EA043336B1 - ANTI-VISTA ANTIBODIES AND ANTIBODY FRAGMENTS, COMPOSITIONS CONTAINING THEIR AND THEIR APPLICATION FOR STIMULATION OF IMMUNITY AND TREATMENT OF CANCER - Google Patents

Description

Related applications

This application claims priority to Provisional Application No. 61/920695, filed December 2013, and Provisional Application No. 62/085086, filed November 26, 2014. All details of the foregoing applications are incorporated herein by reference.

State of the art

Expression of negative regulators of immune checkpoints by cancer cells or immunocytes in the tumor microenvironment can suppress the host immune response against the tumor. To effectively combat cancer, it is desirable to block tumor-mediated suppression of the host immune response. Accordingly, there is a need for new and effective therapeutic agents that inhibit negative regulators of immune checkpoints in the tumor microenvironment that suppress antitumor immune responses.

The essence of the invention

The present invention relates to antibodies and antibody fragments comprising an antigen binding region that binds to the V domain of suppressor of T cell activation (VISTA) Ig. VISTA is a checkpoint regulator that negatively suppresses the immune response. See Wang et al., VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses, J. Exp. Med., 208(3)577-92 (2011). It is expressed on normal neutrophils, monocytes and a subset of human T cells. In addition, cynomolgus monkey cells express VISTA in a manner similar to normal human cells. VISTA is also expressed in peripheral blood cells of people with cancer. Binding of an antibody or antibody fragment to VISTA modulates or enhances the immune response. The antibody fragment may include, for example, a Fab, F(ab') ₂ or scFv antibody fragment. An antibody or antibody fragment may comprise an antibody constant region. An antibody or antibody fragment can bind to VISTA that is expressed on a hematopoietic cell, e.g., a myeloid cell and/or lymphocyte, monocyte or neutrophil, T cell, natural killer (NK) cells, natural killer T (NKT) cells , tumor cell and/or tumor microenvironment (TME). The tumor microenvironment is the cellular environment of the tumor. This may include nearby immune cells, fibroblasts, blood vessels, other cells, signaling molecules, and the extracellular matrix.

An antibody and antibody fragment may include one or more heavy chain hypervariable regions (CDRs) and/or one or more light chain CDRs including one or more CDRs (e.g., all three heavy chain CDRs, all 3 light chain CDRs, or all 6 CDRs) of any of the anti-VISTA antibodies described in this application, including antibodies designated VSTB112 (S2), VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 (S47). In some embodiments, the antibodies or fragments thereof are selected from the group consisting of VSTB112, VSTB95, VSTB116, VSTB50, VSTB53 and VSTB60. In one embodiment of the present invention, the antibody or fragment includes one or more heavy chain CDRs and one or more light chain CDRs of any of the anti-VISTA antibodies described in the present invention. In some embodiments, the antibody or antibody fragment may further comprise at least one heavy chain and at least one light chain of any of the anti-VISTA antibodies described herein. In some embodiments of the present invention, an antibody or antibody fragment includes at least one heavy chain including a heavy chain variable region sequence and/or at least one light chain including a light chain variable region sequence. In some embodiments of the present invention, the antibody includes a human framework region. In some embodiments of the present invention, the antibody is a bivalent antibody. In some embodiments of the present invention, the fragment is an anti-VISTA binding element. In some embodiments, the antibody heavy chain CDRs are shown in SEQ ID NOs: l, 2, and 3, and the light chain CDRs are shown in SEQ ID NOs: 4, 5, and 6. In some embodiments, the heavy chain variable region amino acid sequences are and light chain are shown in SEQ ID NO: 7 and 8, respectively.

The present invention also includes anti-VISTA antibodies, which are substantially similar to the antibodies described in the present invention. For example, in one embodiment of the invention, the antibody or fragment comprises an antibody VH domain comprising a CDR1 VH having an amino acid sequence that is substantially similar to SEQ ID NO: 1, a CDR2 VH having an amino acid sequence that is substantially similar to SEQ ID NO: : 2, and VH CDR3 having an amino acid sequence that is substantially similar to SEQ ID NO: 3, and which further includes an antibody VL domain comprising CDR1 VL having an amino acid sequence that is substantially similar to SEQ ID NO: 4, CDR2 VL having an amino acid sequence that is substantially similar to SEQ ID NO: 5, and CDR3 VL having an amino acid sequence that is substantially similar to SEQ ID NO: 6.

The present invention also provides anti-VISTA antibodies that competitively inhibit, or compete for binding with, the anti-VISTA antibodies described herein.

In some embodiments of the present invention, the anti-VISTA antibodies are part of a conjugate, for example, a conjugate that includes a cytotoxic molecule or other substance described in the present invention. Such molecules are well known in the art.

In some embodiments of the present invention, the antibody or antibody fragment is a monoclonal antibody. In some embodiments of the present invention, the antibody is a chimeric, humanized, or human antibody. In some embodiments of the present invention, the antibody or antibody fragment includes a human constant region. In some embodiments of the present invention, the antibody or antibody fragment is specific for a VISTA epitope, for example, within the amino acid sequence of SEQ ID NO: 9. In some embodiments of the present invention, the antibody or antibody fragment binds to the VISTA epitope with an affinity of at least ^1x10'7 l/mol, for example at least 1x10'8 l/mol, for example at least 1x10'9 l/mol.

In some embodiments of the present invention, modulation of the immune response includes an increase in CD45+ leukocytes, CD4+ T cells and/or CD8+ T cells. In some embodiments of the present invention, modulation of the immune response includes increased production (eg, by a T cell) of cytokines (eg, IFNy, IL-10, TNFa, IL-17), enhanced T cell response, and/or modulated expression of Foxp3.

The present invention also provides compositions comprising an antibody or antibody fragment described herein (eg, an anti-VISTA antibody) and a pharmaceutically acceptable carrier, diluent or excipient. For example, the composition may include a VISTA antagonist comprising an antibody or fragment of such an antibody including an antigen binding region that binds to VISTA, and a vaccine (such as a viral vector vaccine, a bacterial vaccine, a DNA vaccine, a DNA vaccine, a peptide vaccine). In some embodiments of the present invention, the composition is a pharmaceutical composition and binding of the antibody or antibody fragment to VISTA modulates or enhances the immune response.

The present invention also provides methods for treating or preventing cancer comprising administering to an individual (eg, a mammal, such as a human or non-human animal) in need thereof an effective amount of at least one antibody, antibody fragment, or composition described herein. In some embodiments of the present invention, an antibody or antibody fragment binds to VISTA, thereby modulating or enhancing the immunogenic response to cancer. In some embodiments, the cancer is leukemia, lymphoma, myelodysplastic syndrome, and/or myeloma. In some embodiments, the cancer is any type or type of leukemia, including lymphocytic leukemia or myelogenous leukemia, such as, for example, acute lymphoblastic leukemia (ALL), chonic lymphocytic leukemia (CLL), acute myelocytic leukemia (myelogenous leukemia). AML), chronic granulocytic leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocyte leukemia, or adult T-cell leukemia. In some embodiments, the lymphoma is a histocytic lymphoma, and in some embodiments, the cancer is multiple myeloma. In some embodiments of the present invention, the cancer is a solid tumor, such as melanoma or bladder cancer. In some embodiments of the present invention, the cancer is lung cancer (eg, non-small cell lung carcinoma (NSCLC)). Some treatment methods further include administering a vaccine (such as a viral vector vaccine, a bacterial vaccine, a cell-based vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine, or a protein vaccine). The present invention also provides a method of inhibiting the growth of a tumor in an individual in need thereof, comprising administering an effective antibody or antibody fragment or composition described in the present invention. The composition, antibody or fragment or other pharmaceutical substances (eg, vaccine) can be administered by any parenteral or non-parenteral route, for example, intravenously (IV), subcutaneously (SQ) or orally (PO). In some embodiments of the present invention, the composition, antibody, or fragment is administered every three months, weekly, every two weeks, every three weeks, or every four weeks. In some embodiments of the present invention, other pharmaceutical or therapeutic agents are administered together with, before, during, or after the antibodies, fragments, or compositions described in the present invention. The co-administered substance may be administered by the same route as the antibody, fragment or composition, or by a different route. The present invention also includes methods for producing antibodies, fragments or compositions, for example, a method for producing an antibody or fragment described in the present invention, including culturing host cells under conditions to obtain said antibody. The method may further include isolating the antibody. The present invention

- 2 043336 also relates to nucleic acids, for example, isolated nucleic acids, which include a nucleotide sequence encoding antibodies and fragments, expression vectors including such nucleic acids, for example, operably linked to a promoter, and host cells transformed with such expression vector. The present invention also provides kits and finished products comprising a composition containing an anti-VISTA antibody and a container, and further includes a package insert or label indicating that the composition can be used to treat cancer.

The present invention also provides an isolated antibody or fragment of such an antibody, comprising an antigen binding region that binds to the V domain of a suppressor of T cell activation (VISTA) Ig, wherein the antibody comprises a VH domain of an antibody containing a CDR1 VH having the amino acid sequence of SEQ ID NO: 25, CDR2 VH having the amino acid sequence of SEQ ID NO: 26 and CDR3 VH having the amino acid sequence of SEQ ID NO: 27, and which further includes an antibody VL domain containing CDR1 VL having the amino acid sequence of SEQ ID NO: 28, CDR2 VL, having the amino acid sequence of SEQ ID NO: 29, and a CDR3 VL having the amino acid sequence of SEQ ID NO: 30. In some embodiments of the present invention, the antibody or antibody fragment includes one or more humanized or human framework region. In specific embodiments of the present invention, the antibody or antibody fragment includes an antibody VH domain that contains SEQ ID NO: 37 and/or an antibody VL domain that contains SEQ ID NO: 44. In some embodiments of the present invention, the antibody includes a heavy chain constant region ( eg, a human heavy chain constant region) and/or a light chain constant region (eg, a human light chain constant region, such as the light chain constant region set forth in SEQ ID NO: 56). Preferably, the heavy chain constant region is an IgG1 heavy chain constant region (eg, the IgG1 heavy chain constant region is shown in SEQ ID NO: 61). In a specific embodiment of the present invention, the heavy chain constant region of IgG1 has been altered to enhance protease resistance of the antibody. An example of an IgG1 heavy chain constant region that has been modified to enhance protease resistance is the IgG1 heavy chain constant region set forth in SEQ ID NO: 60. In some embodiments of the present invention, the antibody or antibody fragment includes a heavy chain comprising SEQ ID NO: 60 , and a light chain containing SEQ ID NO: 56; or a heavy chain comprising SEQ ID NO: 61 and a light chain comprising SEQ ID NO: 56. In a particular embodiment of the present invention, the antibody or antibody fragment is expressed in a cell that is deficient in fucosylation enzymes (for example, a Chinese hamster ovary cell ( CHO), which is deficient in fucosylation enzymes).

The present invention also provides a composition comprising an antibody or fragment of an antibody comprising an antibody VH domain comprising a CDR1 VH having the amino acid sequence of SEQ ID NO: 25, a CDR2 VH having the amino acid sequence of SEQ ID NO: 26, and a CDR3 VH, having the amino acid sequence of SEQ ID NO: 27, and which further includes an antibody VL domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 28, a VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 30; and a pharmaceutically acceptable carrier, solvent or excipient.

In another embodiment of the present invention, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody or fragment of an antibody that binds to the V domain of suppressor of T cell activation (VISTA) Ig, wherein the antibody comprises An antibody VH domain comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 25, a VH CDR2 having the amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 27, and which further includes an antibody VL domain, comprising CDR1 VL having the amino acid sequence SEQ ID NO: 28, CDR2 VL having the amino acid sequence SEQ ID NO: 29, and CDR3 VL having the amino acid sequence SEQ ID NO: 30. In a particular embodiment of the present invention, the cancer is lung cancer . In yet another embodiment of the present invention, the lung cancer is non-small cell lung carcinoma (NSCLC). In some embodiments of the present invention, the method further includes administering a second anticancer therapy (eg, surgery, chemotherapy, radiation therapy, biological therapy, immunomodulatory therapy, and combinations thereof).

The present invention also provides an antibody or antibody fragment comprising an antigen binding region that binds to the V domain of suppressor of T cell activation (VISTA) Ig, wherein the antibody binds to a conformational epitope in VISTA (eg, human VISTA). In some embodiments of the present invention, the conformational epitope includes or is present within residues 103-111 (NLLTLLDSGL (SEQ ID NO: 62)) and 136-146

- 3 043336 (VQTGKDAPSNC (SEQ ID NO: 63)) human VISTA (SEQ ID NO: 46). In another embodiment of the present invention, the conformational epitope includes or is present within residues 24-36 (LLGPVDKGHDVTF (SEQ ID NO: 64)), 54-65 (RRPIRNLTFQDL (SEQ ID NO: 65) and 100-102 (TMR) of human VISTA (SEQ ID NO: 46) In yet another embodiment of the present invention, the conformational epitope includes amino acid residues in the FG loop of human VISTA (SEQ ID NO: 46).

In addition, the present invention provides a method of enhancing an immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of antibodies that bind to the V domain of suppressor of T cell activation (VISTA) Ig, or a fragment of these antibodies comprising an antigen binding region , which binds to VISTA, thereby enhancing the immune response to cancer. In a specific embodiment of the present invention, the immune response is an antitumor immune response. In another embodiment of the present invention, the invention provides a method of inducing a biological response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody that binds the V domain of suppressor of T cell activation (VISTA) Ig, or a fragment of that antibody comprising an antigen-binding region that binds to VISTA, thereby enhancing the immune response to cancer. Examples of biological responses include activation of monocytes; induction of T cell proliferation and cytokine secretion; antibody-dependent cell-mediated cytotoxicity (ADCC) of VISTA-expressing cells; and antibody-dependent cellular phagocytosis (ADCP) of VISTA-expressing cells.

Brief description of drawings

The patent or set of application materials contains at least one drawing made in color. Copies of this patent or application set with color drawing will be provided upon request.

In fig. 1A-1C are graphs showing VISTA expression on TF1 AML cells, VISTA protein expression by flow cytometry shown in the TF-1 AML cell line.

In fig. 2A-2E are graphs showing staining and gating strategies for establishing human myeloid and lymphoid subsets.

In fig. 3A-3G are graphs showing VISTA expression on human myeloid and lymphoid subsets from one healthy donor.

In fig. 4 are graphs showing VISTA expression on human myeloid and lymphoid subsets from several healthy normal donors.

In fig. 5A and 5B are graphs showing staining and gating strategy for detecting VISTA expression on human monocytes and macrophages.

In fig. 6A-6C are graphs showing the expression of VISTA on human monocytes and macrophages.

In fig. 7A-7E are graphs showing staining and gating strategy for detecting VISTA expression on human T and NK cell subsets.

In fig. 8A-8G are graphs showing VISTA expression on human T and NK cell subsets from one healthy normal donor.

In fig. 9 is a graph showing VTSTA expression on human T and NK cell subsets from several normal donors.

In fig. 10A-10D are graphs showing staining and gating strategy for detecting VISTA expression on subpopulations of human dendritic cells.

In fig. 11A-11C are graphs showing VISTA expression on subpopulations of human dendritic cells and basophils from one healthy donor.

In fig. 12 are graphs showing VISTA expression on subpopulations of human dendritic cells and basophils from several healthy normal donors.

In fig. 13A-13D show analysis of VISTA expression on healthy human peripheral blood cells. VISTA expression profile on healthy human peripheral blood cells using multicolor flow cytometry analysis: Whole blood samples from 2 different individuals were analyzed for VISTA expression on (Fig. 13A) monocytes SSC ^lo CD11b ^h, CD14 ^{h, CD16've} CD33 ^+ve HLA -DR ^+ve CD19' ^ve ) (Fig. 13B) neutrophils (SSC ^h, CD177 ⁺ CD11b ^h, CD14 ^lo CD16 ^+ve CD33 ^+ve HLA-BR' ^ve CD19' ^ve ). Peripheral blood mononuclear cells were isolated using a Ficoll gradient to analyze (FIG. 13C) CD4+ T cells (CD3 ^+ve CD4 ^+ve ) and (FIG. 13B) CD8+ T cells (CD3 ^+ve CD8 ^+ve ).

In fig. 14A-14C show analysis of VISTA expression on peripheral blood cells from a lung cancer patient and a healthy control donor. Expression profile of VISTA on peripheral blood cells of a lung cancer patient using multicolor flow cytometry analysis: FACS representative plot (FIG. 14A) is from one individual. Peripheral blood mononuclear cells were isolated using Ficoll and analyzed for VISTA expression on (Fig. 14B) monocytes (CD14 ⁺ CD11b ⁺ CD33 ⁺ HLABR + CD15 ^- ) and (Fig. 14C) myelo

- 4 043336 ide suppressor cells (CD14- CD11b ⁺ CD33-HLABR-CD15+ CD146+).

In fig. 15A-15C show the expression profile of VISTA in peripheral blood cells from a patient with colon cancer using multicolor flow cytometry analysis: a representative FACS plot (FIG. 15A) is shown from one individual. Peripheral blood mononuclear cells were isolated using Ficoll and analyzed for VISTA expression on (Fig. 15B) monocytes (CD14 ⁺ CD11b ⁺ CB33 ⁺ HLABR+ CD145 ^- ) and (Fig. 15C) myeloid suppressor cells (CD14' CD11b ⁺ CD33-HLABR-CD15+ CD146+).

In fig. 16A, 16B show the expression profile of VISTA on cynomolgus monkey peripheral blood cells using multicolor flow cytometry analysis: whole blood from 4 different macaques was examined for VISTA expression on (Fig. 16A) monocytes (SSC ^lo CD11b ^hl CD14 ^hl HLABR ^hl CD16 ^ve CD19 ^ve and (Fig. 16B) neutrophils CD11b ^hl CD14 ^lo HLA-DR' ^ve CD16' ^ve CD19' ^ve Peripheral blood mononuclear cells from three macaques were isolated using a Ficoll gradient for analysis (Fig. 16C) of CD4+ T cells (TCRa/ e ^+ve CD4 ^+ve ) and (Fig. 16D) CD8+ T cells (TCRa/e ^+ve CD8 ^+ve ).

In fig. 17 is a graph showing the absolute values of VISTA RNA expression in Heme cell lines.

In fig. 18 shows mouse A20 cells that are stably transfected with either GFP or human VISTA. They were incubated with ovalbumin peptide and DO11.10 T cells. CD25 expression by T cells was measured 24 h after the start of incubation. A20-huVISTA cells suppress CD25 expression by T cells, but this readout is significantly restored by incubation with VSTB95.

In fig. 19A-19F are graphs showing the results of the human VISTA ELISA.

In fig. 20A-20F show FACS results of human VISTA showing binding of anti-VISTA antibodies to cells expressing human VISTA.

In fig. 21A-21D show a study of dilution of 6 candidate anti-VISTA antibodies in a mixed lymphocyte culture reaction from 30 mg/ml to 0.0 μg/ml.

In fig. 22A and 22B show dilution studies of 6 candidate anti-VISTA antibodies in the SEB assay (single CPM pulses and IFN-g concentrations) from 30 to 0.0 μg/ml.

In fig. 23 shows a sensogram of a raft using VSTB85 anti-VISTA antibody coated on a Proteon SPR chip and VISTA protein with the indicated competitors running on the chip (competitors are listed in the table).

In fig. 24 shows the experimental design for the MB49 bladder tumor mouse model.

In fig. 25A and 25B show MB49 tumor growth in female C57B1/6 mice. Graphs show tumor growth in individual mice treated with anti-mouse VISTA antibodies (FIG. 25B) or control IgG (FIG. 25A). In fig. 26 shows the amino acid sequence of human VISTA (SEQ ID NO: 46).

In fig. Figure 27 shows a multiple sequence alignment of orthologous VISTA.

In fig. 28 shows regions of human VTSTA bound by VSTB50 and VSTB60 antibodies (top) or VSTB95 and VSTB112 antibodies (bottom) as determined by HDX.

In fig. 29 shows the VISTA epitope associated with VSTB112. VISTA is shown as a cartoon with the threads labeled (above). Residues having at least one atom within 5 A of VSTB112 in the complex are colored blue. Blue and orange regions indicate strand breakage, and blue and green regions indicate the N- and C-termini of the VISTA structure, respectively. The VISTA design sequence is applied in the structure definition (below). Circles below the sequence were used to denote residues that constitute only the backbone contacting VSTB112, triangles denote a contact on the side chain, and squares denote a contact on the side chain resulting in either a hydrogen bond or ionic bond interaction as calculated by PISA . Regions are colored to indicate the CDR having the highest number of atoms linking a given residue to the CDR, colors indicated in FIG. 29. Secondary structural elements as labeled in the MOE program, with yellow arrows indicating β-strands and red boxes indicating α-helices.

In fig. 30 paratope VSTB112 is presented. The VISTA antigen is shown in the illustration and VSTB112 VISTA within 5 angstroms (A) is shown on the surface with the colors used to indicate CDR identity according to the sequence below (top). Contacting frame residues adjacent to the CDR are colored similarly to the corresponding CDR sequence (bottom) of the VSTB112 Fv region. The painted background was specified by CDRs according to Kabat definitions. Circles below the sequence were used to denote the residues that make up the backbone contacting VISTA, triangles denote a contact on the side chain, and squares denote a contact on the side chain resulting in either a hydrogen bond or an ionic bond interaction as calculated by PISA.

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In fig. 31 shows a comparison of epitope regions determined by crystallography and hydrogen-deuterium exchange (HDX). The VISTA construct sequence was used in structure determination. Circles below the sequence were used to denote residues that constituted only the backbone contacting VSTB112, triangles denote a side chain contact, and squares denote a side chain contact resulting in either a hydrogen bond or an ionic bond interaction as calculated by PISA.

In fig. 32 shows activation of CD14+ monocytes in whole PBMCs by VSTB174 (derived from VSTB112). For each part of the experiment, cells were incubated with PBS, control IgG1 antibody, or VSTB174 at 1, 0.1, or 0.01 μg/ml. The left panel shows the CD80 MFI; the right panel shows HLA-DR MFI (studies from two donors with significant results are shown).

In fig. 33 are graphs showing the ADCC activity of VSTB174 directed against K562-VISTA cells.

In fig. 34 are graphs showing the ADCP activity of VSTB174 against K562-VISTA cells. Both antibodies shown have the same Fab, but VSTB174 has an IgG1 Fc and VSTB140 has a silent IgG2 Fc.

In fig. 35 is a graph showing phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAbs against K562-VISTA. Each mAb was tested with 7 half-log doses ranging from 0.0008 to 0.56 μg/ml.

In fig. 36 is a graph showing phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAbs against K562 cells of a myeloma cell line. Each mAb was tested with 7 half-log doses ranging from 0.0008 to 0.56 μg/ml.

In fig. 37 presents a study of the effectiveness of MB49 tumor assessed by VSTB123 1, 5, 7.5 and 10 mg/kg in VISTA-KI female mice. Tumor volumes were approximately 50 mm ³ when dosing began on day 6 postimplantation. VSTB123 is a VSTB112 Fab transplanted into a mouse Fc backbone and bound to human VISTA in VISTA-KI mice.

In fig. 38 is a graph showing that CD14+ cells expressing high/intermediate levels of VISTA were found in 13/13 lung cancer samples, as well as resected lung tissue and peripheral blood from patients.

In fig. 39 shows IHC staining for VISTA in lung cancer using GG8.

Detailed Description of the Invention

The following is a description of exemplary embodiments of the present invention.

The present invention relates to antibodies to a new immunoglobulin family ligand targeting the immunoglobulin V domain suppressor of T cell activation (VISTA) (Genbank: JN602184) (Wang et al., 2010, 2011). VISTA has homology to PD-L1 but displays a unique gene expression profile that is restricted to the hematopoietic compartment. In particular, VISTA is constitutively and highly expressed on CD11b ^hlgh myeloid cells, and expressed at lower levels on CD4+ and CD8+ T cells. The human homologues share approximately 85% homology with mouse VISTAs and have a similar gene expression profile (Lines et al., Cancer Research 74:1924, 2014). VISTA is expressed on antigen presenting cells (APCs), suppressing CD4 ⁺ and CD8 ⁺ T cell proliferation and cytokine production through its cognate receptor independently of PD-1. In the passive EAE (experimental autoimmune encephalomyelitis) disease model, VISTA specific monoclonal antibodies enhance T cell-dependent immune responses and exacerbate disease. VISTA is overexpressed on tumor cells with impaired protective antitumor immunity in tumor-bearing hosts. Studies with human VISTA have confirmed its suppressive function on human T cells (Lines et al., Cancer Research, 74:1924, 2014, Studies from Flies et al. also identified VISTA (named PD-1H) as a potent immune suppressive molecule (Flies et al., 2011).VISTA is described in more detail in 20130177557 A1 and US Patent Nos. 7,919,585 and 8,236,304, all of which are incorporated herein by reference in their entirety.

VISTA is a novel negative checkpoint regulator that suppresses immune responses. As described in Example 12 of this application, treatment with VISTA-specific monoclonal antibodies in mouse tumor models showed a complete reversal of the suppressive nature of the tumor immune microenvironment and enhancement of protective antitumor immunity, thereby demonstrating the potential of the VISTA monoclonal antibody as a new therapeutic agent for cancer immunotherapy.

Antibodies and fragments.

The term antibody means polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, human antibodies and anti-idiotypic (anti-Id) antibodies, as well as fragments, regions and derivatives thereof, prepared by any known method, such as, but not limited to , enzymatic digestion, peptide synthesis or recombinant methods. The anti-VISTA antibodies of the present invention are capable of binding VISTA regions that modulate, regulate or enhance the immune response. In some embodiments

- 6 043336 antibodies of the present invention competitively inhibit one or more anti-VISTA antibodies described in the present description. Methods for determining whether two or more antibodies compete for binding to the same target are known in the art. For example, a competition binding assay can be used to determine whether one antibody blocks the binding of another antibody to a target. Typically, a competition binding assay involves the use of a purified target antigen (eg, PD-1) bound to either a solid substrate or cells, an untagged binding molecule of interest, and a labeled reference binding molecule. Competitive binding is measured by the amount of binding marks to a solid substrate or cells in the presence of the binding molecule of interest. Typically, the binding molecule of interest is present in excess. Typically, when a competing binding molecule is present in excess, it will inhibit the specific binding of a reference binding molecule to a heterogeneous antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. In some embodiments of the present invention, competitive binding is determined using an ELISA competitive inhibition assay. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of animals immunized with an antigen. Monoclonal antibodies contain an essentially homogeneous population of antibodies specific for antigens, where the population contains essentially similar epitope binding sites. Monoclonal antibodies can be produced by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256:495-497 (1975); US Patent No. 4376110; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); and Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory (1988); Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), the entire contents of which are incorporated herein by reference in their entirety. Such antibodies can be of any class of immunoglobulins, including IgG, IgM, IgE, IgA, GILD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the present invention can be cultured in vitro, in situ or in vivo. The present invention also includes fragments, specific regions and variants thereof, including antibody mimetics or comprising antibody regions that copy the structure and/or function of an antibody or specific or region thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to the mammalian VISTA protein. For example, antibody fragments capable of binding to VISTA or regions thereof, including, but not limited to, Fab (eg, by papain cleavage), Fab' (eg, by pepsin cleavage and incomplete reduction), and F(ab') ₂ (eg , by pepsin cleavage), facb (eg, by plasmin cleavage), pFc' (eg, by pepsin or plasmin cleavage), Fd (eg, by pepsin cleavage, incomplete recovery and reaggregation), Fv or scFv (eg, by molecular -biological methods) fragments are included in the present invention (see, for example, Colligan, Immunology, supra). Antibody fragments of the present invention also include those described in Aaron L., Nelson, mAbs 2:1, 77-83 (Jan/Feb 2010), the contents of which are incorporated by reference in their entirety. Such fragments can be obtained by, for example, enzymatic digestion, synthetic or recombinant methods, as known in the art and/or as described in this application. Antibodies can also be produced in various processed forms using antibody genes in which one or more stop codons have been introduced before the natural stop region. For example, a combination of genes encoding the heavy chain F(ab')2 region may be designed to include DNA sequences encoding the CH1 domain and/or the heavy chain hinge. Different regions of antibodies can be linked together chemically using conventional methods or can be produced as a contiguous protein using genetic engineering techniques.

In one embodiment of the present invention, the amino acid sequence of the immunoglobulin chain or portion thereof (e.g., variable region, CDR) has approximately 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value thereof) to the amino acid sequence of the corresponding variable chain sequence described in the present invention. Preferably, 70-100% identical amino acids (e.g., 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value thereof) are determined using a suitable computer algorithm, as is known in the art.

Examples of heavy chain and light chain variable region sequences are provided herein.

Antibodies of the present invention, or specific variants thereof, may include any number of nonessential amino acid residues from an antibody of the present invention, where the amount is selected from the group of integers consisting of 10-100% of the number of nonessential amino acid residues in the anti-TNF antibody. Optionally, this subsequence of nonessential amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,

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210, 220, 230, 240, 250 or more amino acids in a chain or any range or value thereof. Moreover, the number of such subsequences may be any integer selected from the group consisting of 1 to 20, such as at least 2, 3, 4, or 5.

As will be appreciated by those skilled in the art, the present invention includes at least one biologically active antibody of the present invention. A biologically active antibody has a specific activity of at least 20, 30 or 40%, and preferably at least 50, 60, or 70%, and most preferably at least 80, 90 or 95-100% of native (not synthetic), endogenous or related and known antibody. Methods for examining and quantifying enzymatic activity and substrate specificity are well known to those skilled in the art.

Substantial similarity refers to a compound having at least 85% (e.g., at least 95%) identity and at least 85% (e.g., at least 95%) activity from native (non-synthetic), endogenous, or related and known antibodies.

As used herein, the term human antibody refers to an antibody in which substantially each portion of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantially immunogenic in humans, with only minor changes or sequence variations. Similarly, antibodies of primates (monkey, baboon, chimpanzee, etc.), rodents (mice, rats, etc.) and other mammals are designated as such species, subclass, class, subfamily, family of specific antibodies. In addition, chimeric antibodies may include any combination of the above. Such changes or variants are optional and preferably maintain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, a human antibody is different from a chimeric or humanized antibody. This indicates that the human antibody can be produced by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin genes (eg, heavy chain and/or light chain). In addition, when the human antibody is a single chain antibody, it may contain a linker peptide that is not present in native human antibodies. For example, Fv may contain a linker peptide, such as two to about eight glycine or other amino acid residues, that connects the heavy chain variable region and the light chain variable region. Such linker peptides are considered to be of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies may also be used, which are monoclonal, preferably human or humanized, antibodies that have binding specificity for at least two different antigens. In this case, one of the binding specificities will be for at least one VISTA protein, and the other will be for any other antigen. Methods for producing bispecific antibodies are known in the art.

Recombinant production of bispecific antibodies can be based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537 (1983)). See also WO 93/08829, US Patent Nos. 6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902, 5989530, 5959084, 5959083, 5932448, 583 3985, 5821333, 5807706, 5643759, 5601819, 5582996, 5496549, 4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J., 10:3655 (1991), Suresh et al., Methods in Enzymology, 121:210 (1986), each of which are incorporated herein description by reference in their entirety.

In one embodiment of the present invention, the invention relates to bispecific antibodies targeting VISTA and a second target protein (eg, immune checkpoint protein). Exemplary bispecific antibodies include a bispecific antibody targeting VISTA and PD-L1 and a bispecific antibody targeting VISTA and PD-L2.

Human antibodies that are specific for human VISTA proteins or fragments thereof can be raised against a corresponding immunogenic antigen, such as a VISTA protein or a portion thereof (including synthetic molecules such as synthetic peptides).

Other mammalian specific or general antibodies can be induced in a similar manner. The preparation of immunogenic antigens and the production of monoclonal antibodies can be carried out using any suitable method.

For example, a hybridoma is prepared by fusing a suitable immortalized cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243 , P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or the like, or heteromyeloma, fusion products thereof, or any cells or cell fusion derived therefrom, or any other suitable cell line known in the art, See, for example, www .atcc.org, with antibody-producing cells Antibody-producing cells may include isolation or cloning of spleen, peripheral blood, lymph, tonsil, or other immune cells

- 8 043336 (eg B cells), or any other cells expressing constant or variable or framework or hypervariable region (CDR) heavy or light chain sequences. Such antibody-producing cells may be recombinant or endogenous cells and may also be prokaryotic or eukaryotic (eg, mammals such as rodents, horses, sheep, goats, rams, primates). See, for example, Ausubel, supra, and Colligan, Immunology, supra, chapter 2, incorporated herein by reference in its entirety. Antibody-producing cells can also be obtained from peripheral blood, or preferably from the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell may also be used to express heterologous or endogenous nucleic acid encoding an antibody, specific fragment or variants thereof, of the present invention. Fusion cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting or other known methods. Cells that produce antibodies with the desired specificity can be selected using a suitable assay (eg, enzyme-linked immunosorbent assay (ELISA)). Other suitable methods for producing or isolating antibodies with the desired specificity may be used, including, but not limited to, methods that are used to select a recombinant antibody from a library of peptides or proteins (e.g., but not limited to, bacteriophage, ribosome, oligonucleotide, RNA, cDNA or the like, display library; for example, as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK; Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, Calif.; Ixsys. See, for example, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; PCT/ GB94/01422; PCT/GB94/02662; PCT/GB97/01835; WO 90/14443; WO 90/14424; WO 90/14430; PCT/U594/1234; WO 92/18619; WO 96/07754; EP 614989; WO 95/16027; WO 88/06630; WO 90/3809; US patent No. 4704692; PCT/US91/02989; WO 89/06283; EP 371998; EP 550400; EP 229046; PCT/US91/07149; or randomly obtained peptides or proteins: US patents No. 5723323; 5763192; 5814476; 5817483; 5824514; 5976862; WO 86/05803, EP 590689, each of which is incorporated herein by reference in its entirety, or which depend on immunization of transgenic animals (eg, SCID mice, Nguyen et al., Microbiol. Immunol., 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol., 16:95-118 (1996); Eren et al., Immunol., 93:154-161 (1998), each of which is incorporated herein by reference in its entirety, and also reference patents and applications) that are capable of producing a panel of human antibodies as known in the art and/or as described herein. Such methods include, but are not limited to, ribosomal display (Hanes et al., Proc. Natl. Acad. Sci., USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. ., USA, 95:14130-14135 (November 1998)); methods for producing antibodies from a single cell (US Patent No. 5627052, Wen et al., J. Immunol., 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci., USA, 93:7843 -7848 (1996)); gel microplates and flow cytometry (Powell et al., Biotechnol., 8:333-337 (1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth., 182:155-163 (1995); Kenny et al., Bio/Technol., 13:787-790 (1995)); selection of B cells (Steenbakkers et al., Molec. Biol. Reports, 19:125-134 (1994); Jonak et al., Progress Biotech, vol. 5, in vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies can also be used and are known in the art. Typically, a humanized or engineered antibody has one or more amino acid residues from a source that is non-human, such as, but not limited to, mouse, rat, rabbit, nonhuman primate, or other mammal. These human amino acid residues are often referred to as imported residues, which are typically taken from an imported variable, constant, or other domain of a known human sequence. Known human Ig sequences are disclosed, for example, www.ncbi.nlm.nih.gov/entrez/query.fcgi;www.atcc.org/phage/hdb.html, each of which is incorporated by reference in its entirety. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or change binding, affinity, avidity, specificity, half-life or other relevant characteristic known in the art. Typically, part or all of the non-human or human CDR sequences are retained, with part or all of the framework and/or constant regions of the non-human sequences replaced by human or other amino acids. Antibodies can also, if desired, be humanized to maintain high affinity for the antigen and other favorable biological properties, using three-dimensional immunoglobulin models that are known to those skilled in the art. Computer programs exist that illustrate and show possible three-dimensional conformational structures of selected immunoglobulin sequence variants. Examination of these mappings allows analysis of the likely roles of residues in the functioning of a candidate immunoglobulin sequence, i.e. analysis of residues that affect the ability of a candidate immunoglobulin to bind to

- 9 043336 antigen. Thus, framework (FR) residues can be selected and combined from consensus or import sequences to achieve desired antibody characteristics, such as increased affinity for a target antigen. In general, CDR residues are directly and most significantly involved in influencing antigen binding. Humanization or engineering of the antibodies of the present invention can be accomplished using any known method, such as, but not limited to, those described in, for example, Winter (Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); Verhoeyen et al., Science, 239:1534 (1988)); Sims et al., J. Immunol., 151:2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci., USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993); US patents No. 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539, 4816567, each are incorporated herein by reference in their entirety, including references indicated in them.

The anti-VISTA antibody may also, if desired, be produced by immunizing a transgenic animal (e.g., mouse, rat, rabbit, hamster, nonhuman primate, and the like) capable of producing a panel of human antibodies as described herein and/or known from the art. this level of technology. Cells that produce human anti-VISTA antibody can be isolated from such animals and immortalized using suitable methods, such as those described herein.

Transgenic animals that produce a variety of human antibodies that bind to human antigens can be produced by known methods (for example, but not limited to, US patent No. 5770428, 5569825, 5545806, 5625126, 5625825, 5633425, 5661016 and 5789650 issued to Lonberg et al., Jakobovits et al., WO 98/50433, Jakobovits et al., WO 98/24893, Lonberg et al., WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al., WO 94/ 25585, Kucherlapate et al., WO 96/34096, Kucherlapate et al., EP 0463151 B1, Kucherlapate et al., EP 0710719 A1, Surani et al., U.S., pat. No. 5545807, Bruggemann et al., WO 90/ 04036, Bruggemann et al., EP 0438474 B1, Lonberg et al., EP 0814259 A2, Lonberg et al., GB 2272440 A, Lonberg et al., Nature, 368:856-859 (1994), Taylor et al., Int. Immunol., 6(4)579-591 (1994), Green et al., Nature Genetics, 7:13-21 (1994), Mendez et al., Nature Genetics, 15:146-156 (1997), Taylor et al., Nucleic Acids Research, 20(23):6287-6295 (1992), Tuaillon et al., Proc. Natl. Acad. Sci., USA, 90(8)3720-3724 (1993), Lonberg et al., Int. Rev. Immunol., 13(1):65-93 (1995) and Fishwald et al., Nat. Biotechnol., 14(7):845-851 (1996), each of which is incorporated herein by reference in its entirety). Typically, these mice include at least one transgene comprising DNA from at least one human immunoglobulin locus that is, or can be, functionally rearranged. Endogenous immunoglobulin loci in such a mouse may be disrupted or deleted to eliminate the animal's ability to produce antibodies encoded by endogenous genes.

Screening antibodies for specific binding to similar proteins or fragments can be easily achieved using peptide library display. This method involves screening large collections of peptides for individual members having the desired function or structure. Antibody screening of displayed peptide display libraries is known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, often from 5-100 amino acids in length, and often from about 8 to 25 amino acids in length. In addition to direct chemical synthetic methods for preparing peptide libraries, several methods with recombinant DNA have been described. One type involves a displayed peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains a nucleotide sequence that encodes specific displayed peptide sequences. Such methods are described in PCT Patent Publications No. 91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis and recombinant methods. See PCT Patent Publications No. 92/05258, 92/14843 and 96/19256. See also US Pat. Nos. 5,658,754 and 5,643,768. Peptide library display, vector, and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, Calif.) and Cambridge antibody Technologies (Cambridgeshire, UK). See, for example, US patents No. 4704692, 4939666, 4946778, 5260203, 5455030, 5518889, 5534621, 5656730, 5763733, 5767260, 5856456, 5223409, 5403 484, 5571698, 5837500, assigned to Dyax, 5427908, 5580717, 5885793, assigned to Cambridge antibody Technologies; 5750373, assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493 and 5698417.

The antibodies of the present invention can also be produced by using at least one anti-VISTA antibody encoding a nucleic acid to produce transgenic animals, such as goats, cows, sheep, etc., that produce such antibodies in their milk. Such animals can be obtained using known methods. See, for example, but not limited to, US Pat. No. 5,827,690; 5849992; 4873316; 5849992; 5994616; 5565362; 5,304,489, and the like, each of which is incorporated herein by reference.

The anti-VISTA antibodies of the present invention can be produced using transgenic plants according to known methods. See also, for example, Fischer et al., Biotechnol. Appl. Biochem., 30:99-108 (October, 1999); Cramer et al., Curr. Top. Microbol. Immunol., 240:95-118 (1999); and the links provided therein; Ma et al., Trends Biotechnol., 13:522-7 (1995); Ma et al., Plant Physiol., 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans., 22:940-944 (1994); and the links provided therein. Each of the above references is incorporated herein by reference in its entirety.

The antibodies of the present invention can bind to human VISTAs with varying affinities (K _D ). In a preferred embodiment of the present invention, at least one monoclonal antibody of the present invention may further bind to human VISTA with high affinity. For example, a human monoclonal antibody may bind to human VISTA with a K _D equal to or less than about 10 ^-7 M, such as, but not limited to, 0.1-9.9 (or any range or value therein) x 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 - ¹¹ , 10 ^-12 , 10 ^-13 or any range or value therein. In some embodiments of the present invention, an antibody or antibody fragment may bind to human VISTA with an affinity of at least 1x10 ^-7 L/mol, such as at least 1x10 ^-8 L/mol, such as at least 1x10 ^-9 L/mol.

The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York , NY (1992); and the methods described in the present invention). The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions (eg, salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (eg, _KD , _Ka , Kd) are preferably performed with standard antibody and antigen solutions and a standardized buffer.

Nucleic acid molecules.

When using the information provided in the present description, such nucleotide sequences encoding at least 70-100% of the nonessential amino acids of at least one of these fragments, variants or consensus sequences thereof or a introduced vector containing at least one of these sequences, a molecule nucleotide acid of the present invention encoding at least one anti-VISTA antibody comprising all heavy chain variable regions CDR SEQ ID NO: 1, 2 and 3 and/or all light chain variable regions CDR SEQ ID NO: 4, 5 and 6 can be obtained using the methods described in the present invention or known from the prior art.

The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA, obtained by cloning or manufactured synthetically, or any their combinations. DNA can be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand, also known as a non-transcribed strand, or it may be a non-coding strand, also known as an anti-sense strand.

Isolated nucleic acid molecules of the present invention may include nucleic acid molecules containing an open reading frame (ORF), for example, but not limited to, at least one specified portion of at least one CDR, such as CDR1, CDR2 and/or CDR3 of at least one heavy chain or light chain; a nucleic acid molecule containing a coding sequence for an anti-VISTA antibody or fragment, for example, a fragment containing a variable region; and nucleic acid molecules that include a nucleotide sequence different from those described above, but which, due to the degeneracy of the genetic code, still encodes at least one anti-VISTA antibody, as described herein and/or known in the art. It is common for those skilled in the art to obtain such degenerate nucleic acid variants that encode the specific anti-VISTA antibodies of the present invention. See, for example, Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.

As indicated herein, the nucleic acid molecules of the present invention, which includes a nucleic acid encoding an anti-VISTA antibody, may include, but is not limited to, an encoding amino acid sequence of an antibody fragment; the coding sequence of the whole antibody or part thereof; the coding sequence of the antibody, fragment or portion, as well as additional sequences, such as sequences encoding at least one leader signal or fusion peptide, with or without the above additional coding sequences, such as at least one intron, together with additional, non-coding sequences including, but not limited to, 5' and 3' non-coding sequences, such as transcribed, non-transcribed sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (eg, ribosome binding and mRNA stability); additional coding

- 11 043336 sequence that encodes additional amino acids, such that provide additional functionality. Thus, the antibody coding sequence can be fused to a marker sequence, such as a peptide coding sequence, which facilitates purification of the fusion antibodies containing the antibody fragment or portion. Human genes that encode constant (C) regions of antibodies, fragments or regions of the present invention can be obtained from a human fetal liver library using known methods. The C regions of human genes can be obtained from any human cells, including those that express or produce human immunoglobulins. The human CH region can be derived from any known class or isotype of human H chains, including γ, μ, α, δ or ε and their subtypes such as G1, G2, G3 and G4. Because the H chain isotype is responsible for various antibody effector functions, the choice of CH region will be determined by the desired effector functions, such as complement fixation or antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Compositions.

The pharmaceutical compositions disclosed herein are prepared in accordance with standard techniques and are administered in dosages that are selected to treat, for example reduce, prevent or eliminate, or slow or stop the progression of, the condition being treated (See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y., the contents of which are incorporated herein by reference for a general description of methods of administration of various substances for human therapy). Compositions containing the antibodies and substances described herein can be delivered using controlled or sustained release delivery systems (eg, capsules, biodegradable matrices). Examples of sustained release delivery systems for drug delivery that are suitable for administering compositions of the claimed compounds are described in, for example, US Pat. No. US 5,990,092; 5039660; 4452775; and 3,854,480, the entire contents of which are incorporated herein by reference. For the preparation of pharmaceutical compositions from the anti-VISTA antibodies and/or fragments of the present invention, pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, wafers, suppositories and dispersible granules. For example, the compounds of the present invention may be in powder form for reconstitution upon delivery. The solid carrier may be one or more substances which may also act as solvents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, disintegrating agents, or encapsulating material. In powders, the carrier is a finely divided solid that is mixed with the finely divided active substance.

Powders and tablets preferably contain from about one to about seventy percent active ingredient. Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter and the like. Tablets, powders, wafers, lozenges, melting strips, capsules and pills can be used as solid dosage forms containing the active substance suitable for oral administration.

Preparations in liquid dosage form include solutions, suspensions, retention enemas, and emulsions, such as aqueous or water-propylene glycol solutions. For parenteral administration, liquid preparations can be prepared as a solution in an aqueous propylene glycol solution.

The pharmaceutical composition may be in unit dosage form. In this form, the composition is divided into unit doses containing appropriate quantities of the active substance. The unit dosage form may be a packaged preparation, the package containing discrete quantities of unit doses. Dosages may vary depending on the needs of the patient, the severity of the condition being treated, the compound and the route of administration used. Determining the correct dose for a particular situation is known to one skilled in the art.

Also, the pharmaceutical composition may optionally contain other compatible substances, for example pharmaceutical, therapeutic or prophylactic substances. Therapeutic or prophylactic substances include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic compound molecules and organic compound molecules. Examples of classes of such substances (eg, antineoplastic agents) include, but are not limited to, cytotoxins, angiogenesis inhibitors, immunomodulatory substances, immuno-oncology agents, and substances used to relieve pain or to offset the harmful effects of one or more therapeutic substances (eg, a bisphosphonate used to reduce the effects of glucocorticoid hypercalcemia). Angiogenesis inhibitors, substances and therapies that are suitable for use in the compositions and methods described herein include, but

- 12 043336 are not limited to them, angiostatin (plasminogen fragment); antiangiogenic antithrombin III; angiozyme. Bisphosphonates include, but are not limited to, alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate and zoledronate.

Immunomodulatory agents and therapies that are suitable for use in the compositions and methods described herein include, but are not limited to, anti-T cell receptor antibodies, such as anti-CD3 antibodies (eg Nuvion (Protein Design Labs), OKT3 (Johnson & Johnson), or anti-CD20 antibodies Rituxan (IDEC)), anti-CD52 antibodies (eg Campath 1H (Ilex)), anti-CD11a antibodies (eg Xanelim (Genentech)); anti-cytokine or anti-cytokine receptor antibodies and antagonists such as anti-IL-2 receptor antibodies (Zenapax (Protein Design Labs)), anti-IL-6 receptor antibodies (eg MRA (Chugai)), and anti-IL-6 receptor antibodies 12 antibodies (CNTO1275 (Janssen)), anti-TNFa antibodies (Remicade (Janssen)) or TNF receptor antagonist (Enbrel (Immunex)), anti-III-6 antibodies (BE8 (Diaclone) and siltuximab (CNTO32 (Centocor)), and antibodies , which immunospecifically bind to tumor-associated antigens (eg, trastuzimab (Genentech)).

Immuno-oncology agents that are suitable for use in the compositions and methods described in the present invention include, but are not limited to, ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1) , αntu-PD-L1 antibodies and anti-LAG-3 antibodies. The composition is preferably in the form of a dosage unit containing a therapeutically effective amount of the antibody or fragment. Examples of dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules may contain, in addition to the active substance, conventional carriers such as binders, for example gum acacia, gelatin, polyvinylpyrrolidone, sorbitol or tragacanth; fillers, for example calcium phosphate, glycine, lactose, corn starch, sorbitol or sucrose; lubricants, such as magnesium stearate, polyethylene glycol, silicon dioxide or talc; disintegrating agents, for example potato starch, flavoring or coloring agents, or suitable wetting agents. Liquid preparations for oral administration, typically in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs, may contain conventional additives such as suspending agents, emulsifying agents, anhydrous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include gum acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fat, lecithin, methylcellulose, methyl or propyl parahydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

Other general details regarding methods for preparing and using the compounds and compositions described in the present invention are well known in the art. See, for example, US Pat. No. 7,820,169, the contents of which are incorporated in their entirety.

Methods of treatment.

Any person skilled in the art, such as a clinician, can determine the appropriate dosage and route of administration for a particular antibody, fragment or composition to be administered to an individual, taking into account the selected agents, dosage form and route of administration, various patient characteristics and other factors. Preferably, the dose produces no or minimal or no harmful side effects. In standard multiple dosage regimens, the pharmaceutical agent may be administered in a dosage regimen that is designed to maintain a predetermined or optimal plasma concentration in the subject being treated. Antibodies, fragments and compositions can be added in any suitable dosage range or therapeutically effective amount, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 30, 40, 50, 60, 70, 80, 90 and 10 0 mg/kg . In one embodiment of the present invention, the dose of the composition, antibody or fragment administered is 0.1-15 mg/kg per administration.

The antibody or fragment may be administered once, at least once, twice, at least twice, three times, or at least three times a day. The antibody or fragment may be administered once, at least once, twice, at least twice, three times, at least three times, four times, at least four times, five times, at least five times , six times a week or at least six times a week. The antibody or fragment may be administered once a month, at least once a month, twice a month, at least twice a month, three times a month, or at least three times a month. The antibody or antibody fragment may be administered once a year, at least once a year, twice a year, at least twice a year, three times a year, at least three times a year, four times a year , at least four times a year, five times a year, at least five times a year, six times a year or at least six times a year. Anti-VISTA antibodies, fragments or compositions may, for example, be administered via parenteral or non-parenteral routes, including, but not limited to, intravenous, subcutaneous, oral, rectal, intramuscular, intraperitoneal, transmucosal, intradermal, subarachnoid, nasal or externally. It will be apparent to one of ordinary skill in the art that the following dosage forms may contain as active substance either a compound or a corresponding

- 13 043336 pharmaceutically acceptable salt of the compound of the present invention. In some embodiments of the present invention, the dosage forms may contain as the active substance either a compound or a corresponding pharmaceutically acceptable salt of the compound. The anti-VISTA antibodies of the present invention may be administered as part of a combination therapy (eg, with each other or one or more other therapeutic agents). The compounds of the present invention may be administered before, after, or simultaneously with one or more other therapeutic agents. In some embodiments of the present invention, a compound of the present invention or another pharmaceutical substance may be co-administered simultaneously (eg, in parallel) either as separate preparations or as co-formulations. Alternatively, the substances may be administered sequentially, in separate formulations, at appropriate periods of time as determined by a qualified clinician (eg, a time sufficient to ensure concordance of the pharmaceutical effects of the therapies). The compound of the present invention and one or more therapeutic agents may be administered in a single dose or in multiple doses, in a sequence and schedule suitable to achieve the desired therapeutic effect. The present invention also relates to a method of modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient. In some embodiments of the present invention, the compounds and compositions of the present invention are used to treat or prevent cancer. Cancer can include any malignant or benign tumor of any organ or system of the body. Examples include, but are not limited to, the following: breast cancer, digestive/gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, genitourinary cancer, germ cell cancer, gynecological cancer, head and neck cancer, hematological/blood cancer, musculoskeletal cancer, neurological cancer, respiratory tract/thoracic cancer, bladder cancer, colon cancer, rectal cancer, lung cancer, uterine cancer, kidney cancer, pancreatic cancer, liver cancer, cancer stomach, testicular cancer, esophageal cancer, prostate cancer, brain cancer, cervical cancer, ovarian cancer and thyroid cancer. Other types of cancer may include leukemia, melanoma and lymphoma, as well as any type of cancer described in the present invention. In some embodiments of the present invention, the solid tumor is infiltrated with myeloid and/or T cells. In some embodiments, the cancer is leukemia, lymphoma, myelodysplastic syndrome, and/or myeloma. In some embodiments of the present invention, the cancer may be any kind or type of leukemia, including lymphocytic leukemia or myelogenous leukemia, such as, for example, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (myelogenous leukemia). AML), chronic granulocytic leukemia (CML), hairy cell leukemia,

T-cell prolymphocytic leukemia, large granular lymphocyte leukemia, or adult T-cell leukemia. In some embodiments, the lymphoma is histocytic lymphoma, follicular lymphoma, or Hodgkin lymphoma, and in other embodiments, the cancer is multiple myeloma. In some embodiments of the present invention, the cancer is a solid tumor, such as melanoma or bladder cancer. In a specific embodiment of the present invention, the cancer is lung cancer, such as non-small cell lung carcinoma (NSCLC).

The present invention also relates to a method of modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of the following: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL ), B cell, T cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's lymphoma, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphosarcoma, multiple myeloma, Kaposi's sarcoma, colon and rectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/malignant hypercalcemia, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic tumor , cancer associated with bone resorption, cancer associated with bone pain, etc. In some embodiments of the present invention, the solid tumor is infiltrated with myeloid and/or T cells. In a specific embodiment of the present invention, the solid tumor is a lung cancer, such as non-small cell lung carcinoma (NSCLC).

In some embodiments of the present invention, the compounds and therapies described herein are co-administered with a vaccine (such as a viral vector vaccine, a bacterial vaccine, a cell-based vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine, or a protein vaccine). Such vaccines are well known in the art. See, for example, Jeffrey Schlom, Therapeutic Cancer Vaccines: Current Status and Moving Forward, J. Natl. Cancer Inst.; 104:599-613 (2012), the contents of which are incorporated herein by reference in their entirety.

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In some embodiments of the present invention, the compounds and therapies described herein are co-administered with chemotherapy, hormone and biological therapies, and/or bisphosphonates. In some embodiments, the chemotherapy agents include one or more of the following: arboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), doxorubicin (Adriamycin), etoposide (Vepezid), fluorouracil (5 -FU), gemcitabine (Gemzar), irinotecan (Camptosar), paclitaxel (Taxol), topotecan (Gikamtin), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban).

In other embodiments of the present invention, the anti-VISTA compounds and therapies described herein are co-administered with one or more immune checkpoint antibodies, such as, for example, nivolumab, pembrolizumab, tremelimumab, ipilimumab, anti-PD-L1 antibody, anti-PD -L2 antibody, anti-TIM-3 antibody, anti-LAG-3v, anti-OX40 antibody and antiGITR antibody.

In another embodiment of the present invention, the anti-VISTA compounds and therapies described herein are co-administered with the small molecule inhibitor indoleamine 2,3-dioxygenase (IDO). The anti-VISTA compounds and compositions of the present invention can be administered to a subject in need thereof to prevent (including preventing the recurrence of cancer) or treat (eg, inhibit the development or improvement of cancer or one or more symptoms thereof) cancer. Any substance or therapy (eg, chemotherapy, radiation therapy, targeted therapies such as imatinib, sorafenib and vemurabenib, hormone therapy and/or biological therapy or immunotherapy) that is known to be beneficial or that has been or is currently being used to prevent, treat, control development or improvement of cancer or one or more of its symptoms may be used in combination with a compound or composition of the invention described herein. Anticancer agents include, but are not limited to: 5-fluorouracil; activicin; aldesleukin; altretamine; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; azacitidine; Azetepa; azotomycin; batimastat; bicalutamide; bleomycin sulfate; sodium brequinarate; bropyrimine; busulfan; carboplatin; carmustine; carubicin hydrochloride; karzelesin; cedefingol; chlorambucil; cirolemicin; cisplatin; cladribine; Crisnatole mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunomycin hydrochloride; decitabine; dexormaplatin; desaguanine; desaguanine mesylate; diazkwon; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; enloplatin; enpromat; epipropidine; epirubicin hydrochloride; erbulozole; ezorubicin hydrochloride; estramustine; extramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; phasarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocytabine; phosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosin; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-m; interferon alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; acetatelanreotide; letrozole; leuprolide acetate; larozole hydrochloride; lometrexol sodium salt; lomustine; losoxantrone hydrochloride; Masoprokol; meclorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaryl; mercaptopurine; methotrexate; methotrexate sodium; methoprine; meturedepa; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; ormaplatin; paclitaxel; pegaspargase; porfromycin; prednimustine; procarbazine hydrochloride; puromycin; rogletimide; safingol hydrochloride; semustine; simtrazen; sodium sparphosate; sparsomycin; spiromustine; spiroplatin; streptonigrine; streptozocin; sulofenur; talizomycin; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxiron; testolactone; thiamiprin; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate; trimetrexate glucuronate; triptorelin; uramustine; uredepa; Vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglicinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozol; zeniplatin; zinostatin; zorubicin hydrochloride. Targeted therapies include, but are not limited to, tyrosine kinase inhibitors (eg, imatinib, sorafenib, and vemurafenib). The present invention also includes the administration of an anti-VISTA compound of the invention in combination with radiation therapy, including the use of X-rays, gamma rays and other radiation sources to destroy cancer cells. Treatments for cancer are known in the art and described in literature such as Physician's Desk Reference (57th ed., 2003). Anti-VISTA antibodies described in the present invention are also useful, for example, in the treatment of chronic infectious diseases such as HIV, VNV, HCV, and HSV, among others.

Various properties and sequence information for selected anti-VISTA antibodies of the present invention are presented in table. 1A, 1B and 2 of this description.

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Table 1A

CDR sequences of selected fully human or humanized anti-human VISTA antibodies

mAb ID VH family Heavy chain cdrl (Imgt) Heavy chain cdr2 (Imgt) Heavy chain cdr3 (Imgt) Light chain cdrl (Imgt) Light chain cdr2 (Imgt) Light chain cdr3 (Imgt) VSTB50 V GYTFTNYG (SEQ ID NO:1) INPYTGEP (SEQ ID NO:2) AREGYGNYIFPY (SEQ ID NO:3) ESVDTYANSL (SEQ ID NO:4) RAS (SEQ ID NO:5) QQTNEDPRT (SEQ ID NO:6) VSTB53 GYTFTHYT (SEQ ID NO:7) IIPSSGYS (SEQ ID NO:8) ARGAYDDYYDYYAMDY (SEQ ID NO:9) QTIVHSNGNTY (SEQIDNQ:1O) KVS (SEQ ID NO:11) FQASHVPWT(SE QIDNO:12) VSTBGO V GYTFTNYG (SEQIDN0:13) INTYTGES (SEQ ID NO:14) ARDYYGIYVSAY (SEQ ID NO:15) ESVDNYANSF (SEQIDN0:16) RAS (SEQ ID NO:17) QQSHEDPYT (SEQIDN0:18) VSTB95 GFTFRNYG (SEQ ID NO:19) IISGGSYT (SEQ ID NQ:2O) ARIYDHDGDYYAMDY (SEQ ID NO:21) QSIVHSNGNTY (SEQ ID NO:22) KVS (SEQ ID NO:23) FQGSHVPWT (SEQIDNO:24) VSTB112 D GGTFSSYA (SEQIDNO:25) IIPIFGTA (SEQ ID NO:26) ARSSYGWSYEFDY (SEQ ID NO:27) QSIDTR (SEQ ID NO:28) SAS (SEQ ID NO:29) QQSAYNPIT (SEQIDNQ:3O) VSTB116 D GGTFSSYA (SEQIDN0:31) IIPIFGTA(SEQ ID NO:32) ARSSYGWSYEFDY (SEQ ID NO:33) QSINTN (SEQ ID NO:34) AAS (SEQ ID NO:35) QQARDTPIT(SEQ ID NO:36)

Table 1B

Heavy and light chain sequences of selected fully human or humanized anti-human VISTA antibodies

ID Squirrel Heavy-chain AA CDS Light-chain AA CDS VSTB50 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEW MGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCA REGYGNYIFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO:47) DIVMTQTPLSLSVTPGQPASISCRASESVDT YANSLMHWYLQKPGQPPQLLIYRASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCQQTNEDPRTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO:48) VSTB53 QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEW MGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA RGAYDDYYDYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO:49) DIVMTQSPLSLPVTPGEPASISCRSSQTIVH SNGNTYLEWYLQKPGQSPQLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCFQASHVPWTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLS STLT LSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NQ:5O) VSTB60 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMTWVRQAPGQGLEW MGWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCA RDYYGIYVSAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO:51) DIVMTQTPLSLSVTPGQPASISCRASESVD NYANSFMHWYLQKPGQSPQLLIYRASNLE SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCQQSHEDPYTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO:52)

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VSTB95 EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEW VASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR IYDHDGDYYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO:53) DIVMTQSPLSLPVTPGEPASISCRSSQSIVH SNGNTYLEWYLQKPGQSPQLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCFQGSHVPWTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLS STLT LSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO:54) VSTB112 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO:55) DIQMTQSPSSLSASVGDRVTITCRASQSIDT RLNWYQQKPGKAPKLLIYSASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSA YNPITFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLN N FYP REAKVQWKVD N ALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:56) VSTB116 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO:57) DIQMTQSPSSLSASVGDRVTITCRASQSINT NLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAR DTPITFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLN N FYP REAKVQWKVD N ALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:58) VSTB140* QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKD DIQMTQSPSSLSASVGDRVTITCRASQSIDT RLNWYQQKPGKAPKLLIYSASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSA YNPITFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYE KHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:56) TLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFN STFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLS PGK(SEQID NO:59)

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VSTB149* ^A QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPDVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE DIQMTQSPSSLSASVGDRVTITCRASQSIDT RLNWYQQKPGKAPKLLIYSASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSA YN PITFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLN N FYP REAKVQWKVD N ALQSGNSQESVTEQDSKDSTYSLSSTLTLS K ADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:56) QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NQ:60) VSTB174* QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE DIQMTQSPSSLSSASVGDRVTITCRASQSIDT RLNWYQQKPGKAPKLLIYSASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSA YN PITFGQGTKVEI KRTVAAPSVFIFPPSDE QLKSGTASVVCLLN N FYP REAKVQWKVD N ALQSGNSQESVTEQDSKDSTYSLSSTLTLSK QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR ADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:56) EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK(SEQ ID NO:61)

*Constant region sequences in VSTB140, VSTB149 and VSTB174 are underlined.

^Δ Amino acid residues conferring protease resistance in the VSTB149 heavy chain are highlighted in bold.

table 2

Dissociation constant (KD) for selected anti-VISTA antibodies

Sample KD(M) kal(1/Mc) kdl (1/c) S.I. 1.71E-10 1.69E+06 2.89E-04 1.O9E-1O 1.11E+O6 1.21E-04 S40 5.07E-10 1.46E+05 7.40E-05 6.96E-10 1.39E+O5 9.69E-05 S41 6.32E-10 4.82E+05 3.05E-04 3.1OE-1O 7.08E+05 2.19E-04 S42 1.04E-10 1.05E+06 1.09E-04 2.65E-10 5.13E+O5 1.36E-04 S43 2.64E-11 1.25E+06 3.3OE-O5 5.28E-11 1.18E+O6 6.22E-05 S44 2.53E-11 1.23E+06 3.12E-05 6.40E-11 9.93E+O5 6.36E-05 S45 2.35E-11 1.58E+06 3.72E-05 2.58E-11 1.46E+06 3.77E-05 S46 1.06E-10 1.56E+06 1.66E-04 2.96E-10 1.50E+06 4.44E-04 S47 3.56E-10 5.14E+05 1.83E-04 2.52E-1O 5.69E+O5 1.43E-04 S33 8.30E-10 1.23E+06 1.02E-03 1.22E-09 8.96E+O5 1.10E-03 S34 1.08E-09 5.95E+05 6.43E-04 2.8OE-O9 5.20E+05 1.46E-03 S35 8.06E-11 2.08E+06 1.68E-04 1.35E-10 1.78E+O6 2.41E-04 S36 6.29E-11 1.77E+06 1.12E-04 2.9OE-11 1.58E+O6 4.58E-05 S37 2.23E-09 5.10E+05 1.14E-03 4.43E-09 3.94E+O5 1.75E-03 S38 2.26E-09 5.18E+05 1.17E-03 2.O3E-O9 5.37E+O5 1.O9E-O3 S39 5.62E-10 3.97E+05 2.23E-04 3.47E-10 4.15E+05 1.44E-04 S25 1.31E-O9 6.21E+05 8.12E-04 1.1OE-O9 5.65E+O5 6.24E-04

- 18 043336

S26 No binding 3.53E-09 2.38E+05 8.41E-04 S27 1.13E-09 8.86E+05 9.97E-04 1.61E-09 7.12E+05 1.15E-03 S48 3.12E-10 1.24E+06 3.87E-04 1.21E-09 8.78E+05 1.06E-03 S28 2.03E-09 1.08E+06 2.19E-03 2.03E-09 9.30E+05 1.88E-03 S29 3.78E-11 1.42E+06 5.38E-05 8.90E-11 9.06E+05 8.06E-05 S30 No binding No binding S31 Weak connection Weak connection S32 Weak connection Weak connection S15 9.34E-11 6.46E+05 6.04E-05 5.13E-10 3.50E+05 1.80E-04 S16 1.26E-10 5.54E+05 6.99E-05 1.92E-10 4.43E+05 8.53E-05 S17 7.68E-10 9.88E+05 7.59E-04 4.10E-10 7.09E+05 2.91E-04 S18 2.28E-09 4.90E+05 1.12E-03 1.05E-09 3.13E+05 3.29E-04 S19 1.54E-09 1.02E+06 1.58E-03 2.86E-10 7.03E+05 2.01E-04 S20 1.48E-09 6.67E+05 9.85E-04 4.57E-10 6.36E+05 2.91E-04 S21 3.18E-09 3.16E+05 1.00E-03 1.34E-09 2.70E+05 3.60E-04 S22 2.98E-09 1.09E+06 3.25E-03 1.27E-09 1.25E+06 1.59E-03 S6 6.36E-10 5.28E+05 3.36E-04 3.02E-10 5.98E+05 1.80E-04 S7 6.75E-10 1.31E+06 8.87E-04 3.27E-10 1.15E+06 3.75E-04 S8 1.15E-10 1.89E+06 2.18E-04 5.97E-11 1.25E+06 7.48E-05 S9 1.67E-10 1.87E+06 3.11E-04 9.31E-11 1.27E+06 1.18E-04 S10 8.90E-11 1.55E+06 1.38E-04 4.30E-11 1.22E+06 5.27E-05 S12 4.94E-10 1.57E+06 7.76E-04 2.39E-10 1.19E+06 2.86E-04 S13 1.02E-10 1.42E+06 1.44E-04 6.46E-11 9.55E+05 6.17E-05 S14 2.02E-10 1.26E+06 2.55E-04 7.55E-11 1.12E+06 8.43E-05 S1 2.06E-10 1.60E+06 3.29E-04 8.35E-11 1.21E+06 1.01E-04 S2 1.56E-10 9.74E+05 1.52E-04 8.66E-11 7.25E+05 6.28E-05 S3 4.33E-11 9.07E+05 3.93E-05 4.89E-11 7.41E+05 3.63E-05 S4 1.52E-10 8.98E+05 1.36E-04 7.54E-11 6.93E+05 5.23E-05 S49 1.45E-10 1.01E+06 1.46E-04 1.04E-10 7.28E+05 7.60E-05 S5 2.13E-10 1.25E+06 2.67E-04 1.37E-10 8.51E+05 1.17E-04

Examples

Example 1. Analysis of VISTA expression on human hematopoietic cells.

Ways.

Preparation and staining of fresh human PBMCs for VISTA expression.

VISTA expression was studied in freshly isolated human PBMCs (peripheral blood mononuclear cells) from various donors. Anti-human VISTA-biotin (GA-1) was used for staining (5 μg/ml). Mouse IgG1, K-biotin (Clone MOPC-21 at 5 μg/ml) was used as isotype control.

Donor material.

Blood samples were obtained from Biological Specialty Corp. (Colmar, PA) and were collected and analyzed the same day. 10 ml of whole blood containing heparin sulfate was delivered for analysis.

Sample preparation.

Blood was diluted 1:1 in sterile PBS. 22 mL of diluted cord blood was layered onto 20 mL sterile Ficoll-Hypaque (GE Healthcare, cat #17-144003) in 50 mL conical tubes. The tubes were centrifuged at 1800 rpm for 20 min at room temperature. Mononuclear cells at the interface after centrifugation were collected using a 1-mL pipette and pooled into two 50-mL conical tubes. Sterile PBS was added to each tube to reach a volume of 50 ml and the cells were centrifuged at 300 g for 10 min at 4°C. The supernatant was discarded. Cells were resuspended in 50 ml of sterile PBS and the tubes were centrifuged at 300 g for 10 min at 4°C. The supernatant was discarded. Cells were pooled and resuspended in 50 ml sterile PBS until counted.

Staining protocol.

A frozen tube containing 5x10' PBMCs was used to equalize controls and as a staining control. The following reagents and/or consumables were used: FACS staining buffer (BSA) from BD Biosciences (cat #554657) was supplemented with 0.2% EDTA; phosphate buffered saline (PBS) (Gibco cat #14190); 96-hole polypropylene round

- 19 043336 bottom tablet (BD #3077); 1.2 ml polypropylene cassette tubes (Coming #4451); biotinylated anti-VISTA clone GA-1 from ImmunoNext Lot #080612B (used at 5 μg/ml); biotinylated mIGl, K isotype control (Clone MOPC-21); Biolegend, cat #400104 Lot #B116649 (used at 5 μg/ml); anti-human antibodies (see staining table below); Near-infrared live/dead dye (Invitrogen, cat #L10119); and streptavidin reagents, including STP-APC (BD Biosciences cat #554067, Lot #04251) (used at 1:200 dilution in FACS buffer), STP-PE (Biolegend cat #405203, Lot #B139688) (used at 1:200 dilution in FACS buffer), STP-PE Cy7 (showed nonspecific binding in isotype control samples), STP-Q605 (Invitrogen cat #Q10101MP, Lot #53449A) (used at 1:200 dilution in FACS buffer).

Cell surface staining protocol.

Before staining, 1 x 10 ^b cells were transferred to 96-well round-bottom plates and washed with 150 μl PBS. The plates were then centrifuged at 1300 rpm at 4°C for 3 min.

The cells were then washed again in PBS and centrifuged as described above. Live/dead staining was then performed in 50 μl of PBS containing 0.25 μl of near-infrared live/dead dye. After 10 min at room temperature, the wells were washed with 150 μl of FACs staining buffer and centrifuged at 1300 rpm at 4°C for 3 min. The supernatant was discarded.

Cells were blocked with human serum at 1:100 in 50 μl FACS staining buffer. The plates were incubated at 4°C for 15 min. The wells were then washed with 150 μl of FACs staining buffer and centrifuged at 1300 rpm at 14°C for 3 min. The supernatant was discarded.

A mixture containing the following antibodies was then added to each well to stain the surface. The mixtures are described in table. 3-6 below. Each mixture was administered separately from the others depending on the population being studied.

Table 3

Cell line staining

Target Fluorochrome Antigen Mouse Iry sa Human Isotype Clone Supplier To Cat no. Lot No. Titer (µl/10 b cells) FITC/AF48 8 CD19 X mlgGl HIB19 Biolegend 302206 B123019 2 RE CDllb X mlgGl, K ICRF44 BD Bio. 555388 45134 2 RegSRSu5.5 HLA-DR X mlgG2a, K G46-6 BD Bio. 560652 25161 0.5 RE Su7 CD16 X mlgGl, K 3G8 BD Bio. 557744 87825 0.2 ARS Su7 NIR Live/Dead X AF700 CD56 X mlgGl, K B159 BD Bio. 557919 19470 1 APC/AF647 VISTA-Bio X PB/V450 CD3 X mlgGl, K UCHT1 BD Bio. 558117 90926 0.5 Q605 CD14 X mlgG2a, K TiK4 Invitrogen Q10013 1049158 0.2

Table 4

T cell staining

Target Fluorochrome Antigen Mouse Rat Human Isotype Clone Provider Cat no. Lot No. Titer (µl/10 ⁶ Cells) FITC/AF488 CD4 X mlgGl, K RPA-T4 BD Bio. 555346 38460 2 RE VISTA- Bio X RegSR-Su5.5 CD8 X mlgGl, K RPA-T8 BD Bio. 560662 1037 0.5 RE Su7 CD56 X mlgGl, K B159 BD Bio. 557747 47968 0.5 ARS Su7 NIR X AF7OO CD45RO X mlgG2a, K UCHL1 Biolegend 304218 B143062 1 APC/AF647 TCRgd X mlgG, K IN 1 Biolegend 331212 B126473 2 PB/V450 CD45RA X mlgG2b, K HI100 BD Bio. 560363 90928 0.5 Q655 CD3 X mlgG2a S4.1 Invitrogen Q10012 982352 0.5

-20043336

Table 5

DC staining

Target Fluorochrome Antigen Mouse Rat Human Isotype Clone Provider Cat no. Lot No. Titer (µl/10 ⁶ cells) FITC/AF488 Linl X Mix Mix BD Bio. 340546 2152758 5 RE CDllc X mlgGl, K BD Bio. 555392 45123 2 RegSRSu5.5 HLA-DR X mlgG2a, k G46-6 BD Bio. 560652 25161 0.5 ARS Su7 NIR X APC/AF647 CD83 X mlgGl, K HB15 e BD Bio. 551073 57688 2 BV421 CD123 X mlgGl, K 6N6 Biolegend 306017 B148193 0.5 Q605 VISTA-Bio X

Table 6

Myeloid staining

Target Fluorochrome Antigen Mouse Rat Human Isotype Clone Provider Cat no. Lot No. Titer (µl/10 ⁶ cells) FITC/AF488 CD33 X mlgGl NMZ- 4 Biolegend 303304 B100963 3 RE CDllb X mlgGl, K ICRF4 4 BD Bio. 555388 45134 2 ARS Su7 NIR X APC/AF647 VISTA-Bio X Q605 CD45 X mlgGl, K HI30 Invitrogen Q1005 1 880470 1

After surface staining, cells were washed twice as described above with FACS staining buffer and centrifuged at 1300 rpm at 4°C for 5 min. Samples were resuspended in 50 μl of FACS staining buffer containing the appropriate fluorescently labeled streptavidin. Samples were incubated at 4°C for 30 min. Cells were washed with 150 μl of FACS staining buffer and centrifuged at 1300 rpm at 4°C for 5 min. This washing step was repeated before the samples were resuspended in 250 μl of FACS staining buffer. Samples were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) on the same day. Analysis data

Flow cytometry data were reanalyzed using FlowJo Version 9 software to obtain specific phenotypic populations. Geometric mean calculation was used to compare VISTA expression in different cell subpopulations. Each population was normalized to background by subtracting isotype control values from those of anti-VISTA-treated samples. Graphs were generated in Prism and statistics were obtained using either Student's t -value if only two samples were compared, or one-way ANOVA with Bonferroni's test-retest.

Results.

Expression of VISTA on human myeloid and lymphoid subsets.

As shown in FIG. 2A-2E, 3A-3G, 4, 5A, 5B and 6A-6C, VISTA expression on CD14+ monocytes was significantly different from all other populations (p<0.001).

There were no significant differences between other populations. Monocytes expressed the highest levels of VISTA in peripheral blood, with the CD14 + CD16 ^- subpopulation having significantly higher expression levels than CD14 ^and CD16 ⁺ cells. While APCs showed moderate expression of VISTA, lymphoid subsets showed low levels of expression.

VISTA expression on human T and NK subpopulations.

As shown in FIG. 7A-7E, 8A-8G and 9, with NK subsets, CD56l ^o cells showed significantly higher levels of VISTA expression than CD56 ^Hi NK cells. A subset of T cells, CD8 ⁺ memory cells expressed higher levels of expression, although they were not significantly higher than CD8 ⁺ or CD4 ⁺ naïve T cells.

Expression of VISTA on subpopulations of human dendritic cells.

As shown in FIG. 10A-10D, 11A-11C and 12, no significant differences in VISTA expression; DCs and basophils showed low levels of VISTA expression, in plasmacytoid dendritic cells

- 21 043336 (pDCs) are generally higher, but not to a significant extent.

Conclusion.

These results showed expression of VISTA on various subsets of immune cells, and that VISTA is expressed on monocytes at higher levels, with some expression on various subsets of T cells and NK cells, and virtually no expression on B cells.

Example 2. Expression of VISTA on peripheral blood cells.

Ways.

Whole blood staining. Freshly isolated whole blood (100 μl) was stained with the antibody mixture as indicated below by incubation for 30 min at 4°C. Red blood cells (RBCs) were lysed with RBC lysis buffer, and the remaining cells were washed with 1x staining buffer. Cells were resuspended in 200 μl of staining buffer. Data were collected using a MACSQuant flow cytometer and analyzed using FlowJo analysis software.

Staining of peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were isolated from whole blood using a Ficoll gradient.

Freshly isolated 1 x 10 ⁶ PBMCs were stained with a mixture of antibodies in 100 μl of staining buffer. Samples were incubated for 30 min at 4°C and then washed once with staining buffer. Cells were resuspended in 100 μl of staining buffer. Data were collected using a MACSQuant® flow cytometer (Miltenyi Biotec) and analyzed using FlowJo analysis software.

Antibodies used were CD11b, CD33, CD177, CD16, CD15, CD14, CD20, HLADR, CD3, CD4, CD8, CD127, CD69, and FOXP3 antibodies (Biolegend, San Diego, CA). APC-conjugated mouse anti-human VISTA (clone GG8) was from ImmuNext (Lebanon, NH).

Conclusions.

Expression of VISTA on peripheral blood cells of a healthy person.

Whole blood and peripheral blood mononuclear cells were analyzed for VISTA expression using multicolor flow cytometry. As shown in FIG. 15A and 15B, the highest levels of VISTA expression were found on monocytes, followed by neutrophils. Both CD4+ and CD8+ T cells expressed low levels of VISTA, as shown in FIG. 13C and 13D.

Expression of VISTA on peripheral blood cells of cancer patients.

As shown in FIG. 14A-14C, peripheral blood mononuclear cells (PBMCs) from patients with lung cancer were analyzed. In fig. 14A is an exemplary plot showing analysis of CD14+ monocytes and CD15+ myeloid-derived suppressive cells (MDSCs). The results showed that, phenotypically, CD15+ cells are neutrophil-derived MDSCs. In addition, these cells are absent from healthy human blood samples. In fig. 14B is a representative histogram of VISTA expression on monocyte derivatives from healthy individuals and cancer patients, indicating higher levels of VISTA expression on cancer patient cells compared to healthy individuals. Similarly, high levels of VISTA were detected on MDSCs of cancer patients, as shown in FIG. 14C. In fig. 15A is a representative FACS plot showing the presence of neutrophil-derived MDSCs in the blood of patients with colon cancer. In fig. 15B and 15C are representative histograms showing the highest levels of VISTA expression on monocytes from cancer patients compared to healthy donor blood samples. Expression of VISTA on Cynomolgus Macaque Peripheral Blood Cells As shown in FIG. 16A and 16B, flow cytometry analysis of macaque whole blood showed a VISTA gene expression profile similar to human cells. Both monocytes and neutrophils expressed the highest levels of VISTA compared to CD4+ (Fig. 16C) and CD8+ (Fig. 16D) T cells.

Example 3. Expression of VISTA in the heme of malignant cell lines at the RNA and protein levels.

Because VISTA is expressed in the heme of malignancies, anti-VISTA antibodies could potentially target malignant cells to destroy as well as block VISTA and induce antitumor immune responses.

Data include RNA sequencing (RNAseq) analysis of ~140 heme malignant cell lines (some cell lines are repeated in the analysis).

The data is shown in Fig. 17.

RNAseq values are reported as FPKM (fragments per kilobase of exon per million fragments mapped) values.

Essentially this means that all reads occurring in exonic regions of a gene are counted and normalized to both gene length and the total number of reads per sample (accounting for differences between samples). The limit value is 1; above 1 is positive for VISTA expression (at the RNA level), below 1 is negative for VISTA expression.

The results showed that many cell lines are positive at the RNA level, most notably acute myeloid leukemia and chronic myelogenous leukemia. This would be expected since VISTA was expressed at the highest levels in normal myeloid cells and since its function is presumed to attenuate immune responses, including antitumor immune responses.

Example 4. Development of monoclonal antibodies against VISTA.

Phage panning.

Twenty-four phage panning experiments were performed to enrich cynomolgus VISTA-His-reactive phage. Cynomolgus VISTA protein was used for these experiments because it showed better conjugation to biotin than human VISTA proteins. To determine the success of phage experiments, pools of phages from individual panning steps were added to neutravidin plates coated with biotinylated cynomolgus VISTA-His and detected with HRP-conjugated anti-M13 antibody. Individual colonies were selected from phage selection steps and Fabs were prepared in 96-well plates.

Supernatants of expressed Fabs were assayed for binding to biotinylated cynomolgus VISTA-His. This resulted in over 200 hits.

VH and VL regions from Fab plates were amplified, provided for DNA sequencing and exported as FASTA files. When selecting clones to be translated and studied as MABs, clones were selected based on sequence diversity, as well as having limited risks of post-translational modifications and as few hydrophobic residues as possible.

VH and VL from phage clones were subcloned into mammalian IgG1/kappa expression vectors and transfected into HEK293 cells. Antibodies were purified using Sepharose Fast Flow protein A affinity resin. The concentration of phage MABs was determined by quantitative ELISA using Nanodrop measurements. The antibody panel was expressed at high levels. SDS-PAGE analysis showed that each expressed antibody variant was conserved. Coincident maturation of phage antibodies was done by amplifying VH domains from a mixture of polyclonal antibodies from the final panning step for cloning into phage vectors that had diversity in the VL. This resulted in an enriched pool that was selected for with additional diversity in the VL. Phages were selected through 1-2 rounds of stringent panning with the potential to detect very high binding affinity to the VISTA ECD His protein. Solid-phase ELISA of monoclonal Fabs was performed to determine the achievement of maturation. ELISA and expression data were normalized to a reference clone equal to 100% of the primary panning experiment, and the affinity of mature clones with the highest binding signal to the cynomolgus VISTA antigen than the reference clone was determined. This process generated several clones that showed up to 200% binding when screened at low antigen concentration (1 nM), the clones with the highest affinity were sequenced and obtained as MABs.

Obtaining a hybridoma.

One group of BALB/cAnNCrl mice received one intraperitoneal (IP) administration of 50 μg Hu VISTA-Ig recombinant protein (Sino) emulsified in Freund's complete adjuvant followed by a single IP administration of 50 μg Hu VISTA-Ig recombinant protein emulsified in incomplete adjuvant for two weeks. Freund. Two weeks later, mice received a single IP injection of 50 μg of recombinant cynomolgus VISTA-Fc protein emulsified in Freund's incomplete adjuvant. All mice received a final injection of 25 μg human and 25 μg cynomolgus VISTA in PBS at the base of the tail, five days before receiving spleens for fusion. Another group of BALB/cAnNCrl mice received a single IP administration of 50 μg human (Hu) VISTA-His recombinant protein emulsified in Freund's complete adjuvant. Two weeks later, mice received a single IP injection of 50 μg of Hu VISTA-His recombinant protein emulsified in Freund's incomplete adjuvant. Two weeks later, mice received a single IP injection of 50 μg VISTA-His recombinant cynomolgus protein emulsified in Freund's incomplete adjuvant. After two weeks, all mice received a final treatment with 25 μg Hu VISTA-His and 25 μg cynomolgus VISTA-His in PBS, three days before receiving spleens for fusion.

On the day of fusion, mice were sacrificed by CO ₂ asphyxia, and the spleens were removed and placed in 10 ml of cold phosphate-buffered saline. A single cell suspension of splenocytes was prepared by grinding the spleens through a fine sieve with a small pestle and washed with PBS at room temperature. Cells were washed once in PBS and subjected to RBC lysis. Briefly, cells were resuspended in 3 ml of RBC lysis buffer (Sigma #R7757) per spleen and placed on ice for 5 min. Cells were washed again once in PBS at room temperature and labeled for magnetic sorting. According to the instructions, cells were labeled with anti-mouse Thy1.2, anti-mouse CD11b and anti-mouse IgM magnetic microcarriers (Miltenyi Biotec #130-049-101, 130-049-601 and 130-047-301, respectively), then sorted using MS columns with Midi MACS. Negative cell fractions (positive cell fractions were discarded) were fused with FO cells. Fusion was performed in a 1:1 ratio of mouse myeloma cells to viable spleen cells. Briefly, spleen cells and myeloma cells were mixed, pelleted, and washed once in 50 ml PBS. The sediment was resuspended with 1 ml propylene glycol (PEG) solution (2 g PEG molecular

- 23 043336 weighing 4000, 2 ml DMEM, 0.4 ml DMSO) per 10e8 splenocytes at 37°C for 30 s. The cell/fusion mixture was then immersed in a 37°C water bath for approximately 60 s with gentle stirring. The fusion reaction was stopped by adding 37°C DMEM for 1 min. Confluent cells were left undisturbed for 5 min at room temperature and then centrifuged at 150xg for 5 min. Cells were then resuspended in MediumE (StemCell Technologies cat #03805) containing HAT (Sigma cat #H0262) and seeded into a 96-well flat-bottomed polystyrene tissue culture plate (Corning #3997).

Capture ELISA was used to screen hybridoma supernatants for cynomolgus VISTA-specific antibodies. Briefly, plates (Nunc-Maxisorp #446612) were coated at 4 μg/ml for at least 60 min with goat anti-mouse IgG (Fc) antibodies (Jackson #115-006-071) in adsorption buffer (Thermo 28382). The plates were blocked with 200 μl/well of 0.4% (w/v) bovine serum albumin (BSA) in PBS for 30 min at room temperature. The plates were washed once and 50 μl/well of hybridoma supernatant was added and incubated at room temperature for at least 30 minutes. The plates were washed once and 50 μl/well of 0.1 μg/ml cynomolgus VISTA-huIg was added and incubated at room temperature for 30 min. The plates were washed once and 1:40,000 streptavidin HRP (Jackson 016-030-084) in 0.4% BSA/PBS was added to the plates and incubated for 30 min at room temperature. The plates were washed 3 times and then developed using 100 μl/well of TMB Turbo substrate (Thermo Scientific 34022) by incubating for approximately 10 min at room temperature. The reaction was stopped using 25 μl/well of 4N sulfuric acid and absorbance was measured at 450 nm using an automated plate spectrophotometer. Fifteen primary hits were selected for subcloning using limiting dilution and screened in the same primary screening format. All reactive cynomolgus VISTA hybridoma cell lines were cross-screened using human VISTA-Ig to determine cross-specificity. Briefly, plates (Nunc-Maxisorp #446612) were coated at 4 μg/ml with goat anti-mouse Fc (Jackson #115-006-071) in 0.1 M sodium carbonate-bicarbonate buffer, pH 9.4 (Pierce 28382 BupH™) O/ N at 4°C. Without washing, wells were blocked with 200 μl of blocking agent (0.4% BSA (Sigma) (w/v) in PBS (Invitrogen)) overnight at 4°C. After blocking solution was removed, undiluted hybridoma supernatants were incubated on coated plates for 30 min at room temperature. The plates were washed once with PBST (0.02% Tween 20 (Sigma) (w/v) in PBS) and then incubated for 30 min with Hi VISTA-Ig diluted to 100 ng/ml in the block. Plates were washed once with and probed with goat anti-human-Fc-HRP (Jackson #109-036-098) diluted 1:10,000 in block for 30 min at room temperature. The plates were washed again and then developed using 100 μl/well of TMB Turbo substrate (Thermo Scientific 34022) by incubating for approximately 10 min at room temperature. The reaction was stopped using 25 μl/well of 4N sulfuric acid and absorbance was measured at 450 nm using an automated plate spectrophotometer.

Hybridomas that showed responses to both human VISTA and cynomolgus VISTA had three antibody V region sequences cloned. Hybridoma cells were prepared prior to reverse transcriptase (RT) reactions with Invitrogen's Superscript III cells Direct cDNA System kit. Briefly, the culture medium was discarded and the plate was placed on ice and resuspended in 200 μl of cold PBS. Forty microliters were transferred to a MicroAmp fast 96-well PCR reaction plate and the plate was placed on a cold metal base plate, sealed with polymer film and centrifuged at 700 rpm for 3 min. The PBS was discarded and 10 μl of resuspension buffer and 1 μl of lysis enhancer were added to each well. The plates were sealed and incubated at 75°C for 10 min and stored at -80°C.

For the RT reaction, each well contained 5 μl of water, 1.6 μl of 10x DNase buffer, 1.2 μl of 50 mM EDTA, 2 μl of Oligo(dT)20 (50 mM) and 1 μl of 10 mM dNTP mixture. The plate was incubated at 70°C for 5 min, followed by incubation on ice for 2 min, then the following reagents were added to each well; 6 µl 5X RT buffer, 1 µl RNaseOUT™ (40 units/µl), 1 µl Superscript™ III RT (200 units/µl) and 1 µl 0.1 M DTT. The plate was sealed and placed on a reaction thermoblock preheated to 50°C and incubated at 50°C for 50 minutes, followed by inactivation (incubated at 85°C for 5 minutes). The reaction was cooled on ice and single-stranded cDNA was stored at -80°C until further use. To amplify the V region, 20 μl of the PCR reaction mixture was prepared. Each well contained 16.2 μl of water, 2.0 μl of 10x PCR reaction buffer, 0.8 μl of MgSO ₄ (50 mM), 0.4 μl of 10 mM dNTPs, a mixture of 0.15 μl of 100 μm forward primers, 0.05 μl of 100 μm reverse primers, 0.2 μl of HiFi Tag enzyme. The cDNA obtained as described above was transferred (2 μl/well) to the components of the PCR mixture, the plate was sealed and the amplification reaction was carried out; for VH the program was (i) 94°C for 1 min (ii) 94°C for 15 s (iii) 55°C for 30 s (iv) 68°C for 1 min. Steps (ii-iv) were repeated for a total of 35 cycles followed by a final elongation at 68°C for 3 min. For VL the program was (i) 94°C for 1 min (ii) 94°C for 15 s (iii) 55°C for 30 s (iv)

- 24 043336

65°C for 30 s, (v) 68°C for 1 min. Steps (ii-v) were repeated for a total of 35 cycles followed by a final elongation at 68°C for 3 min.

The forward primers were pre-mixed and this mixture was applied in a 3:1 ratio to the reverse primers. PCR products were checked on an agarose gel. Reactions were prepared for infusion cloning by adding an amplifier (In-Fusion HC Cloning Kit, cat #639650, Clontech). Five microliters of the PCR reaction was transferred to a PCR plate, followed by 2 μl of enhancer/well. The plate was sealed and incubated in a thermoblock for reactions (15 min at 37°C and 15 min at 80°C). The delivery vector (vDR243 or vDR301) was prepared by Esp3I digestion; (1.5 μg of vector was digested in 3 μl Tango buffer, 2 l Esp3I and water in 30 μl reaction at 37°C for 2 h).

For infusion cloning, 2 μl of enhancer-treated PCR products were mixed with 100 ng of digested Esp3I vector and 2 μl of 5x infusion enzyme (Clontech). The reaction was carried out in the format of a 96-well PCR plate. The plate was incubated for 15 min at 50°C on a PCR machine and the competent cells were transformed by heat shock for 40 seconds at 42°C without shaking and spread onto LB agar plates with the selected antibiotic and incubated overnight at 37 °C. The next day, colonies were collected into a 96-well deep-well plate containing LB/carbenicillin medium and grown overnight at 37°C. Frozen products were prepared from an overnight culture mixed in equal volumes with 30% w/v. glycerin. The V regions were sequenced using the sequencing primer SPF0052. The sequences were analyzed, one positive well for VH and VL hybridomas was selected, replated into new plates, and grown overnight in ampicillin-supplemented medium. Cloning was then done with miniprep DNA for transfer in small quantities into a 96-well plate. Selected forty-eight mouse hybridoma sequences for both heavy and light chains were adapted by the human framework using in-house software. One human scaffold was selected for each mouse VH or VL. The V region of DNA sequences was obtained by reverse translation. Synthetic DNA sections corresponding to the amino acid sequences of HFA were ordered from Integrated DNA Technologies (Coralville, IA). Cloning was performed into pre-excised vDR149 and vDR157, human IgG1 and human kappa, respectively. Qiagen Endo-free Maxi-prep kits were used to prepare DNA. Expi293 culture (100 ml) was used to express this panel of antibodies.

Example 5: Protocol for In Vitro T Cell Suppression Assay of Human VISTA-IG T.

Mouse A20 cells were stably transfected with either GFP or human VISTA. They were incubated with ovalbumin peptide and DO11.10 T cells. CD25 expression by T cells was measured 24 h after the start of incubation. A20-huVISTA cells suppress CD25 expression by T cells, but this finding was significantly restored by incubation with VSTB95 (Fig. 18).

Example 6: Adaptation of human anti-VISTA antibody frameworks.

Mouse hybridoma sequences for both the heavy and light chains of the human framework were adapted by CDR grafting (Jones et al., Nature, 321:522-525 (1986) using in-house software. The program delineated the V region hypervariable region (CDRs) sequences in according to the definitions of Kabat (Wu, T. T. & Kabat, E. A. (1970), J. Exp. Med., 132, 211-50) and compared the framework regions with human germline genes using Blast. the line with the highest sequence identity to the mouse scaffolds was selected as the acceptor gene for human framework adaptation (HFA). In some cases, closely related human germline genes were selected instead, based on previous experience, with well expressed human scaffolds. DNA sequences for human scaffolds were selected for each of the V regions of mouse VH or VL were obtained by back translation.Synthetic DNA regions corresponding to the amino acid sequences of HFA were obtained from Integrated DNA Technologies (Coralville, IA). Cloning was performed in human IgG1 and human kappa, respectively.

Example 7: Anti-VISTA Antibody Constructs.

Plasmid and information sequence for molecules for cell line development: plasmid constructs were created for anti-VISTA antibodies having VSTB112 variable regions and IgG1K constant regions (VSTB174, new number due to an allotypic change in the constant region), IgG2sigma constant region (VSTB140) or protease-resistant IgG1 constant region (VSTB149).

Lonza vectors.

The pEE6.4 and pEE12.4 Chinese hamster ovary (CHO) expression vector system (Lonza Biologics, PLC) was established by Biologics Research (BR) and Pharmaceutical Development & Manufacturing Sciences (PDMS) as a primary expression system for producing therapeutic mAbs in expression mammalian cell line. Each vector includes a human cytomegalovirus promoter (huCMV-MIE) to drive heavy chain (HC) or light chain (LC) expression and includes an ampicillin resistance gene. The pEE12.4 vector also includes a gene encoding the enzyme glutamine synthetase (GS). Growth conditions that require glutamine synthetase activity place selective pressure on cells to maintain the expression vector (GS Gene Expression System

Manual Version 4.0). pEE6.4 was used to clone the HC gene and pEE12.4 to clone the LC gene as gene signaling vectors. A dual plasmid Lonza gene was created from these two separate Lonza gene vectors.

Amino acid sequence of the heavy chain variable regions of selected VISTA mAbs.

> VSTB112 heavy chain (SEQ GO NO:37)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG

IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDY

WGQGTLVTVSS > VSTB50 heavy chain (SEQ GO NO:38)

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMG

WINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPY WGQGTLVTVSS > VSTB53 heavy chain (SEQ GO NO:39)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMG

YIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDY YAMDYWGQGTLVT VS S > VSTB95 heavy chain (SEQ GO NO:40)

EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVAS

IISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYA

MD YWGQGTT VT VS S

Amino acid sequence of light chain variable regions of selected VISTA mAbs.

>VSTB50 light chain (SEQ GO NO:41)

DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLI

YRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIK >VSTB53 light chain (SEQ GO NO:42)

DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLI

YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEI

K >VSTB95 light chain (SEQ GO NO:43)

DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLI

YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEI

K >VSTB112 light chain (SEQ GO NO:44)

DIQMTQSPSSLSSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASS

LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK >VSTB116 light chain (SEQ GO NO:45)

DIQMTQSPSSLSSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAAS

SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQGTKVEIK

Example 8. Solid-phase ELISA and FACS screening of anti-VISTA antibodies.

These experiments were performed to determine the ability of the resulting antibodies to bind to the human VISTA protein or the cynomolgus VISTA protein in an ELISA, as well as to determine, using FACS screening, the ability of the antibodies to bind to the VISTA protein on the surface of K562 cells (human myelogenous leukemia cell line), expressing human VISTA proteins or cynomolgus VISTA proteins.

Ways.

Brief description of the solid-phase ELISA method.

The plates were coated overnight at 4°C with 1 μg/ml SB0361 (human) or SB0361 (cynomolgus) proteins, which are extracellular VISTA domains from the respective species. Antibodies were diluted to 1 μg/mL as a starting concentration with 1:4 stepwise dilutions for a total of 4 concentrations and incubated at room temperature (RT) for 2 h. Mouse anti-human IgG1-HRP (horseradish peroxidase) was used for detection and incubated for 1 hour at room temperature. All washes were performed using PBS-Tween (0.05%).

Brief description of the FACS method.

2x105 K562-G8 (human) or K562-C7 (cynomolgus) cells were stained with 5 μg/ml of each antibody tested and incubated for 30 min at 4°C. Goat anti-human IgG1-PE (phycoerythrin) antibody was used as a second identifying antibody at a concentration of 5 μg/ml. Cells were run on a BD Fortessa and analyzed using FlowJo software (Tree Star, Inc., Ashlang, OR) for MFI (mean fluorescence intensity) for the live population.

Analysis data/results.

For each antibody, a subjective score (Yes/No) was given for any antibody strongly bound or not for both ELISA and FACS assays for each of the 4 studies. If the antibody returned a No for binding result in any assay, it was repeated to confirm that it was negative. The results are shown in table. 7 below and FIG. 19A-19F and 20A-20F.

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22 Y Y Y Y 23 N N N N 24 N N N N 25 Y Y Y Y 26 N Y N Y 28 Y Y Y Y thirty N N N N 31 N N N N 32 N N N N 33 Y Y Y Y 34 Y Y Y Y 35 Y Y Y Y 36 Y Y Y Y 37 Y Y Y Y 38 Y Y Y Y 39 Y Y N N 40 Y Y Y Y 41 Y Y Y Y 42 Y Y Y Y 43 Y Y Y Y 44 Y Y Y Y 45 Y Y Y Y 46 Y Y Y Y 47 Y Y Y Y 48 Y Y Y Y 49 Y Y Y Y

Example 9 Results of screening for anti-human VISTA antibodies using mixed lymphocyte reaction (MLR) and Staphylococcus enterotoxin activation assay (SEB).

The purpose of this study was to provide data supporting the identification of multiple functional α-VISTA antibodies that enhance cellular immune responses in a mixed lymphocyte response (MLR) assay and a Staphylococcus enterotoxin B (SEB) activation assay. The mixed lymphocyte culture reaction (MLR) is a standard immunoassay that is dependent on MHC class I and II, inappropriate for driving the allogeneic T cell response. Peripheral blood mononuclear cells were isolated from two mismatched individuals, incubated together, and proliferation and cytokine production occurred as a result of the mismatches.

Materials and methods.

10% AB medium was prepared by mixing 500 ml RPMI with 50 ml human AB serum, 5 ml penicillin/streptomycin (10,000 U/ml), 5 ml L-glutamine (100x) and 10 ml HEPES (1 M). The medium was stored for no more than 14 days. 1 mCi of tritium-labeled thymidine was prepared by diluting 0.2 ml of the original thymidine (1 mCi/ml) in 9.8 ml of RPMI.

Soluble VISTA antibodies were diluted to 20 μg/ml in 10% AB serum medium. 100 μl of the appropriate antibody solutions were added to the appropriate wells of a 96-well U-bottom plate (Falcon product #353077 or equivalent). Various cell populations were then added to a final concentration of 10 μg/ml.

Isolation of leukocytes.

Donors were at least 18 years of age, generally healthy, and randomly selected from the local population. Donor blood was transferred from isolated tubes into 50 ml conical tubes. Apply below 15 ml of Ficoll 1077 per 25 ml of blood, carefully so as not to mix with the blood. Cells were centrifuged at 1250 g for 25 min at room temperature without braking. Leukocytes were isolated at the Ficoll-serum interface and the cells were diluted in 40 ml Hanks' balanced salt solution (HBSS). Cells were centrifuged at 453 g (1500 rpm) for 10 min at 4°C. Cells were resuspended in 50 ml HBSS and counted by transferring 500 μl into a separate tube.

Setting up a mixed lymphocyte culture reaction (MLR) in a 96-well plate.

The appropriate number of stimulating cells and immunocompetent cells required for the study was determined based on the number of samples analyzed. The sham population was seeded at 0.5x10 ⁵ cells/well and the immunocompetent population was seeded at LOxlO ⁵ cells/well in a 96-well U-bottom plate. All conditions had to be implemented in triplicate. An appropriate number of stimulating cells were pipetted into a new conical tube and centrifuged as previously described. Cells were resuspended in 10 ml and irradiated with 4000 rad. Cells were centrifuged as described previously and resuspended at conc.

-28043336 tration 1x10 ⁶ /ml in 10% AB serum medium and added 50 µl to the appropriate wells. The required number of immunocompetent cells were isolated and centrifuged as described previously, and resuspended at a concentration of 2x10 ⁶ /ml in 10% AB serum medium and 50 μl was added to the appropriate wells. Cells were incubated for 5 days at 37°C and 5% CO ₂ . On the fifth day, 30 μl of the supernatant was removed to test the production of interferon-gamma (IFN-γ). On the fifth day, 25 μl of a 40 mCi/ml solution of tritium-labeled thymidine was added to each well and incubated for 8 h at 37°C and 5% CO ₂ . Cells were transferred to a 96-well microstincillation plate according to the manufacturer's instructions. Counted using a micro-stincillation detector according to the manufacturer's instructions. IFN-γ concentration was determined by ELISA (eBioscience cat #88-7316-88) using the manufacturer's protocol. Assay Data: The average counts per minute (CPM) or IFN-γ concentration for untreated wells was calculated. The average CPM or IFN-γ value was calculated for each study group. Logarithmic transformation of Log 10 data set. Using 12 MLR multiple scores for each compound, the mean was calculated for the set of 12 study groups for each compound mean score for 12 experiments = Σ[(log ₁ o(average MLR of triplicates for test compound))(loglo(average SRM in triplicate without treatment))]/12.

Acceptable standards.

All study reagents and appropriate controls were tested for endotoxin prior to the study and had levels <0.1 EU/mg. The immunocompetent cells themselves had an average CPM level below 700 CPM, indicating that the cells were quiescent when incubated alone. The CPM for the MLR group was at least 2-fold higher than the CPM for immunocompetent cells incubated alone, indicating that a reaction had occurred and that the donors were mismatched. All MLR studies included a negative control of human IgGl protein. The human IgGl negative control results were not significantly different from untreated samples based on Student's t test.

Screening for anti-VISTA antibodies in MLR.

All compounds were preliminarily screened. Before performing MLR with anti-VISTA antibodies, binding of the antibody also to VISTA-bound cells and, by ELISA, to VISTA protein was confirmed by FACS analysis. Antibodies S26 (VSTB77), S30 (VSTB86), S31 (VSTB88), S32 (VSTB90) and S39 (VSTB74) failed this preliminary screening but remained to be tested in the assay. For the purpose of primary screening, all antibodies were tested at 10 μg/ml in proliferating MLR and IFN-γ parameters were measured (FIGS. 21A-21D and 22A, 22B).

Selection of six leading antibodies.

For the initial screening, six candidates were selected for further analysis: VSTB112 (S2), VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 (S47).

Dilution studies of six candidate MLRs: protocol amendments.

The protocol is identical to that described above, with the modification that antibodies were diluted to the following concentrations: 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 μg/ml.

Determination of 1C ₅₀ values.

Crude indicators of CPM and IFN-γ concentration were used to determine 1C ₅ o for each of the antibodies. 1C ₅ o calculations were determined using the EZ-R stats program. Six individual immunocompetent cells were used to determine 1C ₅ o values. Individual values of CPM and IFN-γ concentration in MLR with dose selection of leading candidates.

Table 8

_1C5o values for both CPM and IFN-γ in MLR

VSTB112 (S2) VSTB116 (S5) VSTB95 (S16) VSTB5O (S41) VSTB53 (S43) VSTB60 (S47) SRM -0.67 -0.78 -0.54 -0.12 -0.33 0.02 Gamma -0.42 -0.16 0.22 0.06 0.27 0.4

**Values are expressed in _log10 antibody concentrations.

Conclusion.

Primary screening showed that multiple VISTA-specific antibodies were able to enhance the MLR cellular immune response. The antibodies were then ranked based on potency and variability and, based on these results, VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, and VSTB60 were selected for evaluation in dose-finding experiments. VSTB60 induced a weaker response than the other five antibodies in dose-finding experiments.

Staphylococcus enterotoxin B (SEB) activation assay.

SEB is a bacterial super-antigen that causes activation of specific νβ ⁺ T cells. Peripheral blood mononuclear cells were isolated and incubated with SEB antigen in culture, which induced sustained cytokine production. This analysis was carried out on five leading candidates.

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Preparation of 10% AB medium, preparation of 1 mCi of tritium-labeled thymidine, preparation of VISTA antibody solution, and isolation of leukocytes were carried out as previously described above, in

MLR.

Preparation of SEB 96-well plate.

The appropriate number of immunocompetent cells required for analysis was determined based on the number of samples analyzed. The population of immunocompetent cells was seeded at 2.0x10 ⁵ cells/well in a 96-well plate with a U-shaped bottom. All conditions had to be implemented in triplicate. Cells were centrifuged as previously described above and resuspended at a concentration of 4x10 ⁶ /ml in 10% AB serum medium and 50 μl was added to the appropriate wells. 50 μl of 10% AB serum medium containing SEB antigen at a concentration of 40 ng/ml was added. In the experiments described, SEB was obtained from Sigma Aldrich (cat #S0812). The final concentration per well was 10 ng/ml. Cells were incubated for 3 days at 37°C and 5% CO2. On the third day, 30 μl of the supernatant was removed for analysis of IFN-γ production. Add 25 μL of 1 mCi/mL tritium-labeled thymidine solution to each well and incubate for 8 h at 37°C and 5% CO2. Cells were transferred to a 96-well micro-stincillation plate according to the manufacturer's instructions. Counted using a micro-stincillation detector according to the manufacturer's instructions. IFN-γ concentration was determined by ELISA (eBioscience cat #88-7316-88) using the manufacturer's protocol.

Protocol: data analysis.

The average counts per minute (CPM) or IFN-γ concentration was calculated for each antibody at all concentrations. Eligibility criteria were performed as previously described. Determination of IC ₅₀ values was carried out as described. Selected CPM and IFN-γ concentrations in SEB dose-finding analysis of leading candidates.

Table 9

IC ₅₀ values for both CPM and IFN-γ in SEB.

VSTB112 (52) VSTB116 (S5) VSTB95 (S16) VSTB50 (S41) VSTB53 (S43) VSTB60 (S47) SRM -1.16 -1.44 -1.12 -0.74 -1.06 Not done Gamma -1.24 -0.35 0.05 1.69 -1.05 Not done

**Values are expressed in _log10 antibody concentrations. Conclusions.

VISTA-specific antibodies enhanced cytokine production and proliferation in a dose-dependent manner in the SEB assay. The IC ₅₀ values from the SEB study were generally similar to the results from the MLR dilution studies.

Example 10 Epitope-specific sorting analysis.

Ways.

The ProteOn XPR36 system (BioRad) was used to perform epitope-binding sorting. ProteOn GLC chips (BioRad, cat #176-5011) were coated with two sets of 6 monoclonal antibodies (mAbs) using the manufacturer's instructions for the amine compound chemistry (BioRad, cat #176-2410). Competing mAbs were preincubated in excess (final concentration 250 nM) with human VISTA (final concentration 25 nM) for 4 h at room temperature and 6 at the same time performed on the chip coated with panels of coated mAbs for a 4 min time association followed by dissociation for 5 min. After each run, the chips were reconstituted with 100 mM phosphoric acid.

Analysis of the resulting data involved grouping all sensorgrams by ligand and using an alignment wizard that automatically performs alignment of the X and Y axes and artifact removal. Interspot correction was then applied to the data.

Non-competitive mAbs were defined as having the same or >A1 binding signal (binding to human VISTA only).

Competitive mAbs were defined as having a binding signal <<A1 signal (ie binding only to human VISTA).

Results.

In the example sensorgram shown in FIG. 23, VSTB85 antibody was coated on a Proteon SPR chip and the VISTA protein was preincubated with the indicated chip competitors. VSTB50 is an example of a non-competitive antibody that was observed as a positive response when the VISTA/VSTB50 complex was performed. GG8, VSTB49, and VSTB51 formed a complex with VISTA without binding to VSTB85 coated on the chip and were, for this reason, qualified as competing for the same binding site on VISTA as VSTB85.

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Table 10

Trial Set #1: Connected to Sensor Trial Set #2: Connected to Sensor

LI L2 L3 L4 L5 L6 LI L2 L3 L4 L5 L6

Samples Group GG8 B85 B95 B104 B112 VIZ B50 B53 B66 B67 IE8 B116 GG8 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1OO.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1O1.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1O2.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1O3.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB104.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1O5.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB106.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB1O7.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB108.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB109.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB11O.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB111.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB112.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB113.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB114.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB115.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB116.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB49.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB51.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB53.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB59.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB65.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB67.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB7O.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB81.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB92.OO1 1 Y Y Y Y Y Y N Y N Y Y Y VSTB95.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB97.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB98.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB99.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB5O.OO1 2 N N N N N N Y N Y N N N VSTB54.001 2 N N N N N N Y N Y N N N VSTB56.001 2 N N N N N N Y N Y N N N VSTB60.001 2 N N N N N N Y N Y N N N VSTB63.OO1 2 N N N N N N Y N Y N N N VSTB66.001 2 N N N N N N Y N Y N N N VSTB73.OO1 2 N N N N N N Y N Y N N N VSTB76.001 2 N N N N N N Y N Y N N N VSTB78.001 2 N N N N N N Y N Y N N N VSTB84.001 2 N N N N N N Y N Y N N N VSTB85.001 3 Y Y Y Y Y Y N Y N Y 1 Y VSTB74.001 4 N N N N N N N N N N N N IE8 5 Y 1 Y Y Y Y N Y N Y Y Y

mAb immobilized on the sensor.

Y = there is competition (signal << than A1 - human VISTA only).

N = no competition (signal > than A1 - human VISTA only).

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I = undefined (signal similar to A1 - human VISTA only).

Example 11 Determination of proteon affinity.

Antibodies were captured on ProteOn chips using anti-IgG Fc coated surfaces. Antibodies were tested for binding to human and cynomolgus (supo) extracellular domains (ECDs) of VISTA at VISTA protein concentrations ranging from 0.39 to 100 nM. Antigens were allowed to bind/bind to the antibody-coated chips for 4 min, after which dissociation was observed for 30 min. The chips were reconstituted by treating twice with 100 mM phosphoric acid for 18 s. All experiments were performed at 25°C and the data were fit to the 1:1 Langmuir binding model.

Example 12. Effect of anti-VISTA treatment in the MB49 mouse model of bladder cancer.

Ways.

C57B1/6 mice were injected with MB49 tumor cells. Once tumors developed, anti-VISTA treatment was started. Tumor growth was then monitored 3 times per week. Mice were sacrificed according to IACUC guidelines once tumors reached 15 mm in any dimension.

For each experiment, a frozen vial of MB49 cells was thawed and grown in RPMI 1640 (+L-Glut) with 10% serum and penicillin/streptomycin antibiotics. After three days in culture, cells were harvested using StemPro Accutase and resuspended in RPMI at a concentration of 5x106 cells/ml and injected at 50 µl per mouse.

Female C57B1/6 mice, 6–8 weeks old, were purchased from the National Cancer Institute. Upon arrival, they were allowed to acclimatize for one day before their right sides were shaved and their tails pierced. Three to five days later they were injected.

Tumor injection (intradermal).

Mice were injected intradermally (i.d.) into their shaved flank with 50 μl of MB49 cell suspension (~250,000 cells). Controlling tumor growth.

Tumor growth was measured using electronic meters firstly at the widest volume (L) and secondly at an angle of 90° to the first dimension (W). Tumor volume was determined as follows:

The tumor was considered established when it reached ~5 mm in diameter (~60 ^mm3 volume). Once created, treatment began. Tumor growth was measured three times a week during treatment and until the experiment was terminated.

Anti-VISTA treatment.

Chimeric 13F3-mIgG2a monoclonal antibodies were administered intraperitoneally at 10 mg/kg. The immunization protocol was three times a week for four weeks.

Killing mice.

In accordance with IACUC guidelines, animals were sacrificed when their tumors reached 15 mm in the longest dimension.

Efficiency analysis.

Mouse tumor volumes were analyzed using Excel for data processing and GraphPad Prism for plotting.

Statistical analysis was performed using macros for R statistical software.

The experimental design is shown in Fig. 24.

Results.

Ch13F3-mIgG2a treatment of female mice resulted in complete tumor rejection (CR) in 70% of animals and partial remission (PR) in 30% (n=7) (Table 13 and FIG. 25B). In contrast, all mIgG2a-treated control mice showed progressive tumor development (6/6) (FIG. 25A). The data showed that anti-VISTA treatment could have a profound effect on tumor growth.

Table 11

Complete remission (CR) versus partial remission (PR)

13F3 lgG2a (n=7) female CR 5 PR 2 up to 32

The sequence of human VISTAs is shown in FIG. 26 and 27, based on Wang et al., 2011, supra, the contents of which are incorporated by reference herein in their entirety.

Example 13 Epitope mapping of anti-VISTA antibodies using hydrogen/deuterium (H/D) exchange assay.

To identify epitopes for the VSTB50, 60, 95 and 112 on human VISTA, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed using the corresponding Fabs. Procedures for H/D exchange used for Fab deviation analysis, ana.

- 32 043336 are logical to those described previously (Hamuro et al., J. Biomol. Techniques, 14:171-182, 2003; Horn et al., Biochemistry, 45:8488-8498, 2006) with some modifications. Fabs were prepared from papain digested IgGs and protein A capture using the Pierce Fab Preparation Kit (Thermo Scientific, cat #44985). The human VISTA protein sequence contained six N-linked glycosylation sites. To improve sequence capture, the protein was deglycosylated with PNGase F. The deglycosylated VISTA protein was incubated in a deuterated aqueous solution for a given time, resulting in the incorporation of deuterium at interchangeable hydrogen atoms. Deuterated VISTA protein was complexed with either Fab VSTB50, VSTB60, VSTB95 or VSTB112 in 46 μl of deuterium oxide (D ₂ O) at 4°C for 30 s, 2, 10 and 60 min. The exchange reaction was quenched by low pH and proteins were destroyed with pepsin. Deuterium levels at specific peptides were monitored from the mass shift on LC-MS. As a reference control, VISTA protein was treated in a similar manner except that it was not complexed with Fab molecules. Fab-bound sites were introduced into regions relatively protected from exchange, and thus containing larger fractions of deuterium than the VISTA reference protein. Approximately 94% of the protein can be mapped to specific peptides.

The HDX-MS VISTA solution comparison variances with VSTB50/VSTB60, and VSTB95/VSTB112 are shown in FIG. 28 above and below respectively. Two groups of epitopes were identified. AntiVISTA VSTB50 recognizes the same epitope as VSTB60 does; VSTB95 binds to a different site epitope, as does VSTB112 on VISTA. Anti-VISTA VSTB50 and 60 share the same epitope, which includes segments, ₁₀₃ NLTLLDSGL ₁₁₁ (SEQ ID NO: 62) and ₁₃₆ VQTGKDAPSNC ₁₄₆ (SEQ ID NO: 63) (Fig. 28 top). Anti-VISTA VSTB95 and 112 appear to target similar epitopes, including segments ₂₇ PVDKGHDVTF ₃₆ (SEQ ID NO: 75) and ₅₄ RRPIRNLTFQDL ₆₅ (SEQ ID NO: 65) (Fig. 28 bottom). There are two other segments showing weak deviations at VSTB95 and 112, including residues 3952 and 118-134. In addition, the levels of decline are not as strong as in the previous sections (27-36 and 54-65) in the differential map. Although one peptide, 100TMR102, showing strong deviations from VSTB95 and 112 is located on the other side of the VISTA surface, it is distant from the epitope regions 27–36 and 54–65. Such deviations may be caused by an allosteric effect. These HDX-MS results determine the level of peptide epitopes for anti-VISTA antibodies. There were no overlapping epitope regions for the two epitope groups. These results were consistent with previous competitive sorting data in which they did not compete with each other.

Example 14 Determination of the structure of the human VISTA ECD:VSTB112 FAB complex by protein crystallography.

To determine the structure of VISTA and to designate the epitope and paratope defining the interaction between the VISTA extracellular domain (ECD) and the Fab fragment of the lead antibody VSTB112, the complex was crystallized and the structure was determined at 1.85 A resolution. The structure of the ECD of human VISTA in complex with the Fab fragment of the antibody VSTB112 was determined in an attempt to determine both the structure of the ECD of VISTA itself and to determine the epitope/paratope for their interaction. The structure revealed VISTA to adopt an IgV loop with a chain topology similar to the TCR Va chain. In addition to the conical disulfide bond bridging the B and F strands at the back and front surfaces of the β-sandwich, the ECD structure showed two additional disulfide bonds, one tethering the CC' loop to the front layer and the second between the A' and G' strands. Although crystalline contacts between VISTA molecules occurred, they were minor and do not provide evidence for a dimer of VISTA ECDs based on this structure. The VSTB112 epitope is shown to include portions of the VISTA BC, CC', and FG loops along with residues of the anterior beta layer (C'CFG) adjacent to those loops. The paratope is biased more toward heavy chain interactions with CDR L3, making minimal contact.

Epitope/paratope defining VISTA interaction.

VSTB112 VSTB112 Fab covers a surface area of 1024.3 A2 upon VISTA ECD binding, with heavy chain surface coverage accounting for 715.3 A2 of the total. Seven hydrogen bonds and 4 salt bridge interactions were formed between VISTA and the light chain of VSTB112, and 10 hydrogen atoms and 2 salt bridge interactions were formed between VISTA and the heavy chain of VSTB112. VSTB112 recognized residues in the leading layer of the C', C, F, and G strands at the ends close to the FG loop, as well as residues in the BC, FG, and CC' loops (Figs. 29 and 30). Interactions with the CC' loop accounted for the majority of contacts with the Fab light chain, with only residues E125 and R127 in the FG loop making additional light chain interactions. Residues 119 to 127, corresponding to the FG loop of VISTA, accounted for 38% of the total 1034.8 A2 surface area closed upon VSTB112 binding. In particular, such a loop is highly polar and included the following sequence IRHHHSEHR- (SEQ ID NO: 76). In addition, W103 in VSTB112 CDR H3 overlapped well against residues H122 and H123 of the VISTA backbone, and H121 VISTA made lateral interactions with the aromatic ring of F55 in CDR H2.

A comparison of epitope regions by crystallography and HDX is shown in FIG. 31.

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Example 15 Activation of T cells and monocytes using anti-VISTA antibodies.

The effect of the functional nature of anti-VISTA antibodies was assessed in two in vitro assays, mixed lymphocyte reaction (MLR) and SEB (Staphylococcus enterotoxin B). Both studies measured T cell proliferation and cytokine production as their primary endpoints, but these effects were due to different mechanisms. In MLR, peripheral blood mononuclear cells (PBMCs) from two different human donors were incubated together, and a major histocompatibility complex (MHC) mismatch between T cells from one donor and dendritic cells from another donor resulted in T cell proliferation and interferon (IFNy) production. . In the SEB study, PBMCs from a single donor were incubated with a bacterial superantigen that directly bound the MHC Class II protein on the surface of antigen presenting cells (APCs) to the T cell receptor (TCR) on T cells, causing T cell activation, proliferation, and production of cytokines. In both studies, VSTB112, which is the primary molecule of VSTB174, showed a dose-dependent induction of T cell proliferation and cytokine production, and was the most active among the candidates (Fig. 21A-21D, Table 12).

Table 12 _____________EC50 values for MLR readout studies_____________

Candidate ECso proliferation (µg/ml) ECso IFNy production (µg/ml) VSTB112 0.21 0.38 VSTB116 0.17 0.69 VSTB95 0.29 1.67 VSTB5O 0.77 1.14 VSTB53 0.47 1.88 VSTB6O 1.04 2.48

VSTB112 (primary VSTB174) was the most active molecule.

Monocyte activation study.

The research data shown in table. 12 were produced with VSTB112, the primary molecule of VSTB174. To better understand the activity of VSTB174, a monocyte activity assay was performed. The results showed that incubation of VSTB174 with whole PBMCs caused increased expression of activation markers (CD80 and HLA-DR) on CD14+ monocytes, indicating the effect of the antibody binding to a subpopulation of immune cells known to express high levels of VISTA (Figure 32). An additional question is whether any antibody that binds to VISTA and has an IgG1 Fc can influence monocyte activation in whole PBMCs. Antibodies VSTB103 and VSTB63 bound to VISTA with high affinity ( _KD 6.36E-10 and 8.30E-10, respectively) and to cells expressing VISTA protein, similar to VSTB112 and VSTB111. VSTB103 was in the same epitope base pair as VSTB112, whereas VSTB63 was in a different epitope base pair; neither antibody promoted monocyte activation. Overall, these results indicated that one mechanism by which VSTB174 could exert its effect on T cell activation/proliferation was through monocyte activation facilitated by NK cells.

Medium Preparation: 500 ml RPMI 1640 (Corning, 10-040-CV) mixed with 50 ml human AB serum (Valley Biomedical, Inc, Lot #3C0405), 5 ml penicillin/streptomycin (Lonza, 17-602E) 10,000 units/ml , 5 ml L-Glutamine (100x) (Gibco, 25030-081) and 10 ml HEPES (1M) (Fisher BP299-100, Lot #-1). The medium was stored for no more than 14 days at 4°C.

Preparation of VISTA solution and control antibodies.

Antibodies were diluted to 2x the required concentration in 10% serum medium AB: VSTB174: lot VSTB174.003.

Add 100 μl of the appropriate antibody solutions to the corresponding wells of a 96-well U-bottom plate (Falcon, 353077). Different cell populations were then added in an amount of 100 μl, the final concentration of each antibody being 1, 0.1 or 0.01 g/ml. CNTO 3930 IgG1 control antibody (Lot 6405, ENDO <0.1 EU/mg) was added at a final concentration of 1 μg/ml.

Isolation of PBMCs.

Donors were at least 18 years of age, generally healthy, and randomly selected from the local population.

Donor blood was transferred from an isolated tube into a 50 ml conical tube.

ml Ficoll 1077 (SIGMA, 10771) was placed carefully under the blood so as not to mix. This was for 25 ml of blood.

Cells were centrifuged at 1250 g for 25 min at room temperature without braking.

Leukocytes were isolated from Ficoll and serum interphase, and the cells were diluted in 40 ml Hanks' balanced salt solution (HBSS).

Cells were centrifuged at 453 g (1500 rpm) for 10 min at 4°C.

Cells were resuspended in 50 ml HBSS and counted by transferring 500 µl into a separate Eppendorf tube.

In addition, the Pan Monocyte isolation kit from Miltenyi was used according to the manufacturer's instructions (cat #130-096-537) to isolate CD14+ cells by negative selection in multiple treatment groups.

Preparation for in vitro cultivation.

The appropriate required number of cells for the study was determined based on the number of samples analyzed. The population of immunocompetent cells was seeded at a concentration of 2.0x105 cells/well in a 96-well plate with a U-shaped bottom. For the CD14 negatively selected population, 0.5x105 cells were seeded. All conditions were carried out in triplicate. Cells were centrifuged as described above and resuspended at a concentration of 2x10 ⁶ /ml for the whole PBMC population and 0.5x10 ⁶ /ml for the negatively selected CD14 population in 10% serum AB medium and 100 μl of test antibody was added to the corresponding well, bringing the total volume in each well up to 200 µl. Cells were incubated for 1, 2, or 3 days at 37°C and 5% CO ₂ .

Antibody staining and flow cytometry.

A 96-well U-bottom plate was centrifuged for 5 min at 453 g and the supernatant was removed.

Cells were washed with 200 μl PBS and centrifuged as in step 5.5.1.

The supernatant was discarded and resuspended in 50 μl of PBS containing the following antibodies.

CD14-APC (clone HCD14) 1:250 (Biolegend cat #325608).

HLA-DR-PE Cy7 (clone L243) 1:250 (Biolegend cat #307616).

CD80-PE (clone 2D10) 1:250 (Biolegend cat #305208).

Hu FcR binding inhibitor (eBioscience cat #14-9161-73).

Incubated for 20 min on ice in the dark.

Add 150 µl PBS and centrifuge as in step 5.5.1. 150 μL PBS buffer was added and analyzed by FACS. Samples were run on a Miltenyi MACSQuant 10-parameter flow cytometer and analyzed using FlowJo 9.7.5 for HLA-DR and CD80 expression on the CD14+ population. Geometric mean fluorescence intensity (MFI), a statistic that defines the average value of a set of numbers, was used as the defining statistic for comparing treatments.

Statistical analysis.

All statistics were performed in Prism GraphPad, version 6. Pairwise comparisons between groups were made at each time point using One-Way ANOVA with Tukey's multiplicity correction. P-values less than 0.05 for all studies and comparisons were considered significant. For all graphs and tables * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001.

Example 16. ADCC and ADCP activities of anti-VISTA antibodies.

VSTB174 had IgG1 Fc that could mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) activities. Both types of studies were performed and showed that VSTB174 could lyse or phagocytose K562-VISTA cells (Fig. 33-34), but not the myeloma cell line K562 primary cells (data not shown). An additional mechanism for activating VSTB174 to modulate the inhibitory effects of VISTA could be the lysis or engulfment of cells expressing high levels of VISTA, thereby removing them from the local microenvironment.

Example 17 Activation of ADCP by additional anti-VISTA antibodies.

An in vitro phagocytosis assay was used to study the enhancement of macrophage-mediated phagocytosis of cells ectopically expressing VISTA by anti-human VISTA mAbs (VSTB173 and VSTB174). Such mAbs have been cloned into different Fc backbones (IgG1 WT (wild type), IgG1 PR (protease resistant), and IgG2σ) and have been postulated to have different activities for enhanced phagocytosis. PR backbones IgG1 and IgG1 were able to bind to Fc receptors and had the potential to induce ADCP, whereas IgG2σ did not bind to Fc receptors and did not mediate ADCP.

Anti-VISTA antibodies were tested in an ADCP assay with K562 primary and K562-VISTA target cells. As shown in FIG. 35 and 36, VSTB174, VSTB149, VSTB173 and VSTB145 enhanced hMac phagocytosis of K562-VISTA cells. VISTA antibodies VSTB140 or VSTB132, and IgG2σ Fc, which did not bind to Fc receptors, did not enhance phagocytosis as expected. VISTA mAbs VSTB174 and VSTB173 with IgG1 Fc showed the most potent phagocytosis than VSTB149 and VSTB145 with IgG1PR Fc (see Tables 13 and 14 for EC ₅₀ values).

Table 13

EC values of ₅₀ anti-human VISTA mAb

Treatment VSTB174 VSTB149 VSTB140 eu ₅₀ 0.0782 0.1142 N.A.

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Table 14

EC50 values of anti-human VISTA mAb

Treatment VSTB173 VSTB145 VSTB132 eu ₅₀ 0.0146 0.1075 N.A.

VSTB174 and VSTB173 showed a slight increase in phagocytosis of K562 primary cells at the highest concentration (Fig. 35 and 36), which could be due to low expression of VISTA K562 cells. Other anti-VISTA antibodies did not enhance phagocytosis of K562 cells.

Negative control antibodies were each tested at two different concentrations in the K562-VISTA phagocytosis assay but did not induce any phagocytosis. These results indicated that phagocytosis mediated by anti-VISTA antibodies is specific and associated with the expression of VISTA antigen by K562-VISTA cells.

Example 18 Activation of ADCC by additional anti-VISTA antibodies.

The following three human anti-VISTA antibodies were tested to study their ability to induce ADCC.

VSTB174(IgG1).

VSTB149(IgG1PR).

VSTB174.LF (IgG1 LF (low fucose)).

Each antibody was assayed at six different concentrations within a single plate, in triplicate over two separate experiments for a total of six data points.

VSTB174, VSTB149, and VSTB174.LF each showed measurable ADCC activity at 10, 1, 0.1, and 0.01 μg/ml, while only the LF antibody showed measurable ADCC activity at 0.001 μg/ml; none of the antibodies showed ADCC at 0.0001 μg/ml. Since each of these antibodies had an IgG1 or IgG1 Fc variant, these results were as expected. The LF antibody showed increased ADCC activity as evidenced by the lower EC ₅₀ value for the LF antibody curve (0.002293 μg/ml) compared to the conventional IgG1 antibody curve (0.02381 μg/ml). The IgG1 antibody PR curve had an EC ₅₀ value similar to the conventional IgG1 curve (0.01846 μg/ml).

Table 15

EC ₅₀ values (μg/mL) of three anti-VISTA antibody studies as determined by ADCC assay

anti-VISTA antibody EC 50 (µg/ml) VSTB174 (IgGl) 0.02381 VSTB149 (IgGl PR) 0.01846 VSTB174.LF (IgGl LF) 0.002293

All human IgG1, human IgG1 PR, and human IgG1 LF showed measurable ADCC mediated cytolysis at antibody concentrations of 10, 1, 0.1, and 0.01 μg/ml, while only LF antibodies showed cytolysis at 0.001 μg/ml antibody concentration. None of the anti-VISTA antibodies showed cytolysis at an antibody concentration of 0.0001 μg/ml.

The LF antibody showed approximately 10-fold greater ADCC-attenuating activity than either of the IgG1 regulatory antibodies or the IgG1 PR antibodies, as indicated by EC ₅₀ values.

Example 19: VSTB174 affinity for human and cynomolgus monkey VISTA.

The affinity of VSTB174 for the extracellular domain (ECD) of human and cynomolgus VISTA was determined using the surface plasmon resonance (SPR) method on a ProteOn instrument. VSTB174 displayed very similar K _D values for each protein, 1.56E-10 M for human ECD VISTA and 8.66E-11 M for cynomolgus VISTA.

Example 20. VISTA antibodies showing efficacy in mouse tumor models.

Mouse strains, reagents and tumor models.

For in vivo studies, human VISTA knockin (VISTA-KI) mice backcrossed into phonon C57B1/6 were used.

Anti-human VISTA antibodies were generated for studies in VISTA-KI mice using the variable region VSTB174 grafted into mouse IgG2a Fc (VSTB123).

MB49 bladder cancer was assessed in VISTA KI mice.

In addition to published studies showing that anti-VISTA antibody therapy inhibited tumor growth in wild-type mice (Le Mercier et al., 2014), an antitumor effect was shown in a hamster mimic antibody in wild-type mice using different dosing regimens and in VISTA-KI mice treated with VSTB123.

Efficacy of in vivo studies in the MB49 VISTA-KI mouse tumor model.

Studies of the effectiveness of MB49 were carried out on VISTA-KI female mice, and VSTB123 was studied in several doses, ranging from 1-10 mg/kg. Mice were injected intradermally with 250,000 MB49 tumor cells on day 0. On day 6, dosing began as shown in FIG. 37 (or 10 mg/kg isotype

Claims (54)

ical control mIgG2a, or the indicated doses of VSTB123; 10 mice/group). VSTB123 was more effective at high doses compared to low doses, as shown in Fig. 37 Doses of 10 and 7.5 mg/kg were equivalent, whereas tumors grew more rapidly in mice given 5 or 1 mg/kg. Example 21 Determination of VISTA expression in human tumors with anti-VISTA antibodies. In fig. Figure 1 shows VISTA expression by an AML tumor cell line—this and the RNAseq expression data in FIG. 17 supported the idea that VISTA was expressed by AML cells and that the anti-VISTA drug was effective by directly targeting these cells for immune modulation or antibody-mediated cytolysis. Data to evaluate VISTA expression in lung cancer were obtained from surgically excised lung cancer specimens. Cells were dissociated and characterized for expression of VISTA and many other markers. The results showed that 13/13 lung tumors (squamous or adenocarcinomas) included CD14+VISTA+ myeloid cells (Fig. 38). Example 22 Determination of VISTA expression in lung tumors using anti-VISTA antibodies. An immunohistochemistry assay was developed using clone GG8, a mouse anti-human VISTA IgG1. Such mAbs were used to study VISTA staining in non-small cell lung carcinoma (NSCLC) FFPE tumor regions. FFPE tumor sections were processed using standard antigen retrieval techniques prior to staining. GG8 mouse anti-human VISTA antibodies were used at a 1:500 dilution. GG8 binding was determined using rabbit anti-mouse polyclonal antibodies followed by HRP anti-rabbit polymer. Subsequent counterstaining with hematoxylin, tumor areas were then assessed. VISTA expression in lung cancer was primarily restricted to the immune infiltrate (an example is shown in FIG. 39) and high levels of VISTA positive cells were present in many lung cancer specimens. Example 23. Structure of the extracellular domain (ECD) of human VISTA in complex with the FAB fragment of VSTB174. VISTA antigen variants were prepared and purified for crystallography. Recombinant his-tagged VSTB174 Fab was internally expressed and purified. Crystals were prepared and used to collect high-resolution data for the VISTA ECD:VSTB174 Fab complex using magnetic bremsstrahlung, and structural analysis was resolved using combinations of homology modeling and electron density analysis (Fig. 29 top). The structure of the VISTA ECD:VSTB174 Fab complex was determined by X-ray crystallography at a resolution of 1.85A, providing the primary structure of VISTA ECD and delineating the VSTB174 epitope and paratope. The ECD VISTA adopted an IgV loop with a topology similar to the CTLA-4 ECD, but retaining a unique G' strand that extended to the anterior layer of the P sandwich. A' and G' are further linked chemically through a disulfide bridge formed between residues C12 in the A' strand and C146 in the G' strand. Six cysteines are found occupied by three intramolecular disulfide bonds and, based on crystal contacts, there is no evidence for dimeric VISTA. VSTB174 recognized residues in the front layer of the C', C, F, and G strands at the ends closest to the FG loop, as well as residues in the BC, FG, and CC' loops. The teachings in all patents, published applications and abstracts cited in the present invention are incorporated by reference in their entirety. While the present invention has been shown and described in detail with reference to an exemplary embodiment of the present invention, it will be apparent to those skilled in the art that various changes in form and description may be made within the scope of the present invention, including the appended patent claims. CLAIM 1. An isolated antibody or fragment of such an antibody, comprising an antigen binding region that binds to the V domain of suppressor of T cell activation (VISTA) Ig, wherein the antibody contains (a) heavy chain hypervariable regions (CDRs) corresponding to SEQ ID NO: 25- 27, and light chain CDRs corresponding to SEQ ID NO: 28-30; or (b) heavy chain CDRs corresponding to SEQ ID NO: 19-21, and light chain CDRs corresponding to SEQ ID NO: 22-24. 2. The antibody or antibody fragment of claim 1, wherein the antibody fragment is a Fab, F(ab')2 or scFv antibody fragment. 3. The antibody or antibody fragment of claim 1, which includes a constant region of a human antibody. 4. An antibody or antibody fragment according to any one of claims 1 to 3, comprising at least one human antibody heavy chain and at least one human antibody light chain. - 37 043336 5. The antibody or antibody fragment of claim 4, wherein the antibody contains the CDR sequences specified in claim 1(a) and contains at least one heavy chain including the heavy chain variable region sequence of SEQ ID NO: 37, or where the antibody contains the CDR sequences specified in claim 1(b), and contains at least one heavy chain including the heavy chain variable region sequence SEQ ID NO: 40. 6. The antibody or antibody fragment of claim 4, wherein the antibody contains the CDR sequences specified in claim 1(a) and contains at least one light chain comprising the light chain variable region sequence of SEQ ID NO: 44, or where the antibody contains the CDR sequences specified in claim 1(b), and contains at least one light chain including the light chain variable region sequence SEQ ID NO: 43. 7. The antibody or antibody fragment of claim 4, wherein the antibody contains the CDR sequences specified in claim 1(a) and contains at least one heavy chain comprising the heavy chain variable region sequence of SEQ ID NO: 37, and at least at least one light chain comprising the light chain variable region sequence of SEQ ID NO: 44, or wherein the antibody contains the CDR sequences specified in claim 1(b) and contains at least one heavy chain comprising the heavy chain variable region sequence of SEQ ID NO: 40, and at least one light chain comprising the light chain variable region sequence of SEQ ID NO: 43. 8. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody is a monoclonal antibody. 9. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody is a humanized antibody. 10. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody binds to a VISTA epitope with a K _D of at least 1x10'9 M. 11. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody binds to a VISTA epitope with a K _D of at least 1x10'8 M. 12. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody binds to a VISTA epitope with a K _D of at least 1x10' ⁷ M. 13. The antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody or antibody fragment modulates an immune response, said modulation including an increase in CD45+ leukocytes, an increase in CD4+ T cells, an increase in CD8+ T cells, or a combination thereof; or a decrease in VISTA-expressing immune cells. 14. An antibody or antibody fragment according to any one of claims 1 to 7, wherein the antibody or antibody fragment modulates an immune response, said modulation including increased cytokine production, increased T cell response, and/or modulation of Foxp3 expression. 15. The antibody or fragment of such an antibody according to claim 1, wherein the antibody or antibody fragment contains the CDR sequences specified in claim 1(a). 16. The antibody or antibody fragment of claim 15, wherein the VH domain of the antibody and the VL domain of the antibody each include one or more humanized or human framework regions. 17. The antibody or antibody fragment of claim 16, wherein the VH domain of the antibody includes SEQ ID NO: 37. 18. The antibody or antibody fragment of claim 16 or 17, wherein the VL domain of the antibody includes SEQ ID NO: 44. 19. The antibody or antibody fragment of any one of claims 15 to 18, wherein the antibody comprises a human antibody heavy chain constant region. 20. The antibody or antibody fragment of claim 19, wherein the human heavy chain constant region is a human IgG1 heavy chain constant region. 21. The antibody or antibody fragment of claim 20, wherein the IgG1 heavy chain constant region comprises the amino acid sequence of the IgG1 heavy chain constant region present in SEQ ID NO: 61. 22. The antibody or antibody fragment of claim 20, wherein the IgG1 heavy chain constant region has been modified to enhance protease resistance of the antibody. 23. The antibody or antibody fragment of claim 22, wherein the IgG1 heavy chain constant region that has been modified to enhance protease resistance of the antibody includes the IgG1 heavy chain constant region amino acid sequence present in SEQ ID NO: 60. 24. An antibody or antibody fragment according to any one of claims 19 to 23, wherein the antibody comprises a human antibody light chain constant region. 25. The antibody or antibody fragment of claim 24, wherein the human antibody light chain constant region comprises the light chain constant region amino acid sequence present in SEQ ID NO: 56. 26. An antibody or antibody fragment according to any one of claims 15 to 25, wherein the antibody includes a heavy chain containing SEQ ID NO: 60 and a light chain containing SEQ ID NO: 56. 27. An antibody or antibody fragment according to any one of claims 15 to 25, wherein the antibody includes a heavy - 38 043336 a chain containing SEQ ID NO: 61, and a light chain containing SEQ ID NO: 56. 28. The antibody or antibody fragment according to any one of claims 15 to 27, wherein the antibody or antibody fragment is expressed in a cell deficient in fucosylation enzymes. 29. The antibody or antibody fragment of claim 28, wherein the cell is a Chinese hamster ovary (CHO) cell. 30. A composition for treating cancer in an individual in need thereof, comprising an antibody or antibody fragment according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier, diluent or excipient. 31. The composition of claim 30, further comprising a vaccine selected from a viral vector vaccine, a bacterial vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine or a protein vaccine. 32. A composition for treating cancer in a subject in need thereof, comprising an antibody or antibody fragment according to any one of claims 15 to 29 and a pharmaceutically acceptable carrier, diluent or excipient. 33. An isolated nucleic acid, which includes a nucleotide sequence encoding an antibody or antibody fragment according to claim 1. 34. An expression vector comprising a nucleic acid according to claim 33, operably linked to a promoter. 35. A host cell transformed with the expression vector of claim 34 to produce the antibody or antibody fragment of claim 1. 36. A method for producing an antibody or antibody fragment according to claim 1, where the method includes culturing host cells according to claim 35 and isolating said antibody or antibody fragment. 37. A kit for the treatment of cancer in an individual in need thereof, comprising a composition according to any one of claims 30-32 and a container and further comprising instructions for use or a label indicating that the composition can be used for the treatment of cancer. 38. A method of treating cancer in a subject in need thereof, wherein the cancer is associated with increased expression of VISTA and/or VTSTA-expressing immune cells, comprising administering to the subject an effective amount of an antibody or antibody fragment according to any one of claims 1 to 14. 39. A method of treating or preventing cancer in an individual in need thereof, wherein said cancer is associated with increased levels of VISTA expression and/or VISTA-expressing immune cells, comprising administering to the individual an effective amount of a composition according to claim 30 or 31. 40. The method of claim 38 or 39, wherein the individual is a mammal. 41. The method of claim 38 or 39, wherein the individual is a human. 42. The method of claim 41, further comprising administering a vaccine selected from a viral vector vaccine, a bacterial vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine, or a protein vaccine. 43. The method of claim 38 or 39, wherein the cancer is lung cancer, bladder cancer, breast cancer, leukemia, lymphoma, myelodysplastic syndrome or myeloma, or a combination thereof. 44. The method of claim 43, wherein the lung cancer is non-small cell lung carcinoma (NSCLC). 45. The method of claim 38 or 39, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome or myeloma, or a combination thereof. 46. The method of claim 45, wherein the leukemia is lymphocytic leukemia or myelogenous leukemia. 47. The method according to claim 45, where the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myelogenous) leukemia (AML), chronic granulocytic leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocyte leukemia, or adult T-cell leukemia. 48. The method of claim 47, wherein the cancer is acute myeloid leukemia (AML). 49. The method of claim 47, wherein the cancer is chronic granulocytic leukemia (CML). 50. The method according to claim 38 or 39, where the cancer is a solid tumor. 51. The method of claim 50, wherein the solid tumor is surrounded by tumor stroma containing myeloid cells, T cells, or a combination of myeloid cells and T cells. 52. The method of claim 50 or 51, wherein the solid tumor is infiltrated with myeloid cells, T cells, or a combination of myeloid cells and T cells. 53. A method of inhibiting tumor growth in a subject in need thereof, comprising administering an effective amount of an antibody or antibody fragment according to claims 1 to 29 or a composition containing said antibody or antibody fragment to the subject in need thereof. 54. The method according to any one of claims 38 to 53, wherein the composition, antibody or antibody fragment is administered parenterally or non-parenterally. -