) said V H comprises the amino acid sequence of SEQ ID NO: 26, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 39; 28) said V H comprises the amino acid sequence of SEQ ID NO: 26, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 40; 29) said V H comprises the amino acid sequence of SEQ ID NO: 29, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 43; 30) said V H comprises the amino acid sequence of SEQ ID NO: 29, and wherein said V L comprises the amino acid sequence of SEQ ID NO: 44; or 31) said V H comprises the amino acid sequence of SEQ ID NO: 30, and wherein said V L comprises the amino acid sequence of SEQ ID NO:

43.

7. The anti-VISTA construct according to claim 6, wherein the V H comprises the amino acid sequence of SEQ ID NO: 30, and wherein the V L comprises the amino acid sequence of SEQ ID NO:

44.

8. The anti-VISTA construct according to any one of claims 1 to 7, wherein the antibody portion is an antibody or an antigen-binding fragment thereof selected from the group consisting of: full-length antibody, bispecific antibody, single-chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, and tetrabody.

9. The anti-VISTA construct according to claim 8, wherein the antibody portion is a full-length antibody.

10. The anti-VISTA construct according to any one of claims 1 to 9, wherein the antibody portion of the anti-VISTA construct has an Fc fragment selected from the group consisting of the following Fc fragment forms: IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof.

11. The anti-VISTA construct according to claim 10, wherein the Fc fragment is selected from the group consisting of: Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

12. The anti-VISTA construct according to claim 10 or claim 11, wherein the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment.

13. The anti-VISTA construct according to any one of claims 10 to 12, wherein the Fc fragment has an extended half-life compared to the corresponding wild-type Fc fragment.

14. The anti-VISTA construct according to any one of claims 1 to 13, wherein the antibody portion of the anti-VISTA construct activates the downstream signaling pathway of VISTA.

15. The anti-VISTA construct according to any one of claims 1 to 14, wherein the anti-VISTA construct is an agonistic antibody of VISTA.

16. The anti-VISTA construct according to claim 14 or claim 15, wherein the antibody portion of the anti-VISTA construct activates or increases the downstream signaling pathway of VISTA by at least about 20%.

17. The anti-VISTA construct according to any one of claims 1 to 13, wherein the anti-VISTA construct is an antagonistic antibody of VISTA.

18. The anti-VISTA construct according to any one of claims 1 to 17, wherein the VISTA is human VISTA.

19. A pharmaceutical composition, the pharmaceutical composition comprising the anti-VISTA construct according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier.

20. An isolated nucleic acid, the isolated nucleic acid encoding the anti-VISTA construct according to any one of claims 1 to 18.

21. A vector, the vector comprising the isolated nucleic acid according to claim 20.

22. An isolated host cell, the isolated host cell comprising the isolated nucleic acid according to claim 20, or the vector according to claim 21.

23. An immunoconjugate, the immunoconjugate comprising the anti-VISTA construct according to any one of claims 1 to 18 linked to a therapeutic agent or a label.

24. A method for producing an anti-VISTA construct, the method comprising: a) Culturing the isolated host cell according to claim 22 under conditions effective for expressing the anti-VISTA construct; and b) Obtaining the expressed anti-VISTA construct from the host cell.

25. A method of treating a disease or disorder in an individual, the method comprising administering to the individual an effective amount of the anti-VISTA construct according to any one of claims 1 to 18, or the pharmaceutical composition according to claim 19.

26. The method according to claim 25, wherein the disease or disorder is related to an immune system disorder.

27. The method according to claim 25 or claim 26, wherein the disease or disorder is related to activated T cells.

28. The method according to claim 27, wherein the activated T cells are CD3+CD25+ T cells and / or CD45+ T cells.

29. The method according to any one of claims 25 to 28, wherein the disease or disorder is related to VISTA-positive cells.

30. The method according to any one of claims 25 to 29, wherein the disease or disorder is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD) or a disorder related to transplantation.

31. The method according to claim 30, wherein the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE).

32. The method according to any one of claims 25 to 31, wherein the anti-VISTA construct is administered intravenously or subcutaneously to the individual.

33. The method according to any one of claims 25 to 32, wherein the anti-VISTA construct is administered at a dose of about 0.001 mg / kg to about 100 mg / kg.

34. The method according to any one of claims 25 - 33, wherein the individual is a human.

35. A kit comprising the anti-VISTA construct according to any one of claims 1 to 18.

36. The anti-VISTA construct according to any one of claims 1 to 18, wherein the anti-VISTA construct inhibits PBMC and / or T cell activation by at least 20%.

37. The anti-VISTA construct according to any one of claims 1 to 19, wherein the anti-VISTA construct reduces the production of IL-17A by at least 30%.

38. The method according to any one of claims 25 to 34, wherein the anti-VISTA construct is administered together with a second agent.

39. The method according to claim 38, wherein the second agent is a VISTA agonist.

40. The method according to claim 38 or claim 39, wherein the second agent is a VISTA ligand.

41. The method according to any one of claims 38 to 40, wherein the second agent is a polypeptide.

42. The method according to claim 40, wherein the VISTA ligand comprises VSIG3 or a fragment thereof.

43. The method according to claim 42, wherein the VSIG3 or a fragment thereof is fused to an Fc domain.

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