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 sequences of anti-VISTA, vectors comprising the nucleic acid molecules, host cells comprising the vectors, anti-VISTA constructs, methods for preparing pharmaceutical compositions containing anti-VISTA constructs, and methods of using anti-VISTA constructs or compositions. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 53-57, and the V L comprises any one of the amino acid sequences of SEQ ID NOs: 58 to 62.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 404,470, filed September 7, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Electronic Sequence Listing Reference The electronic sequence listing (193852000740SEQLIST.xml; size: 60,285 bytes; and creation date: September 6, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to anti-VISTA constructs and uses thereof. [Background technology]

[0004] VISTA (programmed death-1 homolog, also known as PD-1H, VSIR, Dies1, DD1α, and Gi24) is a cell surface inhibitory molecule of the B7 / CD28 gene family expressed on T cells and myeloid cells. VISTA functions as an inhibitory ligand on antigen-presenting cells (APCs) and can regulate T cell responses via an unknown receptor. Additionally, VISTA may also function as an inhibitory receptor on T cells. For example, agonistic VISTA monoclonal antibodies (mAbs) dramatically modulate antigen-specific CD4+ T cell responses and protect mice from graft-versus-host disease (GVHD) and experimental hepatitis. Mice lacking VISTA on a C57BL / 6 background (B6 PD-1H KO) are prone to autoimmune induction, including experimental autoimmune encephalomyelitis and systemic lupus erythematosus, when backcrossed into strains susceptible to systemic lupus erythematosus. VISTA has been shown to be involved in peripheral immune tolerance and negatively regulates T cell activation (see, for example, Sci Transl Med. 2019 Dec 11;11(522)).

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

[0006] In one aspect, the present application provides a heavy chain variable region (V H ) and the light chain variable region (V L and a) V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47, L comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50, and V H comprises any one of the amino acid sequences of SEQ ID NOs: 53 to 57, and V L or b) V H comprises an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 4 to 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, L comprises an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8 to 10, an LC-CDR2 comprising the sequence of any one of SEQ ID NOs: 11 to 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, H comprises any one of the amino acid sequences of SEQ ID NOs: 16 to 30, and V L The present invention provides an anti-VISTA construct comprising the amino acid sequence of any one of SEQ ID NOs: 32 to 44.

[0007] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 53-57, and the V LIn some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 58 to 62. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 53, and V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 55, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 56, and the V L comprises the amino acid sequence of SEQ ID NO:61.

[0008] In some embodiments, V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 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:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0009] In some embodiments, V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 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:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0010] In some embodiments, V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 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.

[0011] In some embodiments, V Hcomprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, 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:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0012] In some embodiments, V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, 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:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0013] In some embodiments, V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, 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.

[0014] In some embodiments, V H comprises 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:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0015] 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:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0016] In some embodiments, V H comprises 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.

[0017] In some embodiments with any of the anti-VISTA constructs described above, V H comprises any one of the amino acid sequences of SEQ ID NOs: 16 to 30, and V L In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 30, and V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 16, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 16, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 16, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 16, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, and the VL In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 18, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 18, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 18, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 18, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 20, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 21, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 22, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 23, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 24, and the V LIn some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 25, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 25, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 25, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 26, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 26, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 26, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 29, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 29, and the V L In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 30, and the V L comprises the amino acid sequence of SEQ ID NO:43.

[0018] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion is an antibody or antigen-binding fragment thereof selected from the group consisting of a full-length antibody, a bispecific antibody, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab'), an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv), an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody. In some embodiments, the antibody is a full-length antibody.

[0019] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion comprises an Fc fragment selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived 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.

[0020] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA antibody portion of the anti-VISTA construct activates a signaling pathway downstream of VISTA.

[0021] In some embodiments according to any of the anti-VISTA constructs described above, the anti-VISTA construct is an agonistic antibody to VISTA.

[0022] In some embodiments of any of the anti-VISTA constructs described above, the antibody portion of the anti-VISTA construct activates or increases a signaling pathway downstream of VISTA by at least about 20%.

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

[0024] In some embodiments according to any of the anti-VISTA constructs described above, the anti-VISTA construct is human VISTA.

[0025] In some embodiments of any of the anti-VISTA constructs described above, the anti-VISTA construct inhibits activation of PBMCs and / or T cells by at least 20%.

[0026] 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%.

[0027] In another aspect, the application provides a pharmaceutical composition comprising any of the anti-VISTA constructs described above and a pharmaceutically acceptable carrier.

[0028] In another aspect, the application provides an isolated nucleic acid encoding any of the anti-VISTA constructs described above.

[0029] In another aspect, the present application provides a vector comprising any of the above-described isolated nucleic acids.

[0030] In another aspect, the present application provides an isolated host cell comprising any of the above-described isolated nucleic acids and / or vectors.

[0031] In another aspect, the application provides an immunoconjugate comprising any of the above-described anti-VISTA constructs linked to a therapeutic agent or label.

[0032] In another aspect, the application includes: a) culturing any of the isolated host cells described above under conditions effective to express an anti-VISTA construct; and b) obtaining the expressed anti-VISTA construct from the host cell.

[0033] Another aspect of the present application provides methods of treating a disease or condition in an individual, the method comprising administering to the individual an effective amount of any of the anti-VISTA constructs described above or any of the pharmaceutical compositions described above. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition 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 condition is associated with VISTA-positive cells. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the anti-VISTA construct is administered to the individual intravenously or subcutaneously. 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 in conjunction 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, the VSIG3 or a fragment thereof is fused to an Fc domain.

[0034] In another aspect, the application provides a kit comprising any one of the anti-VISTA constructs described above. [Brief explanation of the drawings]

[0035] [Figure 1] 1 shows an SDS-PAGE gel of exemplary anti-VISTA antibody VH / VL combinations. The gel was run under non-reducing conditions.

[0036] [Figure 2A] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing human VISTA using fluorescence activated cell sorting (FACS). [Figure 2B] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing human VISTA using fluorescence activated cell sorting (FACS). [Figure 2C] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing human VISTA using fluorescence activated cell sorting (FACS). [Figure 2D] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing human VISTA using fluorescence activated cell sorting (FACS). [Figure 2E] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing human VISTA using fluorescence activated cell sorting (FACS).

[0037] [Figure 3A] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS). [Figure 3B] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS). [Figure 3C] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS). [Figure 3D]Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS). [Figure 3E] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS). [Figure 3F] Figure 1 shows the binding activity of 9E9 and 20E4 parental and humanized anti-VISTA antibodies to Jurkat cells expressing mouse VISTA using fluorescence activated cell sorting (FACS).

[0038] [Figure 4A] Binding of humanized 9F9 anti-VISTA antibody to human VISTA by Octet is shown. [Figure 4B] Binding of humanized 9F9 anti-VISTA antibody to human VISTA by Octet is shown.

[0039] [Figure 4C] Binding of humanized 20E4 anti-VISTA antibody to human VISTA by Octet is shown.

[0040] [Figure 5A] Binding of humanized 9F9 anti-VISTA antibody to mouse VISTA by Octet is shown. [Figure 5B] Binding of humanized 9F9 anti-VISTA antibody to mouse VISTA by Octet is shown.

[0041] [Figure 5C] Binding of humanized 20E4 anti-VISTA antibody to mouse VISTA by Octet is shown.

[0042] [Figure 6A]This shows activation of the VISTA signaling pathway by different concentrations of humanized 9F9 anti-VISTA antibody in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z-expressing cells (human VISTA is fused to hCD3z expressed on the cells). [Figure 6B] This shows activation of the VISTA signaling pathway by different concentrations of humanized 9F9 anti-VISTA antibody in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z-expressing cells (human VISTA is fused to hCD3z expressed on the cells). [Figure 6C] This shows activation of the VISTA signaling pathway by different concentrations of humanized 9F9 anti-VISTA antibody in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z-expressing cells (human VISTA is fused to hCD3z expressed on the cells).

[0043] [Figure 6D] Figure 1 shows activation of the VISTA downstream pathway by different concentrations of humanized 20E4 anti-VISTA antibody in the presence of anti-CD3 antibody (OKT3) in Jurkat-NFKb-GFP / hVISTA-hCD3z-expressing cells. Human VISTA is fused to hCD3z, and activation of the VISTA pathway stimulates the FITC signal in the cells.

[0044] [Figure 7] 1 shows an outline of the experimental protocol for a mouse graft-versus-host therapy model.

[0045] [Figure 8] 1 shows the change in body weight over time in graft-versus-host model mice treated with isotype control and mice treated with 9F9 or 20E4 anti-VISTA antibodies.

[0046] [Figure 9]The extent of skin denudation in graft-versus-host mouse model mice injected with isotype control and mice treated with 9F9 or 20E4 anti-VISTA antibodies is shown.

[0047] [Figure 10] Figure 1 shows the levels of human CD45+ cells in the blood of graft-versus-host model mice treated with isotype control and 9F9 or 20E4 anti-VISTA antibodies. Samples were taken on days 14 and 37.

[0048] [Figure 11] Shown are forward and side scatter staining of CD45+ cells in the blood of mice injected with isotype control and treated with human 9E9 or 20E4 anti-VISTA antibodies. Samples were taken on days 14 and 37.

[0049] [Figure 12] 1 shows an outline of the experimental protocol for a mouse graft-versus-host therapy model.

[0050] [Figure 13] The extent of skin denudation in graft-versus-host mouse model mice injected with isotype control and mice injected with 20E4 or VL3 anti-VISTA antibodies is shown.

[0051] [Figure 14A] The percentage of human CD45+ cells in the different treatment groups on day 13 is shown.

[0052] [Figure 14B] The percentage of human CD45+ cells in the different treatment groups on day 22 is shown.

[0053] [Figure 15A] 1 shows inhibition of PBMC and T cell activation by 9F9 anti-VISTA antibody.

[0054] [Figure 15B]Inhibition of PBMC and T cell activation by 20E4 anti-VISTA antibody is shown. * indicates p<0.05, ** indicates p<0.01.

[0055] [Figure 16] Figure 1 shows the efficacy of 9F9 and 20E4 hIgG1 and hIgG2 antibodies in an imiquimod (IMQ)-induced psoriasiform BALB / c-hVISTA mouse model. * indicates p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0056] This application provides novel anti-VISTA constructs that specifically bind to VISTA, methods for preparing the anti-VISTA constructs, and methods for using the constructs (e.g., methods for treating diseases or conditions). Exemplary anti-VISTA constructs include agonist antibodies that can bind to and activate VISTA.

[0057] I. Definition The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0058] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains are respectively referred to as "V H " and "V LThe variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs comprising HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are interposed between adjacent sections known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant regions of their heavy chains. The five major 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. Some major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgG1 (α1 heavy chain), or IgG2 (α2 heavy chain). Chimeric Fc regions (e.g., IgG2 / 4 mixtures) are also contemplated herein.

[0059] The term "antigen-binding fragment," as used herein, refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of an antibody comprising 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 comprise the complete antibody structure. An antigen-binding fragment can bind to the same antigen as bound by the parent antibody or parent antibody fragment (e.g., the parent scFv). In some embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0060] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0061] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv fragment consisting of a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising an antibody domain. H Domains and V LThe scFv further comprises a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0062] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified by Kabat et al., J.Biol.Chem.252:6609-6616(1977);Kabat et al.,USDept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991);Chothia et al.,J.Mol.Biol.196:901-917(1987);Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997);MacCallum et al.,J.Mol.Biol.262:732-745(1996);Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008);Lefranc MPet al.,Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and the definitions include overlapping or subsets of amino acid residues when compared against each other. However, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues comprising the CDRs as defined by each of the above references are set forth below in Table 1 for comparison.The contents of the references cited in this paragraph, in which CDR prediction algorithms and interfaces are known in the art, including, for example, 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), are incorporated herein by reference in their entirety for use in this application and for possible inclusion in one or more claims herein. In some embodiments, the CDR sequences provided herein are based on the IMGT definition. For example, CDR sequences can be determined by the VBASE2 tool (http: / / www.vbase2.org / vbase2.php; see also Retter I, Althaus HH, Munch RM, MMuller M W: VBASE2, an integrative V gene database. Nucleic Acids Res. 2005 Jan 1;33(Database issue):D671-4, which is incorporated herein by reference in its entirety). [Table 1]

[0063] The phrases "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the antibody sequences in Kabat et al., supra. When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or hypervariable region (HVR) of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) or may also contain multiple inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues in a particular antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0064] Unless otherwise indicated herein, the numbering of residues in immunoglobulin heavy chains is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0065] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0066] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Most frequently, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0067] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be produced using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

[0068] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the compared polypeptide, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for assessing alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared. However, for purposes herein, percent 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).

[0069] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent 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 compared, and multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. As an example, the amino acid sequences TKLEIK and TALGIE share 50% homology. Generally, comparisons are performed by aligning the two sequences to maximize homology.

[0070] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant region domain is the C H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or C L ) domain.

[0071] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0072] The "CH1 domain" (also called the "C1" for "H1" domain) typically spans from about amino acid 118 to about amino acid 215 (EU numbering system).

[0073] The "hinge region" is generally defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions.

[0074] The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically spans from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact, native IgG molecules. It has been speculated that the carbohydrates may provide an alternative for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0075] The "CH3 domain" (also referred to as the "C2" domain) comprises the stretch from the C-terminal residue of the Fc region to the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 for IgG).

[0076] The terms "Fc region" or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain and include native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as extending from amino acid residue position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. In some cases, the C-terminal glycine (residue 446 according to the EU numbering system) following the Fc region may also be removed. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0077] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Further preferred FcRs are those that bind IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Included herein are fetal Fc receptors (FcRNs). Other FcRs, including those identified hereafter, are also encompassed by the term "FcR" herein.

[0078] As used herein, the term "epitope" refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0079] As used herein, a first antibody or fragment thereof "competes" for binding of a second antibody or fragment thereof to a target antigen if the first antibody or fragment thereof inhibits target antigen binding of the second antibody or fragment thereof by at least about 50% (e.g., by at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof. Vice versa. A high-throughput process for "binning" antibodies based on mutual competition is described in PCT Publication No. WO 03 / 48731.

[0080] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding, between a target and an antibody or antibody portion that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to this target with greater affinity, avidity, more readily, and / or with a longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤ 10 -12 Dissociation constant of M (K D). In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but is not required to include, exclusive binding. Antibody or antigen-binding domain binding specificity can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ testing, and peptide scanning.

[0081] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with all other components from its production environment.

[0082] 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 contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from association with all components associated with the production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are present in a form that is different from the form or setting in which they are found in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules contained within cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0083] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0084] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to 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. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0085] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked to them. Such vectors are referred to herein as "expression vectors."

[0086] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0087] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0088] The term "immunoconjugate" includes reference to the covalent attachment of a therapeutic agent or detectable label to an antibody, such as an antibody portion described herein. The attachment can be direct or indirect via a linker (such as a peptide linker).

[0089] As used herein, "treatment" or "treating" refers to an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of this application contemplate any one or more of these aspects of treatment.

[0090] The term "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic trait, or the reduction or cessation of the incidence, degree, or likelihood of that trait. "Reducing" or "inhibiting" refers to the decrease, lowering, or prevention of an activity, function, and / or amount compared to a reference. In certain embodiments, "reducing" or "inhibiting" refers to the ability to produce an overall reduction of 20% or more. In other embodiments, "reducing" or "inhibiting" refers to the ability to produce an overall reduction of 50% or more. "Reducing" or "inhibiting" refers to the ability to produce an overall reduction of 75%, 85%, 90%, 95%, or more.

[0091] As used herein, a "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy sample and / or a non-disease sample. In some examples, a reference can be obtained from an untreated sample. In some examples, a reference is obtained from a non-disease or untreated sample from an individual. In some examples, a reference is obtained from one or more healthy individuals who are not individuals or patients.

[0092] As used herein, "delaying the onset of disease" means to postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of the disease. This delay can be for various lengths of time, depending on the medical history and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the disease.

[0093] "Preventing" as used herein includes providing protection against the onset or recurrence of a disease in an individual who may be predisposed to the disease but has not yet been diagnosed with the disease.

[0094] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0095] An "effective amount" of an agent refers to an amount effective to obtain a desired therapeutic or prophylactic result over a period of time at multiple dosages required. The specific dose may vary depending on one or more of the particular agent selected, the dosage 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 in which it is delivered.

[0096] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained therein is in a form that allows it to be effective and which does not contain any ingredients that are unacceptably toxic to the individual to whom the formulation is administered. Such formulations are sterile.

[0097] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents, which together comprise a "pharmaceutical composition" administered to an individual. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0098] A "sterile" preparation is sterile or free or essentially free of viable microorganisms and their spores.

[0099] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0100] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to the administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered within a time interval of about 60 minutes or less, such as about any of 30, 15, 10, 5, or 1 minutes.

[0101] The term "sequential" is used herein to refer to the administration of two or more therapeutic agents, where administration of one or more agents is continued after the administration of one or more other agents is discontinued. For example, the administration of two or more therapeutic agents is separated by a time interval of more than about 15 minutes, such as about 20, 30, 40, 50, or 60 minutes, or for one, two, three, one, two, two weeks, one month, or more.

[0102] As used herein, "in conjunction with" refers to the administration of one therapeutic modality in addition to another. Thus, "in conjunction with" refers to the administration of one therapeutic modality to an individual before, during, or after the administration of another therapeutic modality.

[0103] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product that contain information about the therapeutic product's indications, uses, dosages, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0104] An "article of manufacture" is any manufactured article (e.g., package or container) or kit that includes at least one reagent, e.g., a pharmaceutical agent for the treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0105] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0106] Reference herein to "about" a value or parameter includes (and describes) variations on that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0107] As used herein, "not" a value or parameter generally means and describes "other than" a value or parameter. For example, "a method is not used to treat disease type X" means that the method is used to treat disease types other than X.

[0108] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0109] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the content clearly dictates otherwise.

[0110] II. Anti-VISTA Constructs The present application provides anti-VISTA constructs comprising an anti-VISTA antibody portion that specifically binds to VISTA as described herein.

[0111] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; and the VH comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47. Anti-VISTA constructs are provided, including variants comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the VL of any one of SEQ ID NOs: 58-62, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0112] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L 1 to 3, HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 4 to 6, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and VH comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 4 to 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7,L comprises an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8 to 10, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11 to 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, H includes a variant comprising an amino acid sequence having at least about 80% sequence identity (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to any one of the amino acid sequences of SEQ ID NOs: 16 to 30, and V L Anti-VISTA constructs are provided, comprising the 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% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0113] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0114] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0115] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0116] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0117] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0118] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0119] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0120] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H includes the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0121] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H includes the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0122] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H includes the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0123] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H includes the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0124] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H includes the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0125] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0126] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0127] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0128] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0129] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0130] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0131] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0132] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0133] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 58, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0134] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0135] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0136] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0137] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V L An anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0138] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V LAn anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0139] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V L An anti-VISTA construct is provided, comprising 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.

[0140] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V L An anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0141] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V LAn anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0142] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and V L An anti-VISTA construct is provided, comprising 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.

[0143] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises 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 An anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0144] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises 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 LAn anti-VISTA construct is provided, comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0145] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises 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 An anti-VISTA construct is provided, comprising 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.

[0146] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0147] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0148] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0149] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0150] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0151] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0152] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0153] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0154] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0155] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0156] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0157] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0158] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0159] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0160] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0161] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0162] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0163] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0164] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0165] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0166] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0167] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0168] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0169] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0170] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0171] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0172] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0173] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0174] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0175] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0176] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0177] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0178] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0179] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0180] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0181] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0182] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0183] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0184] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0185] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0186] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0187] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0188] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0189] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0190] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0191] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0192] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0193] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0194] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0195] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0196] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0197] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0198] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0199] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0200] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0201] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0202] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0203] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0204] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0205] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0206] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0207] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0208] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0209] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0210] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0211] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0212] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0213] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0214] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0215] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0216] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0217] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0218] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0219] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0220] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0221] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0222] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0223] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0224] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0225] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0226] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0227] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0228] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0229] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0230] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0231] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0232] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0233] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0234] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0235] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0236] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0237] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0238] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0239] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0240] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0241] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0242] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0243] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0244] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0245] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0246] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0247] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0248] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0249] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0250] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0251] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0252] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0253] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0254] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0255] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0256] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0257] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0258] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0259] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0260] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0261] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0262] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0263] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0264] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0265] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0266] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0267] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0268] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0269] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0270] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0271] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0272] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0273] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0274] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0275] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0276] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0277] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0278] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0279] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0280] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0281] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0282] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0283] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0284] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0285] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0286] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0287] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0288] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0289] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0290] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0291] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0292] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0293] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0294] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0295] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0296] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0297] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0298] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0299] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0300] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0301] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0302] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0303] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0304] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0305] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0306] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0307] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0308] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0309] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0310] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0311] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0312] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0313] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0314] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0315] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0316] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0317] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0318] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0319] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0320] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0321] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0322] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0323] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0324] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0325] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0326] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0327] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0328] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0329] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; LAnti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0330] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0331] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0332] In some embodiments, the heavy chain variable region (VH ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 36, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0333] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0334] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity;L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0335] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0336] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 40, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0337] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0338] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0339] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V Hcomprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0340] In some embodiments, the heavy chain variable region (V H ) and the light chain variable region (V L and an anti-VISTA antibody portion comprising V H comprises the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; L Anti-VISTA constructs are provided, comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0341] In some embodiments, the anti-VISTA construct activates or increases signaling pathways downstream of VISTA by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more than a reference anti-VISTA construct. In some embodiments, the reference anti-VISTA construct is 1E8. In some embodiments, the reference anti-VISTA construct is a parent antibody (e.g., 9F9 or 20E4 disclosed herein).

[0342] In some embodiments, the constructs include full-length antibodies, bispecific antibodies, single-chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv), V H H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody, or an antibody or antigen-binding fragment thereof selected from the group consisting of: an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.

[0343] In some embodiments, the anti-VISTA antibody portion is a full-length antibody.

[0344] In some embodiments, the anti-VISTA antibody portion 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 portion 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.

[0345] In some embodiments, the anti-VISTA antibody portion of the anti-VISTA construct activates a signaling pathway downstream of VISTA. In some embodiments, the anti-VISTA construct is an agonist antibody to VISTA. In some embodiments, the antibody portion of the anti-VISTA construct activates or increases a signaling pathway downstream of VISTA by at least about 20%. In some embodiments, the antibody portion of the anti-VISTA construct increases a signaling pathway downstream of VISTA by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a reference construct (e.g., a corresponding construct that does not activate VISTA, e.g., a corresponding construct comprising a reference agonist anti-VISTA antibody such as a parent VISTA antibody). In some embodiments, the anti-VISTA construct is an antagonist antibody to VISTA.

[0346] In some embodiments, the anti-VISTA antibody portion binds to both human VISTA and mouse VISTA. In some embodiments, the anti-VISTA antibody portion binds to human VISTA. In some embodiments, the anti-VISTA antibody portion binds to mouse VISTA. In some embodiments, the anti-VISTA antibody portion does not bind to mouse VISTA.

[0347] In some embodiments, the anti-VISTA construct comprises or is an anti-VISTA fusion protein. In some embodiments, the anti-VISTA construct comprises an anti-VISTA antibody portion (e.g., an anti-VISTA scFv) and a second portion. In some embodiments, the second portion comprises a half-life extending moiety. In some embodiments, the half-life extending moiety is an albumin binding moiety (e.g., an albumin binding antibody portion). In some embodiments, the anti-VISTA antibody portion and the half-life extending moiety are linked via a linker (e.g., a peptide linker, e.g., a GS linker).

[0348] In some embodiments, the anti-VISTA construct comprises or is an anti-VISTA immunoconjugate comprising an anti-VISTA antibody moiety (e.g., any of the VISTA antibody moieties described herein) and a second agent. In some embodiments, the second agent is a therapeutic agent. In some embodiments, the second agent is a label.

[0349] VISTA V domain Ig suppressor of T cell activation (VISTA) (also known as PD-1H, Gi24, Dies-1, or DD1α) is a more recently identified cell surface co-inhibitory molecule of the CD28 / B7 gene family. VISTA has been reported to function as an inhibitory ligand on antigen-presenting cells and regulate T cell responses, and neutralization of VISTA by either gene knockout or antagonistic antibodies can enhance T cell immune responses against tumors in mouse models. VISTA also appears to play an important role in regulating inflammation and autoimmune diseases, as shown in mouse models of graft-versus-host disease (GVHD), acute hepatitis, encephalitis, lupus, asthma, and psoriasis. VISTA may also function as a co-inhibitory receptor on T cells. VISTA agonist mAbs dramatically modulate antigen-specific CD4 T cell responses and protect mice from GVHD, acute hepatitis, and asthma (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)). Increased VISTA expression in prostate cancer patients has also been shown to be associated with resistance to ipilimumab (a CTLA-4 mAb). Furthermore, targeting VISTA has been shown to synergize with other non-redundant pathways, such as PD-1 blockade, and may result in optimal tumor elimination in experimental mouse models (see Liu et al., Proc. Natl. Acad. Sci. USA 112, 6682-6687 (2015)). Thus, VISTA is a key molecule in regulating immune responses and a potential target for immunotherapy.

[0350] The VISTA gene is located at 10q22.1. This gene is conserved in chimpanzees, cows, mice, rats, chickens, zebrafish, and frogs. 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).

[0351] a) Antibody affinity The binding specificity of an antibody moiety can be determined experimentally by methods well known in the art, including, but not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ test, and peptide scan.

[0352] In some embodiments, the K D is about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M, or about 10-7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and VISTA is D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, VISTA is human VISTA.

[0353] In some embodiments, the K on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is on is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 4M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is on is about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 In some embodiments, the VISTA is a human VISTA.

[0354] In some embodiments, the K off is about 1 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -2 s -1 , about 10 -2 s -1~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 , or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and VISTA is off is 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 In some embodiments, the VISTA is a human VISTA.

[0355] In some embodiments, the binding affinity of an anti-VISTA antibody portion or anti-VISTA construct is higher (e.g., K ) than that of an existing anti-VISTA antibody (e.g., an anti-human VISTA antibody, e.g., 1E8). D value is smaller).

[0356] b) Chimeric or Humanized Antibodies In some embodiments, the anti-VISTA antibody portion is a chimeric antibody. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region of murine origin) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0357] In some embodiments, the anti-VISTA antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains, wherein 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 also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0358] Humanized antibodies and methods for producing humanized antibodies 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 78,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0359] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of 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 (somatically matured) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al. al., J. Biol. Chem. 271:22611-22618 (1996)).

[0360] It will be understood that humanization of murine-derived antibodies is a common and routinely used technique. Accordingly, it will be understood that any and all humanized formats of the anti-VISTA antibodies disclosed in the Sequence Listing may be used in preclinical or clinical settings. When a humanized format of any of the referenced anti-VISTA antibodies, or their antigen-binding regions, is used in such preclinical or clinical settings, it is expected that such humanized format will have the same or similar biological activity and profile as the original, non-humanized format of the antibody.

[0361] c) Human antibodies In some embodiments, the anti-VISTA antibody portion is a human antibody (known as a human domain antibody, or human DAb). Human antibodies can be produced using a variety of techniques well 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.

[0362] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes K-MMOUSE® technology; and U.S. Patent Application Publication No. US2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0363] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have also 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 produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0364] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0365] d) Library-derived antibodies The anti-VISTA antibodies described herein can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are also described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., 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 previously described, see, for example, U.S. Patent No. 7,371,849.

[0366] In a particular phage display method, V is used as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). H and V LGene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage. Phages typically display antibody fragments as either scFv or Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self or self antigens without any immunization. Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0367] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0368] e) Substitutions, insertions, deletions, and variants In some embodiments, antibody variants having one or more amino acid substitutions are provided. Targeted sites for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are shown in Table 2 under the heading "Exemplary Substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 2]

[0369] Amino acids can be classified into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain directionality: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0370] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0371] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have an alteration (e.g., improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have substantially retained a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0372] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made to HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting from the secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Often, CDR-H3 and CDR-L3 are specifically targeted.

[0373] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such modifications do not substantially reduce the antigen-binding ability of the antibody. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in an HVR. Such modifications may be outside of the "hot spots" or CDRs of an HVR.

[0374] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, contact points between the antibody and antigen can be identified from a crystal structure of the antigen-antibody complex. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they have desired properties.

[0375] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0376] f) glycosylation variants In some embodiments, the anti-VISTA antibody portion is modified to increase or decrease the extent to which the construct is glycosylated. Glycosylation sites can be added or deleted from an antibody conveniently by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0377] If the antibody moiety comprises an Fc region, the carbohydrate attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are generally located at the C of the Fc region. H The oligosaccharide is N-linked to Asn297 in the 2 domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody moiety can be made to generate antibody variants with improved certain properties.

[0378] In some embodiments, an 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 an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose can be determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycostructures (e.g., complex, mixed, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located near position 297 in the Fc region (Fc region residues according to EU numbering), although Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Application Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Published examples of "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. al. J. Mol. Biol. 336:1239-1249(2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545(1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the alpha-1,6-fucosyltransferase gene FUT8 has been knocked out (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).

[0379] In some embodiments, the anti-VISTA antibody portion comprises a bisected oligosaccharide, e.g., a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants comprising at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0380] g) Fc region variants In some embodiments, the anti-VISTA antibody portion comprises an Fc fragment.

[0381] 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, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues within an IgG or Fc region follows 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.

[0382] In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof, hi some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0383] In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).

[0384] 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 an L235A mutation. In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.

[0385] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody moiety, thereby creating 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 includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0386] In some embodiments, Fc fragments retain some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody portion is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063(1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502(1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and thereby lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 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, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods well known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0387] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc fragment comprises a N297A mutation. In some embodiments, the Fc fragment comprises a N297G mutation.

[0388] Certain antibody variants with improved or reduced binding to FcRs have also 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)).

[0389] 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.

[0390] In some embodiments, the antibody portion comprises an Fc region comprising one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and / or 334 of the Fc region (residues according to EU numbering).

[0391] In some embodiments, modifications are made to the Fc region that result in altered (either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0392] In some embodiments, the Fc fragment has one or more mutations at Thr250, Met252, Ser254, The256, Thr307, Glu380, Met428, His433, and / or Asn434.

[0393] In some embodiments, the antibody partial variant comprises a variant Fc region containing one or more amino acid substitutions that alter half-life and / or binding to fetal Fc receptor (FcRn). Antibodies with extended half-lives and improved binding to fetal Fc receptor (FcRn), responsible for transport of maternal IgG to the fetus, have been described in US2005 / 0014934A1 (Hinton et al.) (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region containing one or more substitutions that alter binding of the Fc region to FcRn. Such Fc variants include those containing substitutions at one or more Fc region residues, for example, substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0394] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0395] h) Cysteine ​​Engineered Antibody Variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMabs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. Substituting these residues with cysteine ​​places reactive thiol groups at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as described further herein. In some embodiments, any one or more of the following residues can be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibody moieties can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0396] i) Antibody derivative In some embodiments, the antibody moieties described herein can be further modified to include additional nonproteinaceous moieties that are well known in the art and readily available. Suitable moieties 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 / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for diagnosis under a given condition, etc.

[0397] In some embodiments, the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biological activity" or "biologically active" refers to the ability to exhibit biological activity that performs a specific function in the body. For example, the term can refer to association with a specific biological molecule, such as a protein or DNA, or even the promotion or inhibition of the activity of such a biological molecule. In some embodiments, biologically active proteins or fragments thereof include, but are not limited to, proteins and polypeptides administered to patients as active pharmaceutical ingredients for the prevention or treatment of a disease or condition, as well as proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and polypeptides administered to patients to prevent disease, such as vaccines.

[0398] III. Preparation method In some embodiments, methods for preparing anti-VISTA constructs or antibody portions that specifically bind to VISTA are provided, as well as compositions, such as polynucleotides, nucleic acid constructs, vectors, host cells, or media, produced during the preparation of the anti-VISTA constructs or antibody portions. The anti-VISTA constructs or antibody portions or compositions described herein can be prepared by a number of processes, as generally described below and more specifically in the Examples.

[0399] Antibody Expression and Production The antibodies described herein may be prepared using any method known in the art, including those described below and in the Examples.

[0400] Monoclonal antibodies Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of individual antibodies. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methodology (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal (e.g., a hamster or llama) is immunized as described above to elicit lymphocytes that produce, or have the capacity to produce, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). See also Example 1 for immunization in camels.

[0401] The immunizing agent typically includes an antigenic protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0402] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused original myeloma cells. For example, if the original myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma usually contains hypoxanthine, aminopterin, and thymidine, substances that prevent the growth of HGPRT-deficient cells (HAT medium).

[0403] Preferred immortalized myeloma cells are those that fuse efficiently, support stable, high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, mouse myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 cells (and their derivatives, e.g., X63-Ag8-653), available from the American Type Culture Collection, Manassas, Va., USA, are particularly preferred. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0404] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0405] The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0406] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. A cell sorter may also be used. Additionally, hybridoma cells may be grown in vivo as mammalian tumors.

[0407] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0408] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 and above. DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA is incorporated into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin protein, such as E. coli, simian COS cells, HEK cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize the monoclonal antibody in the recombinant host cells. Reviews on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0409] 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). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications have described the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0410] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create chimeric bivalent antibodies containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0411] The monoclonal antibodies described herein can be monovalent, and methods for their preparation are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at a point within the Fc region to prevent cross-linking of the heavy chain. Alternatively, relevant cysteine ​​residues can be substituted with other amino acid residues or removed to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be performed using routine techniques well known in the art.

[0412] Chimeric or hybrid antibodies also can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Suitable agents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0413] Nucleic acid molecules encoding antibody portions In some embodiments, a polynucleotide encoding any one of the anti-VISTA constructs or antibody portions described herein is provided. In some embodiments, a polynucleotide prepared using any one of the above methods is provided. In some embodiments, a 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, a nucleic acid molecule comprises both a polynucleotide encoding a heavy chain 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 the heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain.

[0414] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides from one nucleic acid molecule or from two separate nucleic acid molecules, in some embodiments, for example when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked to each other.

[0415] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody moiety (e.g., an anti-VISTA antibody moiety) comprises a nucleotide sequence encoding a leader sequence that, when translated, will be positioned at the N-terminus of the heavy or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0416] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0417] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art, hi some embodiments, the nucleic acid molecule is in an expression vector suitable for expression in a selected host cell.

[0418] Nucleic Acid Constructs In some embodiments, a nucleic acid construct is provided that includes any one of the polynucleotides described herein. In some embodiments, a nucleic acid construct is provided that is prepared using any of the methods described herein.

[0419] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide, hi some embodiments, the polynucleotide corresponds to a gene and the promoter is the wild-type promoter for that gene.

[0420] vector In some embodiments, vectors are provided that include any polynucleotide encoding the heavy and / or light chain of any one of the antibody portions (e.g., anti-VISTA antibody portions) or nucleic acid constructs described herein. In some embodiments, vectors prepared using any of the methods described herein are provided. Also provided are vectors that include a polynucleotide encoding any of the anti-VISTA constructs, such as antibodies, scFvs, fusion proteins, or other forms of constructs described herein (e.g., anti-VISTA scFvs). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector includes a first polypeptide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, for example, when the antibody is an scFV, the heavy and light chains are expressed as part of a single polypeptide.

[0421] 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 and second vectors are transfected into host cells in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, a molar or mass ratio of the first and second vectors of 5:1 to 1:5 is transfected into host cells. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain to the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain to the vector encoding the light chain.

[0422] In some embodiments, a vector optimized for expression of a polypeptide in CHO or CHO-derived cells or in NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0423] host cell In some embodiments, a host cell is provided that comprises any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, a host cell is provided that is prepared using any of the methods described herein. In some embodiments, the host cell is capable of producing any of the antibody portions described herein under fermentation conditions.

[0424] In some embodiments, 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 (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures well known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13, and FUT8 CHO cells; PER.C6® cells (Crucell); HEK cells, and NSO cells. In some embodiments, antibody portions described herein (e.g., anti-VISTA antibody portions) can be expressed in yeast. See, e.g., U.S. Patent Application Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains of the antibody portion. For example, in some embodiments, CHO cells produce polypeptides that have higher levels of sialylation than the same polypeptides produced in 293 cells.

[0425] Introduction of one or more nucleic acids into a desired host cell can be achieved 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 rd ed. Cold Spring Harbor Laboratory Press, 2001. The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

[0426] The present application also provides host cells comprising any of the polynucleotides or vectors described herein. In some embodiments, the present invention provides host cells comprising an anti-VISTA antibody. Host cells capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtillis) and yeast (e.g., S. cerevisae, S. pombe; or K. lactis).

[0427] 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), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0428] Culture medium In some embodiments, a medium is provided that comprises any of the antibody portions, polynucleotides, nucleic acid constructs, vectors, and / or host cells described herein, hi some embodiments, a medium prepared using any of the methods described herein is provided.

[0429] In some embodiments, the medium contains hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the medium is serum-free. In some embodiments, the medium contains serum. In some embodiments, the medium is D-MEM or RPMI-1640 medium.

[0430] In some embodiments, the medium is chemically defined, hi some embodiments, the medium is specifically derived for a particular cell line (e.g., CHO GS cells).

[0431] Antibody purification Anti-VISTA constructs can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and the constant region of an antibody. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind the constant region and purify anti-VISTA constructs containing an Fc fragment. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, may also be suitable for purifying certain polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying certain polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying certain polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0432] V. Treatment method Also provided herein are methods of treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), or modulating an immune response in an individual (inhibiting the proliferation and / or activation of T cells, e.g., CD3+CD25+ T cells or CD45+ T cells, etc.), which methods comprise administering to an individual (e.g., a mammal such as a human) an anti-VISTA construct as described herein.

[0433] In some embodiments, methods are provided for treating a disease or condition or modulating an immune response (e.g., inhibiting proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual, comprising administering to the individual an effective amount of an anti-VISTA construct described herein. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), or discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0434] In some embodiments, methods are provided for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising administering to the individual an effective amount of an anti-VISTA construct (e.g., any of the anti-VISTA constructs disclosed herein).

[0435] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual comprises combining a heavy chain variable region (VH) and a light chain variable region (V L 30), wherein the VH comprises an amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and L

[0013] Methods are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 44, or a variant thereof, having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-associated condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0436] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual comprises combining a heavy chain variable region (VH) and a light chain variable region (V L53), wherein the VH comprises an amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and L

[0013] Methods are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 58, or a variant thereof, wherein the amino acid sequence has at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0437] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual comprises combining a heavy chain variable region (VH) and a light chain variable region (V L 55), wherein the VH comprises an amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and L

[0013] Methods are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 60, or a variant thereof, wherein the amino acid sequence has at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-associated condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0438] In some embodiments, a method of treating a disease or condition or modulating an immune response (e.g., inhibiting proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual comprises combining a heavy chain variable region (VH) and a light chain variable region (V L 56), wherein the VH comprises an amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and L

[0013] Methods are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 61, or a variant thereof, having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-associated condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0439] In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a corresponding construct that does not activate VISTA (e.g., an isotype control). In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 5%, 10%, 15%, 20%, or 25% compared to a corresponding construct comprising a reference agonist anti-VISTA antibody (e.g., 1E8). In some embodiments, the cells are VISTA-positive cells. In some embodiments, the cells are CD3+CD25+ T cells. In some embodiments, the cells are CD45+ T cells.

[0440] In some embodiments, methods of modulating a cell (e.g., an immune cell) are provided, comprising contacting the immune cell with an anti-VISTA construct (e.g., any of the anti-VISTA constructs described herein). In some embodiments, the cell is a T cell (CD4 and / or CD8 T cell). In some embodiments, the cell is a CD3+CD25+ T 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 is performed in vitro.

[0441] In some embodiments, methods are provided for genome editing a cell (e.g., an immune cell), 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 having a disease or condition described herein.

[0442] In some embodiments, the subject is a mammal (eg, a human).

[0443] In some embodiments, the individual has elevated serum levels of antinuclear antibodies (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of anti-dsDNA antibodies (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of IFNα (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated urinary protein levels (e.g., urinary protein levels that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual).

[0444] Dosage and Administration Methods of Anti-VISTA Constructs The dosing regimen (e.g., specific dosage and frequency) of an anti-VISTA construct used to treat a disease or disorder described herein administered to an individual can vary depending on the particular anti-VISTA construct, the mode of administration, and the type of disease or condition being treated. In some embodiments, an effective amount of an anti-VISTA construct is an amount effective to alleviate at least one symptom of a disease or condition. In some embodiments, an effective amount of an anti-VISTA construct is an amount sufficient to extend the overall survival of an individual (e.g., a human). In some embodiments, an effective amount of an anti-VISTA construct is an amount sufficient to result in a clinical benefit of greater than about any of 50%, 60%, 70%, 80%, or 90% among a population of individuals treated with the anti-VISTA construct.

[0445] In some embodiments, an effective amount of an anti-VISTA construct is an amount that slows or inhibits the progression of a disease or condition (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an individual not receiving treatment. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an infectious disease. In some embodiments, the disease or condition is psoriasis.

[0446] In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of antinuclear antibodies by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of anti-dsDNA antibodies by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of IFNα by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces serum levels of IL-17A by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct). In some embodiments, an effective amount of an anti-VISTA construct reduces urinary protein levels by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, or 90% compared to a reference individual (e.g., an individual having the same disease or condition but not treated with the anti-VISTA construct).

[0447] In some embodiments, an effective amount of an anti-VISTA construct is an amount that reduces (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%) the side effects (e.g., autoimmune response) of a condition (e.g., transplant) compared to an individual not receiving treatment.

[0448] In some embodiments of any of the above aspects, the effective amount of the anti-VISTA construct is in the range of about 0.001 μg / kg to about 100 mg / kg of total body weight, e.g., about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg.

[0449] In some embodiments of any of the above aspects, the effective amount of the anti-VISTA construct in a human is the equivalent dose of 0.5 mg in a mouse.

[0450] In some embodiments of any of the above aspects, the anti-VISTA construct is administered weekly. In some embodiments of any of the above aspects, the anti-VISTA construct is administered every other week. In some embodiments, the anti-VISTA construct is administered weekly for at least about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 weeks.

[0451] Anti-VISTA constructs can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the anti-VISTA construct is contained 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.

[0452] Combination therapy The present application also provides methods of administering an anti-VISTA construct to an individual to treat a disease or condition, the method comprising administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy to treat the disease or condition. In some embodiments, the disease or condition is psoriasis.

[0453] In some embodiments, the anti-VISTA construct is administered concurrently with a second agent or therapy. In some embodiments, the anti-VISTA construct is administered simultaneously 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 before a second agent or therapy. In some embodiments, the anti-VISTA construct is administered after a second agent or therapy. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as a second agent or therapy. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form than a second agent or therapy. In some embodiments, the anti-VISTA construct is administered in the same unit dosage form as a second agent or therapy. In some embodiments, the anti-VISTA construct is administered in a different unit dosage form than a second agent or therapy. In some embodiments, the second agent or therapy is a VISTA agonist. In some embodiments, the second agent or therapy is a VISTA ligand.

[0454] In some embodiments, the second agent or therapy is a polypeptide. In some embodiments, the polypeptide is VSIG3 or a fragment thereof. VSIG3 (V-Set and immunoglobulin domain containing 3) is a ligand for VISTA. VSIG3 is known to inhibit T cells through inhibitory function (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.

[0455] In some embodiments, the second agent or therapy is a small molecule (eg, a small molecule inhibitor).

[0456] VI. Use of anti-VISTA constructs Also provided herein is the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., 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). The use comprises administering to an individual (e.g., a mammal such as a human) a medicament comprising an anti-VISTA construct described herein.

[0457] In some embodiments, use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells) in an individual is provided, comprising administering to the individual an effective amount of an anti-VISTA construct described herein. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0458] In some embodiments, there is provided the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition in an individual or modulating an immune response (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising administering to the individual an effective amount of an anti-VISTA construct (e.g., any of the anti-VISTA constructs disclosed herein).

[0459] In some embodiments, the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising a heavy chain variable region (VH) and a light chain variable region (V L30), wherein the VH comprises an amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and L Uses are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 44, or a variant thereof, comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-associated condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0460] In some embodiments, the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising a heavy chain variable region (VH) and a light chain variable region (V L 53), wherein the VH comprises an amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and LUses are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 58, or a variant thereof, comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0461] In some embodiments, the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising a heavy chain variable region (VH) and a light chain variable region (V L 55), wherein the VH comprises an amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and LUses are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising an amino acid sequence of SEQ ID NO: 60, or a variant thereof, comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0462] In some embodiments, the use of an anti-VISTA construct disclosed herein in the manufacture of a medicament for treating a disease or condition or modulating an immune response in an individual (e.g., inhibiting the proliferation and / or activation of PBMCs or T cells, e.g., CD3+CD25+ T cells or CD45+ T cells), comprising a heavy chain variable region (VH) and a light chain variable region (V L 56), wherein the VH comprises an amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and LUses are provided which comprise administering to an individual an effective amount of an anti-VISTA construct comprising the amino acid sequence of SEQ ID NO: 61, or a variant comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the disease or condition is associated with dysregulation of the immune system. In some embodiments, the disease or condition is an autoimmune disease, inflammation, infection, graft-versus-host disease (GvHD), or a transplant-related condition. In some embodiments, the autoimmune disease is selected from cutaneous lupus, rheumatoid arthritis, psoriasis, autoimmune bowel disease, systemic lupus erythematosus (SLE), and discoid lupus erythematosus (DLE). In some embodiments, the disease or condition is psoriasis.

[0463] In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a corresponding construct that does not activate VISTA (e.g., an isotype control). In some embodiments, anti-VISTA constructs used to modulate an immune response or modulate immune cells (e.g., T cells) in an individual prevent T cell proliferation by at least about 5%, 10%, 15%, 20%, or 25% compared to a corresponding construct comprising a reference agonist anti-VISTA antibody (e.g., 1E8). In some embodiments, the cells are VISTA-positive cells. In some embodiments, the cells are CD3+CD25+ T cells. In some embodiments, the cells are CD45+ T cells.

[0464] In some embodiments, methods are provided for using an anti-VISTA construct (e.g., any of the anti-VISTA constructs described herein) to modulate a cell (e.g., an immune cell), comprising contacting the immune cell with the anti-VISTA construct. In some embodiments, the cell is a T cell (CD4+ and / or CD8 T+ cell). In some embodiments, the cell is a CD3+CD25+ T 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 is performed in vitro.

[0465] In some embodiments, methods are provided for genome editing a cell (e.g., an immune cell), 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 having a disease or condition described herein.

[0466] In some embodiments, the subject is a mammal (eg, a human).

[0467] In some embodiments, the individual has elevated serum levels of antinuclear antibodies (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of anti-dsDNA antibodies (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated serum levels of IFNα (e.g., serum levels of antinuclear antibodies that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual). In some embodiments, the individual has elevated urinary protein levels (e.g., urinary protein levels that are at least about 20%, 40%, 60%, 80%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a healthy individual).

[0468] Dosage and Administration Methods of Anti-VISTA Constructs The dosing regimen (e.g., specific dosage and frequency) of an anti-VISTA construct used to treat a disease or disorder described herein administered to an individual can vary depending on the agent comprising the particular anti-VISTA construct, the mode of administration, and the type of disease or condition being treated. In some embodiments, an effective amount of an agent comprising an anti-VISTA construct is an amount effective to alleviate at least one symptom of a disease or condition. In some embodiments, an effective amount of an agent comprising an anti-VISTA construct is an amount sufficient to extend the overall survival of an individual (e.g., a human). In some embodiments, an effective amount of an agent comprising an anti-VISTA construct is an amount sufficient to provide a clinical benefit in greater than about any of 50%, 60%, 70%, 80%, or 90% among a population of individuals treated with the agent comprising an anti-VISTA construct.

[0469] In some embodiments, an effective amount of an agent comprising an anti-VISTA construct is an amount that slows or inhibits the progression of a disease or condition (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%) compared to an individual not receiving treatment. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an infectious disease. In some embodiments, the disease or condition is psoriasis.

[0470] In some embodiments, an effective amount of an agent comprising an anti-VISTA construct reduces serum levels of antinuclear antibodies by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a reference individual (e.g., an individual having the same disease or condition but not treated with an agent comprising an anti-VISTA construct). In some embodiments, an effective amount of an agent comprising an anti-VISTA construct reduces serum levels of anti-dsDNA 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 condition but not treated with an agent comprising an anti-VISTA construct). In some embodiments, an effective amount of an agent comprising an anti-VISTA construct reduces serum levels of IL-17A by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% as compared to a reference individual (e.g., an individual having the same disease or condition but not being treated with an agent comprising an anti-VISTA construct). In some embodiments, an effective amount of an agent comprising an anti-VISTA construct reduces urinary protein levels by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 90% as compared to a reference individual (e.g., an individual having the same disease or condition but not being treated with an agent comprising an anti-VISTA construct).

[0471] In some embodiments, an effective amount of a drug comprising an anti-VISTA construct is an amount that reduces (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%) the side effects (e.g., autoimmune response) of a condition (e.g., transplant) compared to an individual not receiving the drug.

[0472] In some embodiments of any of the above aspects, the effective amount of the agent comprises an amount of anti-VISTA construct in the range of about 0.001 μg / kg to about 100 mg / kg of total body weight, e.g., about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg.

[0473] In some embodiments of any of the above aspects, the effective amount of the agent in a human is a dose of the agent comprising an anti-VISTA construct that is equivalent to 0.5 mg of the anti-VISTA construct in a mouse.

[0474] In some embodiments of any of the above aspects, the agent comprising the anti-VISTA construct is administered weekly. In some embodiments of any of the above aspects, the agent comprising the anti-VISTA construct is administered every other week. In some embodiments, the agent comprising the anti-VISTA construct is administered weekly for at least about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 weeks.

[0475] Agents comprising anti-VISTA constructs can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, sustained release formulations of the agent can be used. In some embodiments, the agent is administered intravenously. In some embodiments, the agent is administered intraperitoneally. In some embodiments, the agent is administered intravenously. In some embodiments, the agent is administered intraperitoneally. In some embodiments, the agent is administered intramuscularly. In some embodiments, the agent is administered subcutaneously. In some embodiments, the agent is administered intravenously. In some embodiments, the agent is administered orally.

[0476] Combination therapy The present application also provides the use of an anti-VISTA construct in the manufacture of a medicament to be administered to an individual to treat a disease or condition, further comprising administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy to treat the disease or condition. In some embodiments, the disease or condition is psoriasis.

[0477] In some embodiments, the agent comprising an anti-VISTA construct is administered concurrently with a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered simultaneously with a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered sequentially with a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered before a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered after a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered in the same unit dosage form as a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered in a different unit dosage form than a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered in the same unit dosage form as a second agent or therapy. In some embodiments, the agent comprising an anti-VISTA construct is administered in a different unit dosage form than a second agent or therapy. In some embodiments, the second agent or therapy is a VISTA agonist. In some embodiments, the second agent or therapy is a VISTA ligand.

[0478] In some embodiments, the second agent or therapy is a polypeptide. In some embodiments, the polypeptide is VSIG3 or a fragment thereof. VSIG3 (V-Set and immunoglobulin domain-containing 3) is a ligand for VISTA. VSIG3 is known to inhibit T cells through inhibitory function (e.g., Xie et al., Front. Immunol., 12:625808 (2021)). In some embodiments, the polypeptide is a VSIG3-Fc fusion.

[0479] In some embodiments, the second agent or therapy is a small molecule (eg, a small molecule inhibitor).

[0480] VII. Compositions, Kits and Articles of Manufacture Also referred to herein is any one of the anti-VISTA constructs or anti-VISTA antibody portions described herein, a nucleic acid encoding the antibody portion, a vector comprising a nucleic acid encoding the antibody portion, or a host cell comprising the nucleic acid or vector.

[0481] Suitable formulations of the anti-VISTA constructs described herein can be obtained in the form of a lyophilized formulation or aqueous solution by mixing an anti-VISTA construct or anti-VISTA portion having the desired purity with, optionally, a pharmaceutically acceptable carrier, excipient, or stabilizer (see Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or The lyophilized formulation may comprise a protein such as an immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, or other carbohydrate, including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted with an appropriate diluent at a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the individual being imaged, diagnosed, or treated herein.

[0482] Formulations to be used for in vivo administration must be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0483] Also provided are kits comprising any one of the anti-VISTA constructs or anti-VISTA antibody moieties described herein, which may be useful in any of the methods of modulating or treating cellular compositions described herein.

[0484] In some embodiments, kits are provided that include anti-VISTA constructs that specifically bind to VISTA.

[0485] In some embodiments, the kit further comprises a device capable of delivering the anti-VISTA construct into an individual. For example, one type of device for parenteral administration is a syringe used to inject pharmaceutical compositions into the subject's body. In certain applications, an inhalation device can also be used.

[0486] In some embodiments, the kit further comprises a therapeutic agent for treating a disease or condition, eg, an infectious disease, an autoimmune disease, or a transplant.

[0487] The kits of the present application are placed in suitable packaging, including, but not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic pouches), etc. The kits may optionally include additional components, such as buffers and instructional information.

[0488] Accordingly, the present application also provides an article of manufacture. The article of manufacture may include a container and a label or package insert on o...

Claims

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

2. The V Ｈ comprises the amino acid sequence of any one of SEQ ID NOs: 53 to 57, Ｌ The anti-VISTA construct of claim 1, wherein said construct comprises the amino acid sequence of any one of SEQ ID NOs: 58 to 62.

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

61.

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

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

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

43.

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

44.

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

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

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

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

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

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

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

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

16. 16. The anti-VISTA construct of 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 of any one of claims 1 to 13, which is an antagonist antibody of VISTA.

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

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

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

21. A vector comprising the isolated nucleic acid of claim 20.

22. 22. An isolated host cell comprising the isolated nucleic acid of claim 20 or the vector of claim 21.

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

24. 1. A method of producing an anti-VISTA construct, comprising: a) culturing the isolated host cell of claim 22 under conditions effective to express the anti-VISTA construct; b) obtaining the expressed anti-VISTA construct from the host cell.

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

26. 26. The method of claim 25, wherein the disease or condition is associated with dysregulation of the immune system.

27. 27. The method of claim 25 or 26, wherein the disease or condition is associated with activated T cells.

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

29. The method of any one of claims 25 to 28, wherein the disease or condition is associated with VISTA-positive cells.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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