Anti-VISTA antibodies and fragments

Abstract

The present invention relates to novel antibodies and fragments that bind to V-domain Ig inhibitor of T cell activation (VISTA), and methods of making and using the same. Methods of use include methods of treating cancer, including leukemia, lymphoma, solid tumors and melanoma.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 61 / 920,695 filed on December 24, 2013 and U.S. Provisional Application No. 62 / 085,086 filed on November 26, 2014, the entire teachings of which are incorporated herein by reference. Background Art

[0002] In the tumor microenvironment, the expression of immune checkpoint negative regulators by cancer cells or immune cells can inhibit the host's immune response against the tumor. To effectively fight cancer, it is necessary to block tumor-mediated inhibition of the host's immune response. Therefore, there is a need for novel and effective therapeutic agents that inhibit immune checkpoint negative regulators in the tumor microenvironment, wherein the immune checkpoint negative regulators inhibit anti-tumor immune responses. Summary of the invention

[0003] The present invention relates to antibodies and antibody fragments comprising an antigen binding region of a V domain Ig inhibitory factor (VISTA) that binds to T cell activation. VISTA is a checkpoint regulator that negatively inhibits immune responses. 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 in normal human neutrophils, monocytes, and T cell subsets. In addition, cynomolgus monkey cells express VISTA in a pattern similar to that of normal human cells. VISTA is also expressed in peripheral blood cells of cancer patients.

[0004] The antibody or antibody fragment binds to VISTA to regulate or promote an immune response. Antibody fragments may include, for example, Fab, F(ab')2 or scFv antibody fragments. Antibodies or antibody fragments may include antibody constant regions. Antibodies or antibody fragments may bind to VISTA expressed in hematopoietic cells (e.g., bone marrow cells) and / or lymphocytes, monocytes or neutrophils, T cells, natural killer (NK) cells, natural killer T (NKT) cells, tumor cells and / or in the tumor microenvironment (TME). The tumor microenvironment is the cellular environment of the tumor. It may include surrounding immune cells, fibroblasts, blood vessels, other cells, signaling molecules and extracellular matrix.

[0005] The antibody or antibody fragment may include one or more heavy chain complementary determining 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 anti-VISTA antibody described herein, including antibodies named VSTB112 (S2), VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43), and VSTB60 (S47). In some embodiments, the antibody or its fragment is selected from the group consisting of: VSTB112, VSTB95, VSTB116, VSTB50, VSTB53, and VSTB60. In one embodiment, the antibody or fragment includes one or more heavy chain CDRs and one or more light chain CDRs of any anti-VISTA antibody described herein. In some embodiments, the antibody or antibody fragment may further include at least one heavy chain and at least one light chain of any anti-VISTA antibody described herein. In some embodiments, the antibody or antibody fragment comprises at least one heavy chain comprising a heavy chain variable region sequence and / or at least one light chain comprising a light chain variable region sequence. In some embodiments, the antibody comprises a human framework region. In some embodiments, the antibody is a full antibody. In some embodiments, the fragment is an anti-VISTA binding member. In some embodiments, the heavy chain CDR of the antibody is represented by SEQ ID NO: 1, 2 and 3, and the light chain CDR is represented by SEQ ID NO: 4, 5 and 6. In some embodiments, the heavy chain and light chain variable region amino acid sequences are represented by SEQ ID NO: 7 and 8, respectively.

[0006] The present invention also encompasses anti-VISTA antibodies that are substantially similar to the antibodies described herein. For example, in one embodiment, the antibody or fragment comprises an antibody VH domain, the antibody VH domain comprising a VH CDR1 having an amino acid sequence substantially similar to SEQ ID NO: 1, a VH CDR2 having an amino acid sequence substantially similar to SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence substantially similar to SEQ ID NO: 3, and the antibody or fragment further comprises an antibody VL domain, the antibody VL domain comprising a VL CDR1 having an amino acid sequence substantially similar to SEQ ID NO: 4, a VL CDR2 having an amino acid sequence substantially similar to SEQ ID NO: 5, and a VLCDR3 having an amino acid sequence substantially similar to SEQ ID NO: 6.

[0007] The present invention also relates to anti-VISTA antibodies that competitively inhibit the anti-VISTA antibodies described herein or compete for binding with the anti-VISTA antibodies described herein.

[0008] In some embodiments, the anti-VISTA antibody is part of a conjugate, such as a conjugate comprising a cytotoxic molecule described herein or another agent. Such molecules are well known in the art.

[0009] In some embodiments, the antibody or antibody fragment is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody or antibody fragment comprises a human constant region. In some embodiments, the antibody or antibody fragment is specific for an antigenic determinant of VISTA, such as within the amino acid sequence SEQ ID NO:9. In some embodiments, the antibody or antibody fragment is at least 1×10 -7 Liter / mole (e.g. at least 1×10 -8 Liter / mole, for example at least 1×10 -9 Liter / mol) affinity binds to the antigenic determinant of VISTA.

[0010] In some embodiments, regulating immune response comprises increasing CD45+ leukocytes, CD4+ T cells and / or CD8+ T cells. In some embodiments, regulating immune response comprises increasing (e.g., T cell) cytokine (e.g., IFNγ, IL10, TNFα, IL-17) production, increasing T cell response and / or regulating Foxp3 expression.

[0011] The present invention also relates to the following composition, which comprises an antibody or antibody fragment described herein (e.g., an anti-VISTA antibody) and a pharmaceutically acceptable carrier, diluent or excipient. For example, the composition may include a VISTA antagonist, which comprises an antibody or antibody fragment thereof, which comprises an antigen binding region bound to VISTA; and a vaccine (such as a viral vector vaccine, a bacterial vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine). In some embodiments, the composition is a pharmaceutical composition, and the antibody or antibody fragment is bound to VISTA to regulate or promote an immune response.

[0012] The present invention also relates to a method for treating or preventing cancer, comprising administering an effective amount of at least one antibody, antibody fragment or composition described herein to a subject (eg, a mammal, such as a human or non-human animal) in need thereof.

[0013] In some embodiments, the antibody or antibody fragment is bound to VISTA to regulate or promote the immunogenic response to cancer. In some embodiments, cancer is leukemia, lymphoma, myelodysplastic syndrome and / or myeloma. In some embodiments, cancer can be any type or type of leukemia, including lymphocytic leukemia or myeloid leukemia, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, T cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T cell leukemia. In some embodiments, lymphoma is histiocytic lymphoma, and in some embodiments, cancer is multiple myeloma. In some embodiments, cancer is a solid tumor, such as melanoma or bladder cancer. In some embodiments, cancer is lung cancer (eg, non-small cell lung cancer (NSCLC)). Some treatment methods further comprise 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 relates to a method of inhibiting tumor growth in an individual in need thereof, the method comprising administering an effective antibody or antibody fragment or composition as described herein.

[0014] The composition, antibody or fragment or other agent (eg, vaccine) may be administered by any parenteral or enteral method (eg, intravenous (IV), subcutaneous (SQ), or oral (PO)).

[0015] In some embodiments, the composition, antibody or fragment is administered quarterly, weekly, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, other drugs or therapeutic agents are co-administered before, during, or after the antibodies, fragments, and compositions described herein. The co-administered agent can be administered by the same route as the antibody, fragment, or composition or by a different route.

[0016] The present invention also includes methods for preparing antibodies, fragments and compositions, such as methods for preparing antibodies or fragments described herein, which comprise culturing host cells under conditions for preparing the antibodies. The method may further comprise isolating the antibodies. The present invention also relates to nucleic acids (e.g., isolated nucleic acids) comprising nucleotide sequences encoding antibodies and fragments, expression vectors comprising such nucleic acids (e.g., which are operably linked to a promoter), and host cells transformed with such expression vectors.

[0017] The present invention also relates to kits and articles of manufacture comprising a composition comprising an anti-VISTA antibody and a container, and further comprising a package insert or label indicating that the composition can be used to treat cancer.

[0018] The present invention also provides an isolated antibody or antibody fragment thereof, comprising an antigen binding region that binds to a V domain Ig inhibitor of T cell activation (VISTA), wherein the antibody comprises an antibody VH domain, the antibody VH domain comprising a VH CDR1 having an amino acid sequence of SEQ ID NO: 25, a VH CDR2 having an amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having an amino acid sequence of SEQ ID NO: 27, and the antibody further comprises an antibody VL domain, the antibody VL domain comprising a VL CDR1 having an amino acid sequence of SEQ ID NO: 28, a VL CDR2 having an amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody or antibody fragment comprises one or more humanized or human framework regions. In a specific embodiment, the antibody or antibody fragment comprises an antibody VH domain comprising SEQ ID NO: 37 and / or an antibody VL domain comprising SEQ ID NO: 44. In certain embodiments, the antibody comprises a heavy chain constant region (e.g., a human heavy chain constant region) and / or a light chain constant region (e.g., a human light chain constant region, such as the light chain constant region present in SEQ ID NO: 56). The heavy chain constant region is preferably an IgG1 heavy chain constant region (e.g., an IgG1 heavy chain constant region present in SEQ ID NO: 61). In a specific embodiment, the IgG1 heavy chain constant region is modified to improve the protease resistance of the antibody. An example of an IgG1 heavy chain constant region modified to improve protease resistance is an IgG1 heavy chain constant region present in SEQ ID NO: 60. In certain embodiments, the antibody or antibody fragment comprises a heavy chain comprising SEQ ID NO: 60 and a light chain comprising SEQ ID NO: 56; or a heavy chain comprising SEQ ID NO: 61 and a light chain comprising SEQ ID NO: 56. In a specific embodiment, the antibody or antibody fragment is expressed in a cell lacking fucosylation (e.g., a Chinese hamster ovary (CHO) cell lacking fucosylation).

[0019] The present invention also relates to a composition comprising an antibody or an antibody fragment thereof, wherein the antibody or the antibody fragment thereof comprises an antibody VH domain, wherein the antibody VH domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 25, a VH CDR2 having an amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having an amino acid sequence of SEQ ID NO: 27, and the antibody or the antibody fragment thereof further comprises an antibody VL domain, wherein the antibody VL domain comprises a VL CDR1 having an amino acid sequence of SEQ ID NO: 28, a VL CDR2 having an amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 30; and a pharmaceutically acceptable carrier, diluent or excipient.

[0020] In another embodiment, the present invention relates to a method for treating cancer in an individual in need thereof, the method comprising administering an effective amount of an antibody or antibody fragment that binds to a V domain Ig inhibitor of T cell activation (VISTA) to an individual, wherein the antibody comprises an antibody VH domain, the antibody VH domain comprising a VH CDR1 having an amino acid sequence of SEQ ID NO: 25, a VH CDR2 having an amino acid sequence of SEQ ID NO: 26, and a VH CDR3 having an amino acid sequence of SEQ ID NO: 27, and the antibody further comprises an antibody VL domain, the antibody VL domain comprising a VL CDR1 having an amino acid sequence of SEQ ID NO: 28, a VL CDR2 having an amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 30. In a specific embodiment, cancer is lung cancer. In another embodiment, lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the method further comprises administering a second cancer treatment (e.g., surgery, chemotherapy, radiotherapy, biological therapy, immunomodulatory therapy, and combinations thereof).

[0021] The present invention also provides an antibody or an antibody fragment thereof, comprising an antigen binding region that binds to a V domain Ig inhibitory factor (VISTA) of T cell activation, wherein the antibody binds to a conformational antigenic determinant in VISTA (e.g., human VISTA). In some embodiments, the conformational antigenic determinant comprises residues 103-111 (NLTLLDSGL (SEQ ID NO: 62)) and 136-146 (VQTGKDAPSNC (SEQ ID NO: 63)) of human VISTA (SEQ ID NO: 46) or is present in such residues. In another embodiment, the conformational antigenic determinant comprises residues 24-36 (LLGPVDKGHDVTF (SEQ ID NO: 64)), 54-65 (RRPIRDLTFQDL (SEQ ID NO: 65)) and 100-102 (TMR) of human VISTA (SEQ ID NO: 46) or is present in such residues. In another embodiment, the conformational epitope comprises amino acid residues in the FG loop of human VISTA (SEQ ID NO: 46).

[0022] In addition, the present invention relates to a method of increasing an immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or antibody fragment thereof that binds to a V-domain Ig inhibitor of T-cell activation (VISTA), comprising an antigen binding region that binds to VISTA, thereby promoting an immune response to cancer. In a specific embodiment, the immune response is an anti-tumor immune response.

[0023] In another embodiment, the present invention provides a method of inducing a biological response in an individual in need thereof, comprising administering to an individual a therapeutically effective amount of an antibody or an antibody fragment thereof that binds to a V-domain Ig inhibitor of T cell activation (VISTA), which comprises an antigen binding region that binds to VISTA, thereby promoting an 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 cells expressing VISTA; and antibody-dependent cellular phagocytosis (ADCP) of cells expressing VISTA. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] Figures 1A-1C : Curve showing VISTA expression on TF1 AML cells. Expression of VISTA protein obtained by flow cytometry is shown in the TF-1 AML cell line.

[0026] Figure 2A-2E : Curves of staining and gating strategies for identification of human myeloid and lymphoid subsets.

[0027] Figures 3A-3G : Curves showing the expression of VISTA on human bone marrow and lymphoid subsets from healthy normal donors.

[0028] Figure 4 : Curves showing the expression of VISTA on human bone marrow and lymphoid subsets from multiple healthy normal donors.

[0029] Figure 5A-5B : Curves showing the staining and gating strategy for identifying VISTA expression on human monocytes and macrophages.

[0030] Figures 6A-6C : Curves showing the expression of VISTA on human monocytes and macrophages.

[0031] Figures 7A-7E : Curves showing the staining and gating strategy for identifying VISTA expression on human T and NK cell subsets.

[0032] Figures 8A-8G : Curves showing the expression of VISTA on human T and NK cell subsets from healthy normal donors.

[0033] Fig. 9 : Curves showing the expression of VISTA on human T and NK cell subsets from multiple healthy normal donors.

[0034] Figures 10A-10D : Curves showing the staining and gating strategy for identifying VISTA expression on human dendritic cell subsets.

[0035] Figures 11A-11C : Curves showing the expression of VISTA on human dendritic cell subsets and basophils from healthy normal donors.

[0036] Fig.12 : Curves showing the expression of VISTA on human dendritic cell subsets and basophils from multiple healthy normal donors.

[0037] Figures 13A-13D : Analysis of VISTA expression on healthy human peripheral blood cells. Characterization of VISTA expression on healthy human peripheral blood cells obtained using multicolor flow cytometry analysis: Analysis of mononuclear cells (SSC) from whole blood samples from 2 different individuals lo， CD11b hi CD14 hi CD16 -ve CD33 +ve HLA-DR +ve CD19-ve )( Fig.13A ), neutrophils (SSC hi CD177 + CD11b hi CD14 lo CD16 +ve CD33 +ve HLA-DR -ve CD19 -ve )( Fig. 13B ) were isolated using Ficoll gradients to analyze CD4+ T cells (CD3 +ve CD4 +ve )( Fig. 13C ) and CD8+ T cells (CD3 +ve CD8 +ve )( Fig.13D ).

[0038] Figures 14A-14C : Analysis of VISTA expression on peripheral blood cells from lung cancer patients and healthy control donors. Characterization of VISTA expression on peripheral blood cells from lung cancer patients using multicolor flow cytometry analysis: Representative FACS plots from one individual are shown ( Fig.14A ). Peripheral blood mononuclear cells were separated by Ficoll and the mononuclear cells (CD14+CD11b+CD33+HLADR+CD15-) were analyzed ( Fig. 14B ) and bone marrow-derived suppressor cells (CD14-CD11b+CD33-HLADR-CD15+CD16+)( Fig. 14C ) on VISTA expression.

[0039] Figures 15A-15C : Characterization of VISTA expression in peripheral blood cells from patients with colon cancer using multicolor flow cytometry analysis: Representative FACS plots from one individual are shown ( Fig.15A ). Peripheral blood mononuclear cells were separated by Ficoll and the mononuclear cells (CD14+CD11b+CD33+HLADR+CD15-) were analyzed ( Fig. 15B ) and bone marrow-derived suppressor cells (CD14-CD11b+CD33-HLADR-CD15+CD16+)( Fig. 15C ) on VISTA expression.

[0040] Figures 16A-16D : Characterization of VISTA expression on peripheral blood cells in cynomolgus macaques using multicolor flow cytometry: Analysis of mononuclear spheres (SSC) from whole blood of four different monkeys lo CD11b hi CD14hi HLA-DR hi CD16 -ve CD19 -ve )( Fig.16A ) and neutrophil CD11b hi CD14 lo HLA-DR -ve CD16 -ve CD19 -ve ( Fig. 16B ) were used to separate peripheral blood mononuclear cells from three monkeys for analysis of CD4+ T cells (TCRα / β +ve CD4 +ve )( Fig. 16C ) and CD8+T cells (TCRα / β +ve CD8 +ve )( Fig.16D ).

[0041] Fig.17 : Shows the absolute expression values ​​of VISTA RNA in hemoglobin cell lines.

[0042] Fig.18 Mouse A20 cells were stably transfected with GFP or human VISTA. They were incubated with ova peptide and DO11.10 T cells. CD25 expression of T cells was measured 24 hours after the start of incubation. A20-huVISTA cells suppressed CD25 expression of T cells, but this readout was significantly restored by incubation with VSTB95.

[0043] Figures 19A-19F : Curves showing human VISTA ELISA results.

[0044] Figures 20A-20F : Human VISTA FACS results showing anti-VISTA antibodies bound to cells expressing human VISTA.

[0045] Figures 21A-21D : Dilution study of 6 candidate anti-VISTA antibodies from 30 μg / ml to 0.0 μg / ml in mixed lymphocyte reaction.

[0046] Figures 22A-22B : Dilution study of 6 candidate anti-VISTA antibodies from 30 μg / ml to 0.0 μg / ml in SEB analysis (individual CPM counts and IFN-g concentrations).

[0047] Fig.23: Sensorgrams obtained using anti-VISTA antibody VSTB85 and VISTA protein coated on Proteon SPR chips, with the specified competitors (competitors listed in Table 16) passed over the chip.

[0048] Fig.24 : Experimental design of the MB49 mouse bladder tumor model.

[0049] Figures 25A-25B : MB49 tumor growth in female C57BL / 6 mice. The curves illustrate the expression of anti-mouse VISTA antibody ( Fig.25B ) or control IgG ( Fig.25A )-treated individual mice for tumor growth.

[0050] Fig.26 : The amino acid sequence of human VISTA (SEQ ID NO: 46).

[0051] Fig. 27 : Multiple sequence alignment of VISTA orthologs.

[0052] Fig.28 : Regions of human VISTA bound to VSTB50 and VSTB60 antibodies (top) or VSTB95 and VSTB112 antibodies (bottom) as determined by HDX.

[0053] Fig.29 : VISTA antigenic determinant binding to VSTB112. (Top) VISTA is shown in a sketch with the strands labeled. The residues with at least one atom in the residue are dyed blue. Blue and orange balls highlight chain breaks, and cyan and green balls mark the N-terminus and C-terminus of the VISTA structure, respectively. (Below) The sequence of the VISTA construct used in the structure determination. As calculated by PISA, the circle under the sequence is used to indicate the residues that only contact the main chain with VSTB112, the triangle indicates the side chain contact, and the square indicates the side chain contact that produces hydrogen bonds or salt bridge interactions. The shape is dyed to indicate the CDR with the maximum number of atoms in contact with a given residue, wherein the CDR color is defined as in Figure 59. The secondary structural elements are defined as in the program MOE, wherein the yellow arrow represents the β strand and the red rectangle indicates the α helix.

[0054] Fig.30 : VSTB112 paratope. (Top) VISTA antigen is shown in an illustrative manner, and at 5 angstroms of VISTA VSTB112 in the surface is displayed with colors to indicate the CDR consistency specified in the following sequence. The contact framework residues adjacent to the CDR are similar to the corresponding CDR (bottom) sequence dyeing in the VSTB112 Fv region. The colored background specifies the CDR defined according to Kabat. As calculated by PISA, the circle under the sequence is used to indicate the residues that only make the main chain contact with VISTA, the triangle indicates the side chain contact, and the square indicates the side chain contact that produces hydrogen bond or salt bridge interaction.

[0055] Fig.31 : Comparison of antigenic determinant regions identified by crystallography and hydrogen deuterium exchange (HDX). Sequences of VISTA constructs used in structure determination. As calculated by PISA, circles under the sequence are used to indicate residues that only make main chain contacts with VSTB112, triangles indicate side chain contacts, and squares indicate side chain contacts that produce hydrogen bonds or salt bridge interactions.

[0056] Fig.32 : Activation of CD14+ monocytes in whole PBMCs by VSTB174 (derived from VSTB112). In each part of this experiment, cells were incubated with PBS, IgG1 control antibody, or VSTB174 at 1, 0.1, or 0.01 μg / ml. The left panel shows CD80 MFI; the right panel shows HLA-DR MFI (two donors tested and representative results shown).

[0057] Fig.33 : Curve showing the ADCC activity of VSTB174 against K562-VISTA cells.

[0058] Fig.34 : Curve showing ADCP activity of VSTB174 against K562-VISTA cells. The two antibodies depicted have the same Fab, but VSTB174 has an IgG1 Fc and VSTB140 has an Fc-silent IgG2.

[0059] Fig.35 : Curves showing phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAb against K562-VISTA. Each mAb was tested at 7 half-log doses ranging from 0.0008 μg / ml to 0.56 μg / ml.

[0060] Fig.36 : Curves showing phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAbs against the myeloma cell line K562 cells. Each mAb was tested at 7 half-log doses ranging from 0.0008 μg / ml to 0.56 μg / ml.

[0061] Fig.37 MB49 tumor efficacy study evaluating VSTB123 1, 5, 7.5 and 10 mg / kg in female VISTA-KI mice. Tumor volume was approximately 50 mm when dosing began on day 6 post-implantation 3 VSTB123 is a VSTB112 Fab grafted onto a mouse Fc backbone and binds to human VISTA in VISTA-KI mice.

[0062] Fig.38 : The curve shows that CD14+ cells expressing high / moderate levels of VISTA were found in 13 / 13 lung cancer samples as well as in patients' distal lung tissues and peripheral blood.

[0063] Fig.39 : IHC staining of VISTA in lung cancer using GG8. DETAILED DESCRIPTION

[0064] A description of illustrative embodiments of the invention follows.

[0065] The present invention relates to antibodies to a novel immunoglobulin family ligand (Genbank: JN602184) named V-domain immunoglobulin inhibitor of T cell activation (VISTA) (Wang et al., 2010, 2011). VISTA has homology with PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. Specifically, VISTA is constitutively and highly expressed on CD11b high On bone marrow cells and, at lower levels, on CD4 + and CD8 + T cells. The human homolog shares approximately 85% homology with mouse VISTA and has a similar expression pattern (Lines et al., Cancer Research 74:1924, 2014). VISTA expressed on antigen presenting cells (APCs) inhibits CD4 T cells independently of PD-1 via the cognate receptor. + and CD8 +T cell proliferation and cytokine production. In the passive EAE (experimental autoimmune encephalomyelitis) disease model, VISTA-specific monoclonal antibodies increase T cell-dependent immune responses and aggravate the disease. Overexpression of VISTA on tumor cells weakens the protective anti-tumor immunity of hosts with tumors. Studies on human VISTA have determined its inhibitory function on human T cells (Lines et al., Cancer Research 74: 1924, 2014). Studies from Flies et al. also identified VISTA (called PD-1H) as an effective immunosuppressive molecule (Flies et al., 2011). VISTA is further described in detail in U.S. Publication Application US20130177557A1 and U.S. Patent Nos. 7,919,585 and 8,236,304, all of which are incorporated herein by reference in their entirety.

[0066] VISTA is a novel checkpoint negative regulator that suppresses immune responses. As described in Example 12 herein, treatment with VISTA-specific monoclonal antibodies in a murine tumor model has been shown to reverse the suppressive characteristics of the tumor immune microenvironment and increase protective anti-tumor immunity, thus demonstrating the potential of VISTA monoclonal antibodies as novel therapeutic agents for cancer immunotherapy.

[0067] Antibodies and fragments of the present invention

[0068] The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, human antibodies, and anti-idiotype (anti-Id) antibodies, as well as fragments, regions, or derivatives thereof provided by any known technology, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant technology. The anti-VISTA antibodies of the present invention are capable of binding to a portion of VISTA that regulates, modulates, or enhances an immune response. In some embodiments, the antibodies competitively inhibit one or more of the anti-VISTA antibodies described herein. Methods for determining whether two or more antibodies compete for binding to the same target are known in the art. For example, a competitive binding assay can be used to determine whether an antibody blocks the binding of another antibody to a target. Typically, a competitive binding assay includes the use of a purified target antigen (e.g., PD-1) bound to a solid substrate or cell, an unlabeled test binding molecule, and a labeled reference binding molecule. Competitive inhibition is measured by determining the amount of a marker bound to a solid surface or cell in the presence of a test binding molecule. Usually the test binding molecule is present in excess. Typically, when a competing binding molecule is present in excess, it will inhibit specific binding of a reference binding molecule to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. In some embodiments, competitive inhibition is determined using a competitive inhibition ELISA assay.

[0069] Polyclonal antibodies are heterogeneous groups of antibody molecules derived from the serum of animals immunized with an antigen. Monoclonal antibodies contain a substantially homogeneous group of antibodies to a specific antigen, the group containing substantially similar antigenic determinant binding sites. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., ed., 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., ed., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), the contents of which are incorporated herein by reference in their entirety. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD, and any subclass thereof. The fusion tumor producing the monoclonal antibody of the present invention may be cultured in vitro, in situ, or in vivo.

[0070] The present invention also encompasses digestion fragments, designated portions thereof, and variants thereof, including antibody mimetics or antibody portions comprising structures and / or functions that mimic antibodies or designated fragments or portions thereof, including single-chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to mammalian VISTA proteins. For example, the present invention encompasses antibody fragments or portions thereof that can bind to VISTA, including, but not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments. The antibody fragments of the present invention also include antibody fragments described in Aaron L. Nelson, mAbs 2:1, 77-83 (January / February 2010), the contents of which are incorporated by reference in their entirety.

[0071] Such fragments can be prepared, for example, by enzymatic cleavage, synthesis or recombinant techniques known in the art and / or as described herein. Antibodies can also be prepared in various truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. The various portions of the antibody can be chemically joined together by conventional techniques, or can be prepared as contiguous proteins using genetic engineering techniques.

[0072] In one embodiment, the amino acid sequence of an immunoglobulin chain or portion thereof (e.g., a variable region, CDR) has about 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 therein) to the amino acid sequence of the corresponding variable sequence chain described herein. Preferably, 70-100% amino acid identity (e.g., 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) is determined using a suitable computer algorithm known in the art.

[0073] Examples of heavy and light chain variable region sequences are provided herein.

[0074] The antibodies or variants of the present invention specify that the variants may comprise any number of contiguous amino acid residues from the antibodies of the present invention, wherein the number is selected from the group of integers consisting of 10-100% of the number of contiguous residues in the anti-TNF antibody. Optionally, the length of this subsequence of contiguous 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, 210, 220, 230, 240, 250 or more amino acids or any range or value therein. In addition, 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.

[0075] It will be appreciated by those skilled in the art that the present invention includes at least one biologically active antibody of the present invention. The specific activity of a biologically active antibody is 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 a natural (non-synthetic), endogenous or related and known antibody. Methods for analyzing and quantifying measurements of enzyme activity and substrate specificity are well known to those skilled in the art.

[0076] Substantially similar means that the compound is at least 85% (eg, at least 95%) identical to a natural (non-synthetic), endogenous or related and known antibody and has at least 85% (eg, at least 95%) of its activity.

[0077] As used herein, the term "human antibody" refers to antibodies in which substantially all portions of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinges (VL, VH)) are substantially non-immunogenic in humans, with only minor sequence changes or variations. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees and the like), rodents (mice, rats and the like) and other mammals refer to antibodies specific to such species, subgenus, genus, subfamily, family. In addition, chimeric antibodies may include any combination of the above. Such changes or variations may, as appropriate and preferably, retain or reduce immunogenicity in humans or other species relative to unmodified antibodies. Thus, human antibodies are different from chimeric or humanized antibodies. It is noted that human antibodies may be prepared by non-human animals or prokaryotic or eukaryotic cells that are capable of functionally expressing rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. In addition, when the human antibody is a single-chain antibody, it may include a connecting peptide that is not present in a natural human antibody. For example, Fv may comprise a connecting peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such connecting peptides are believed to be of human origin.

[0078] Bispecific, heterospecific, heterobinding or similar antibodies may also be used, which are single strains, preferably human or humanized antibodies with binding specificity for at least two different antigens. In the present invention, one of the binding specificities is for at least one VISTA protein, and the other is for any other antigen. Methods for preparing bispecific antibodies are known in the art. Recombinant preparation of bispecific antibodies can be based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (Milstein and Cuello, Nature 305: 537 (1983)). See also WO 93 / 08829; U.S. Patents Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO 91 / 00360, WO 92 / 00373, EP03089; Traunecker et al., EMBO J. 10:3655 (1991); Suresh et al., Methods in Enzymology 121:210 (1986), each of which is incorporated herein by reference in its entirety.

[0079] In one embodiment, the present invention relates to a bispecific antibody targeting VISTA and a second target protein (e.g., an immune checkpoint protein). Exemplary bispecific antibodies include bispecific antibodies targeting VISTA and PD-L1 and bispecific antibodies targeting VISTA and PD-L2.

[0080] Human antibodies or fragments thereof specific for human VISTA protein can be raised against an appropriate immunogenic antigen, such as a VISTA protein or portion thereof (including synthetic molecules, such as synthetic peptides).

[0081] Other specific or universal mammalian antibodies may be similarly raised. Immunogenic antigen preparation and monoclonal antibody preparation may be performed using any suitable technique.

[0082] For example, fusion tumors are 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, NIH3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or an analog thereof, or a heteromyeloma, a fusion product thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line known in the art, see, e.g., www.atcc.org) with an antibody-producing cell. Antibody producing cells may include isolated or selected spleen, peripheral blood, lymph, tonsil or other immune cells (e.g., B cells) or any other cells expressing heavy or light chain constant or variable or architecture or complementary determining region (CDR) sequences. Such antibody producing cells may be recombinant or endogenous cells, and may also be prokaryotic or eukaryotic cells (e.g., mammals such as rodents, horses, sheep, goats, sheep, primates). See, e.g., Ausubel above and Colligan above, Immunology, Chapter 2, which are incorporated herein by reference in their entirety.

[0083] Antibody-producing cells can also be obtained from peripheral blood or preferably spleen or lymph nodes of humans or other suitable animals immunized with relevant antigens. Any other suitable host cells can also be used to express heterogeneous or endogenous nucleic acids encoding antibodies of the present invention, their specified fragments or variants. Fusion cells (hybridomas) or recombinant cells can be separated using selective culture conditions or other suitable known methods and selected by limiting dilution or cell sorting or other known methods. Cells producing antibodies with desired specificity can be selected by suitable analysis (e.g., enzyme-linked immunosorbent assay (ELISA)).

[0084] As is known in the art and / or as described herein, other suitable methods for preparing or isolating antibodies with the requisite specificity may be used, including, but not limited to, from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA or analogs thereof, display libraries; e.g., 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; WO90 / 14443; WO90 / 14424; WO90 / 14430; PCT / U594 / 1234; WO92 / 18619; WO96 / 07754; EP 614989; WO95 / 16027; WO88 / 06630; WO90 / 3809; U.S. Patent No. 4,704,692; PCT / US91 / 02989; WO89 / 06283; EP 371998; EP 550 400; EP 229 046; PCT / US91 / 07149; or randomly generated peptides or proteins - U.S. Patent Nos. 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862; WO 86 / 05803, EP 590 689, each of which is incorporated herein by reference in its entirety) to select recombinant antibodies or rely on methods of immunizing transgenic animals capable of producing a repertoire of human antibodies (e.g., 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 and related patents and applications are incorporated herein by reference in their entirety).Such techniques include, but are not limited to, ribosome 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)); single cell antibody preparation technology (U.S. Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gel droplet 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)); B cell selection (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 BV, Amsterdam, Netherlands (1988)).

[0085] Methods for engineering or humanizing non-human or human antibodies may also be used and are well known in the art. Generally, humanized or engineered antibodies have one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" 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, at www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html, each of which is incorporated herein by reference in its entirety.

[0086] As known in the art, such imported sequences can be used to reduce immunogenicity or reduce, increase or change binding, affinity, avidity, specificity, half-life or any other suitable characteristics. In general, some or all non-human or human CDR sequences are maintained, and some or all of the non-human sequences of the framework and / or constant region are replaced with human or other amino acids. The antibodies can also be humanized using three-dimensional immunoglobulin models known to those skilled in the art as appropriate, while maintaining high affinity and other favorable biological properties for the antigen. Computer programs that illustrate and present possible three-dimensional conformational structures of selected candidate immunoglobulin sequences can be used. Detecting these presentations allows the analysis of the possible role of residues in the function of candidate immunoglobulin sequences, that is, the analysis of residues that affect the ability of candidate immunoglobulins to bind to their antigens. In this way, framework (FR) residues can be selected from common and imported sequences and combined to achieve desired antibody characteristics (such as increased affinity for the target antigen). In general, CDR residues are directly and most substantially involved in affecting antigen binding. Humanizing or engineering the antibodies of the invention can be performed 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); U.S. Pat. Nos. 5,723,323, 5,976,862, and 6,874,736. No. 5,824514, No. 5,817483, No. 5,814476, No. 5,763,192, No. 5,723,323, No. 5,766,886, No. 5,714,352, No. 6,204,023, No. 6,180,370, No. 5,693,762, No. 5,530,101, No. 5,585,089, No. 5,225,539; No. 4,816,567, each of which is incorporated herein by reference in its entirety, including the references cited therein.

[0087] As described herein and / or known in the art, anti-VISTA antibodies may also be produced by immunizing transgenic animals (e.g., mice, rats, rabbits, hamsters, non-human primates, and the like) of a lineage capable of producing human antibodies. Cells producing human anti-VISTA antibodies may be isolated from such animals and immortalized using a suitable method, such as the method described herein.

[0088] Transgenic animals that produce a repertoire of human antibodies that bind to human antigens can be prepared by known methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016, and 5,789,650 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 0463 151B1, Kucherlapate et al. EP 0710 719A1, Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO 90 / 04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259A2, Lonberg et al. GB 2 272 440A, 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). Generally, these mice contain at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged or capable of functional rearrangement. The endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the ability of the animal to produce antibodies encoded by the endogenous genes.

[0089] Screening for antibodies or fragments that specifically bind to similar proteins can be conveniently achieved using peptide display libraries. This method includes screening a large number of peptides for individual members with desired functions or structures. Antibody screening of peptide display libraries is well known in the art. The length of the presented peptide sequence can be 3 to 5000 or more amino acids, usually 5-100 amino acids long, and usually about 8 to 25 amino acids long. In addition to direct chemical synthesis methods for producing peptide libraries, several recombinant DNA methods have been described. One type includes presenting peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence encoding the specific peptide sequence presented. Such methods are described in PCT Patent Publications Nos. 91 / 17271, 91 / 18980, 91 / 19818, and 93 / 08278. Other systems for generating peptide libraries have the aspects of in vitro chemical synthesis and recombinant methods. See PCT Patent Publication Nos. 92 / 05258, 92 / 14843, and 96 / 19256. See also U.S. Pat. Nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors, and screening kits are available from suppliers such as Invitrogen (Carlsbad, Calif.) and Cambridge antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Patents Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5,856,456 to Dyax; 5,223,409, 5,403,484, 5,571,698, 5,837,500 to Cambridge No. 5,427,908, No. 5,580,717, and No. 5,885,793 to antibody Technologies; No. 5,750,373, No. 5,618,920, No. 5,595,898, No. 5,576,195, No. 5,698,435, No. 5,693,493, and No. 5,698,417 to Genentech.

[0090] The antibodies of the present invention can also be prepared using at least one nucleic acid encoding an anti-VISTA antibody to obtain transgenic animals (such as goats, cows, sheep, and the like) that produce such antibodies in milk. Such animals can be provided using known methods. See, for example, but not limited to, U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489 and similar patents, each of which is incorporated herein by reference in its entirety.

[0091] The anti-VISTA antibodies of the present invention can also be prepared 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 references cited 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 references cited therein. Each of the above references is incorporated herein by reference in its entirety.

[0092] The antibodies of the present invention can bind to human VISTA with a wide range of affinities (KD). In preferred embodiments, at least one human monoclonal antibody of the present invention can optionally bind to human VISTA with high affinity. For example, the human monoclonal antibody can be equal to or less than about 10 -7 KD of M (such as (but not limited to) 0.1-9.9 (or any range or value therein) × 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 or any range or value therein) binds to human VISTA. In some embodiments, the antibody or antibody fragment can be 1×10 -7 Liter / mole (e.g. at least 1×10 -8 Liter / mole, for example at least 1×10 -9 Liter / mol) affinity binds to human VISTA.

[0093] The affinity or avidity of an antibody for an antigen can be experimentally determined using any suitable method. (See, e.g., Berzofsky et al., "Antibody-Antigen Interactions," Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and methods described herein). The affinity of a particular antibody-antigen interaction measured may vary if measured under different conditions (e.g., salt concentration, pH). Therefore, the measurement of affinity and other antigen binding parameters (KD, Ka, Kd) is preferably performed using standardized solutions of antibodies and antigens and standardized buffers.

[0094] Nucleic acid molecules

[0095] Using the information provided herein, such as a nucleotide sequence encoding at least 70-100% of the contiguous amino acids of at least one of a specified fragment, variant or common sequence thereof, or a deposited vector comprising at least one of such sequences, nucleic acid molecules of the present invention encoding at least one anti-VISTA antibody can be obtained using methods described herein or known in the art, the anti-VISTA antibody comprising all of the heavy chain variable CDR regions of SEQ ID NOs: 1, 2 and 3 and / or all of the light chain variable CDR regions of SEQ ID NOs: 4, 5 and 6.

[0096] The nucleic acid molecules of the present invention may be in the form of RNA (such as mRNA, hnRNA, tRNA or any other form) obtained by cloning or prepared by synthetic methods or in the form of DNA (including but not limited to cDNA and genomic DNA). The DNA may be three-stranded, two-stranded or single-stranded or any combination thereof. Any portion of at least one strand of the DNA or RNA may be a coding strand, also known as a sense strand, or it may be a non-coding strand, also known as an antisense strand.

[0097] The isolated nucleic acid molecules of the present invention may include nucleic acid molecules comprising an open reading frame (ORF), such as, 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 or light chain; nucleic acid molecules comprising coding sequences of anti-VISTA antibodies or fragments (e.g., fragments comprising variable regions); and nucleic acid molecules comprising nucleotide sequences different from those described above but still encoding at least one anti-VISTA antibody as described herein and / or known in the art due to the degeneracy of the genetic code. It is conventional for those skilled in the art to produce such degenerate nucleic acid variants encoding specific anti-VISTA antibodies of the present invention. See, for example, Ausubel et al. above, and such nucleic acid variants are included in the present invention.

[0098] As indicated herein, nucleic acid molecules of the invention comprising nucleic acids encoding anti-VISTA antibodies may include, but are not limited to, nucleic acid molecules encoding amino acid sequences of antibody fragments; coding sequences of the entire antibody or a portion thereof; coding sequences of antibodies, fragments or portions thereof and other sequences, such as coding sequences of at least one signal leader sequence or fusion peptide in the presence or absence of the above-mentioned other coding sequences, such as at least one intron, and other non-coding sequences, including, but not limited to, non-coding 5' and 3' sequences, such as transcribed untranslated sequences that play a role in transcription, mRNA processing (including splicing) and polyadenylation signals (e.g., ribosome binding and stability of mRNA); other coding sequences encoding other amino acids, such as coding sequences that provide other functions. Thus, the sequence encoding the antibody may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of a fusion antibody comprising an antibody fragment or portion.

[0099] Human genes encoding the constant (C) regions of the antibodies, fragments and regions of the invention can be derived from human fetal liver libraries by known methods. Human C region genes can be derived from any human cell, including human cells that express and produce human immunoglobulins. Human C H The C region can be derived from any known class or isotype of human H chain, including γ, μ, α, δ or ε and subtypes such as G1, G2, G3 and G4. Because H chain isotypes are responsible for various effector functions of antibodies, C H The choice of region will be guided by the desired effector function, such as complement fixation or activity in antibody-dependent cellular cytotoxicity (ADCC).

[0100] Composition

[0101] The pharmaceutical compositions disclosed herein are prepared according to standard procedures and administered at doses selected to treat (e.g., alleviate, prevent, or eliminate the treated condition, or slow or interrupt its progression) (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, NY, the contents of which are incorporated herein by reference for general descriptions of methods for administering various agents for human therapy). Compositions comprising the disclosed antibodies and agents can be delivered using controlled or sustained release delivery systems (e.g., capsules, biodegradable matrices). Examples of extended release delivery systems for drug delivery suitable for administering the disclosed compounds are described, e.g., in U.S. Pat. Nos. 5,990,092; 5,039,660; 4,452,775; 3,854,480, the entire teachings of which are incorporated herein by reference.

[0102] To prepare a pharmaceutical composition from the anti-VISTA antibodies and / or fragments of the present invention, a pharmaceutically acceptable carrier may be a solid or a liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible particles. For example, the compounds of the present invention may be in powder form for reconstitution during delivery. The solid carrier may be one or more substances, which may also serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material. When in powder form, the carrier is a finely powdered solid that forms a mixture with the finely powdered active ingredient.

[0103] Powders and tablets preferably contain about 1 to about 70% active ingredient. Suitable carriers are 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, cachets, lozenges, fast-melt strips, capsules and pills can be used as solid dosage forms containing the active ingredient suitable for oral administration.

[0104] Liquid form preparations include solutions, suspensions, retention enemas, and emulsions, for example, water or aqueous propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in aqueous polyethylene glycol solution.

[0105] The pharmaceutical composition may be in unit dosage form. In such a form, the composition is subdivided into unit doses containing appropriate amounts of active ingredients. The unit dosage form may be a packaged preparation containing individual amounts of unit doses. The dosage may vary depending on the patient's requirements, the severity of the condition being treated, the compound used, and the route of administration. Determining the appropriate dosage for a particular situation is within the skill of the art.

[0106] In addition, if necessary, the pharmaceutical composition may contain other compatible agents, such as drugs, therapeutic agents or prophylactic agents. Therapeutic agents or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules and organic molecules. Examples of such agents (e.g., anticancer agents) include, but are not limited to, cytotoxins, angiogenesis inhibitors, immunomodulators, tumor immunotherapy agents, and agents for pain relief or to counteract the toxic effects of one or more therapeutic agents (e.g., the use of bisphosphonates to reduce the hypercalcemic effects of glucocorticoids).

[0107] Angiogenesis inhibitors, agents and therapies suitable for use in the compositions and methods described herein include, but are not limited to, angiostatin (a fragment of plasminogen); anti-angiogenic antithrombin III; angiozyme. Bisphosphonates include, but are not limited to, alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate and zoledronate.

[0108] Immunomodulators and therapies 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 (e.g., Nuvion (Protein Design Laboratories), OKT3 (Johnson & Johnson), or anti-CD20 antibodies Rituxan (IDEC)), anti-CD52 antibodies (e.g., CAMPATH1H (Ilex)), anti-CD11a antibodies (e.g., Xanelim (Genentech)); anti-cytokine or anti-cytokine receptor antibodies and antagonists, such as anti-IL-2 receptor antibodies (Zenapax (Protein Design Laboratories), or anti-CD20 antibodies (Rituxan (IDEC)); Design Laboratories), anti-IL-6 receptor antibodies (e.g., MRA (Chugai)) and anti-IL-12 antibodies (CNTO1275 (Janssen)), anti-TNFα antibodies (Remicade (Janssen)) or TNF receptor antagonists (Enbrel (Immunex)), anti-IL-6 antibodies (BE8 (Diaclone)) and siltuximab (CNTO32 (Centocor)), and antibodies that immunospecifically bind to tumor-associated antigens (e.g., trastuzimab (Genentech)).

[0109] Tumor immunotherapy agents suitable for the compositions and methods described herein include, but are not limited to, ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), anti-PD-L1 antibodies, and anti-LAG-3 antibodies.

[0110] The composition is preferably prepared 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, in addition to the active ingredient, tablets and capsules may contain conventional carriers, such as binders, such as gum arabic, gelatin, polyvinyl pyrrolidone, sorbitol or tragacanth; fillers, such as calcium phosphate, glycine, lactose, corn starch, sorbitol or sucrose; lubricants, such as magnesium stearate, polyethylene glycol, silicon dioxide or talc; disintegrants, such as potato starch; flavorings or coloring agents; or acceptable wetting agents. Oral liquid preparations, usually in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs, may contain conventional additives, such as suspending agents, emulsifiers, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include gum arabic, almond oil, ethanol, partially fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methylcellulose, methyl or propyl paraben, propylene glycol, sorbitol or sorbic acid.

[0111] Other general details regarding methods of making and using the compounds and compositions described herein are well known in the art. See, e.g., U.S. Pat. No. 7,820,169, the entire contents of which are incorporated herein.

[0112] Treatment

[0113] Those skilled in the art (e.g., clinicians) may determine the appropriate dose and route of administration of a particular antibody, fragment, or composition to a subject by considering the selected agent, pharmaceutical formulation, and route of administration, various patient factors, and other considerations. The dose preferably does not cause or produce minimal or no adverse side effects. In a standard multiple dosing regimen, a pharmacological agent may be administered over a dose schedule designed to maintain a predetermined or optimal plasma concentration in the subject being treated. Antibodies, fragments, and compositions may be added in any appropriate dose range or therapeutically effective amount, such as 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg In some embodiments, the composition, antibody or fragment is administered at a dosage of 0.1-15 mg / kg.

[0114] The antibody or fragment may be administered once, at least once, twice, at least twice, three times, or at least three times per 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, or at least six times per 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.

[0115] Anti-VISTA antibodies, fragments and compositions can be administered, for example, via parenteral or enteral methods, including but not limited to intravenous, subcutaneous, oral, rectal, intramuscular, intraperitoneal, transmucosal, transdermal, intrathecal, nasal or surface administration. A general technician should recognize that the following dosage forms may include a compound or a corresponding pharmaceutically acceptable salt of a compound of the present invention as an active ingredient. In some embodiments, the dosage form may include a compound or a corresponding pharmaceutically acceptable salt of a compound as an active ingredient.

[0116] The anti-VISTA antibodies of the present invention can be administered as part of a combination therapy (e.g., administered together with each other or with 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, the compounds of the present invention and other therapeutic agents may be administered simultaneously (e.g., synchronously) in the form of separate formulations or combined formulations. Alternatively, the medicament may be administered sequentially in the form of separate compositions within an appropriate time range (e.g., time sufficient to overlap the drug effects of the therapy) determined by a skilled clinician. The compounds of the present invention and one or more other therapeutic agents may be administered in a single dose or multiple doses in an order and schedule suitable for achieving the desired therapeutic effect.

[0117] The present invention also provides a method for regulating or treating at least one malignant disease of a cell, tissue, organ, animal or patient. In some embodiments, the compounds and compositions of the present invention are used to treat or prevent cancer. Cancer may include any malignant or benign tumor of any organ or body system. Examples include, but are not limited to, the following: breast cancer, digestive tract cancer, gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, urogenital cancer, germ cell cancer, gynecological cancer, head and neck cancer, blood / blood cancer, muscle bone cancer, nerve cancer, respiratory tract / thoracic cancer, bladder cancer, colon cancer, rectal cancer, lung cancer, endometrial cancer, kidney cancer, pancreatic cancer, liver cancer, stomach cancer, testicular cancer, esophageal cancer, prostate cancer, brain cancer, cervical cancer, ovarian cancer and thyroid cancer. Other cancers may include leukemia, melanoma and lymphoma and any cancer described herein. In some embodiments, solid tumors are infiltrated with bone marrow and / or T cells. In some embodiments, cancer is leukemia, lymphoma, myelodysplastic syndrome and / or myeloma. In some specific examples, cancer can be any kind or type of leukemia, including lymphocytic leukemia or myeloid leukemia, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, T cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T cell leukemia. In some specific examples, lymphoma is histiocytic lymphoma, follicular lymphoma or Hodgkin's lymphoma (Hodgkin lymphoma), and in some specific examples, cancer is multiple myeloma. In some specific examples, cancer is a solid tumor, such as melanoma or bladder cancer. In one embodiment, cancer is lung cancer, such as non-small cell lung cancer (NSCLC).

[0118] The present invention also provides a method of regulating 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 myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma (Kaposi's In some embodiments, the solid tumor is infiltrated with bone marrow and / or T cells. In a specific embodiment, the solid tumor is lung cancer, such as non-small cell lung cancer (NSCLC).

[0119] In some embodiments, 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, e.g., 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 in their entirety.

[0120] In some embodiments, the compounds and therapies described herein are co-administered with agents and / or bisphosphonates used for chemotherapy, hormone therapy, and biological therapy. In some embodiments, the agents used for chemotherapy include one or more of the following: arboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), doxorubicin (Adriamycin), etoposide (VePesid), 5-fluorouracil (5-FU) , gemcitabine (Gemzar), irinotecan (Camptosar), paclitaxel (Taxol), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban).

[0121] In other embodiments, the anti-VISTA compounds and therapies described herein are co-administered with one or more of the following immune checkpoint antibodies, such as nivolumab, pembrolizumab, tremelimumab, ipalimumab, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-TIM-3 antibodies, anti-LAG-3v, anti-OX40 antibodies, and anti-GITR antibodies.

[0122] In another embodiment, the anti-VISTA compounds and therapies described herein are co-administered with a small molecule inhibitor of indoleamine 2,3-dioxygenase (IDO).

[0123] The anti-VISTA compounds and compositions of the present invention can be administered to individuals in need to prevent (including preventing the recurrence of cancer) or treat (e.g., control or improve cancer or one or more symptoms thereof) cancer. Any agent or therapy (e.g., chemotherapy, radiation therapy, targeted therapy (such as imatinib, sorafenib, and vemurafenib), hormone therapy, and / or biological therapy or immunotherapy) can be used in combination with the compounds or compositions of the present invention described herein. Anticancer agents include, but are not limited to, 5-fluorouracil; acivicin; aldesleukin; altretamine; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; azacitidine; azetepa; azotomycin; batimastat; bicalutamide; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; carboplatin; carmustine; carubicin hydrochloride; hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate mesylate; diaziquone; docetaxel; cranberries; cranberries hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate;duazomycin, edatrexate, eflornithine hydrochloride, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, fazarabine, fenretinide, floxuridine, fludarabine phosphate phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II or rIL2), interferon α-2a; interferon α-2b; interferon α-m; interferon α-n3; interferon β-I a; interferon γ-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril;mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; ormaplatin; paclitaxel; pegaspargase; porfromycin; prednimustine; procarbazine hydrochloride; puromycin; rogletimide; safingol hydrochloride; semustine; simtrazene; sparlfosate sodium; sparsomycin; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate; trimetrexate glucuronide glucuronate; triptorelin; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;Vincain sulfate (vinglycinatesulfate); vinleurosine sulfate (vinleurosine sulfate); vinorelbine tartrate (vinorelbinetartrate); vinrosidine sulfate (vinrosidine sulfate); vinzolidine sulfate (vinzolidine sulfate); vorozole (vorozole); zeniplatin (zeniplatin); zinostatin (zinostatin); zorubicin hydrochloride (zorubicin hydrochloride). Targeted therapy includes (but is not limited to) tyrosine kinase inhibitors (such as imatinib, sorafenib and vemurafenib). The present invention also encompasses the administration of the anti-VISTA compounds of the present invention in combination with radiation therapy, which includes the use of x-rays, gamma rays and other radiation sources to kill cancer cells. Cancer treatment is known in the art and is described in literature such as Physician's Desk Reference (57th Edition, 2003). ;

[0124] The anti-VISTA antibodies described herein are also useful, for example, for the treatment of chronic infectious diseases (such as HIV, HBV, HCV, and HSV, among others).

[0125] Various properties and sequence information used to select anti-VISTA antibodies of the present invention are provided in Tables 1A, 1B and 2 herein.

[0126] Table 1A: CDR sequences of selected fully human or humanized anti-human VISTA antibodies

[0127]

[0128]

[0129] Table 1B: Heavy and light chain sequences of selected fully human or humanized anti-human VISTA antibodies

[0130]

[0131]

[0132]

[0133] *The constant region sequences in VSTB140, VSTB149 and VSTB174 are underlined.

[0134] Δ The amino acid residues in the heavy chain of VSTB149 that confer protease resistance are indicated in bold.

[0135] Table 2: Dissociation constants (K) of selected anti-VISTA antibodies D )

[0136]

[0137]

[0138] Example

[0139] Example 1: Analysis of VISTA expression on human hematopoietic cells

[0140] method:

[0141] Preparation and staining of fresh human PBMCs for VISTA expression

[0142] The expression of VISTA was tested on freshly isolated human PBMCs (peripheral blood mononuclear cells) from several donors. Anti-human VISTA-biotin (GA-1) was used for staining (5 μg / ml). Mouse IgG1, K-biotin (pure MOPC-21, 5 μg / ml) was used as an isotype control.

[0143] Donor material

[0144] Blood samples were obtained from Biological Specialty, Inc. (Colmar, PA) and analyzed the same day. 10 ml of whole blood containing heparin sulfate was delivered by express delivery for analysis.

[0145] Sample preparation

[0146] Blood was diluted 1:1 with sterile PBS. In a 50ml conical tube, 22ml of diluted cord blood was layered on 20ml sterile Ficoll-Hypaque (GE Healthcare catalog number 17-144003). The tube was centrifuged at 1800rpm for 20 minutes at room temperature. Mononuclear cells at the interface after centrifugation were collected using a 1ml pipette and merged in two 50ml conical tubes. Sterile PBS was added to each tube to make the volume up to 50ml and the cells were centrifuged at 300g for 10 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 50ml sterile PBS and the tube was spun at 300g for 10 minutes at 4°C. The supernatant was discarded. The cells were merged and resuspended in 50ml sterile PBS and then counted.

[0147] Staining solution: Use a 5×10 7 A cryopreserved vial of PBMCs was prepared for compensation control and used as a staining control.

[0148] The following reagents and / or consumables were used:

[0149] FACS staining buffer (BSA) from BD Biosciences (Cat. No. 554657) supplemented with 0.2% EDTA; phosphate buffered saline (PBS) (Gibco Cat. No. 14190); 96-well polypropylene round bottom plates (BD #3077); 1.2 ml polypropylene tubes (Corning #4451); biotinylated anti-VISTA clone GA-1 from ImmunoNext lot 080612B (used at 5 μg / ml); biotinylated mIgG1,K isotype control (clone MOPC-21), Biolegend Cat. No. 400104, lot B116649 (used at 5 μg / ml); anti-human antibodies (see staining method in the table below); near infrared live / dead cell dye (Invitrogen, Cat. No. L10119); and streptavidin reagents, including STP-APC (BD Biosciences catalog #554067, lot #04251) (used at a dilution of 1:200 in FACS buffer), STP-PE (Biolegend catalog #405203, lot #B139688) (used at a dilution of 1:200 in FACS buffer), STP-PE Cy7 (shows non-specific binding in isotype control samples), STP-Q605 (Invitrogen catalog #Q10101MP, lot #53449A) (used at a dilution of 1:200 in FACS buffer).

[0150] Cell surface staining protocol

[0151] Before staining, 1×10 6 The cells were transferred into a 96-well round bottom plate and washed with 150 μl PBS. The plate was then centrifuged at 1300 rpm for 3 minutes at 4°C.

[0152] Subsequently, cells were washed again with PBS and centrifuged as described above.

[0153] Live / dead cell staining was then performed in 50 μl of PBS containing 0.25 μl of near-infrared live / dead cell dye. After 10 minutes at room temperature, the wells were washed with 150 μl of FACs staining buffer and centrifuged at 1300 rpm for 3 minutes at 4° C. The supernatant was discarded.

[0154] Cells were blocked with human serum at 1:100 in 50 μl FACS staining buffer. Plates were incubated for 15 minutes at 4°C. Wells were then washed with 150 μl FACs staining buffer and centrifuged at 1300 rpm for 3 minutes at 4°C. Supernatant was discarded.

[0155] A cocktail containing the following antibodies was then added to each well for surface staining: The cocktails are described in Tables 3-6 below. Each cocktail was used separately from the others depending on the population of interest.

[0156] Table 3: Lineage staining

[0157]

[0158] Table 4: T cell staining

[0159]

[0160] Table 5: DC staining

[0161]

[0162] Table 6: Bone marrow staining

[0163]

[0164] After surface staining, cells were washed twice with FACS staining buffer as previously described and centrifuged at 1300 rpm for 5 minutes at 4°C. The samples were resuspended in 50 μl of FACS staining buffer containing appropriately fluorescently labeled streptavidin. The samples were incubated at 4°C for 30 minutes. Cells were washed with 150 μl of FACS staining buffer and centrifuged at 1300 rpm for 5 minutes at 4°C. This washing step was repeated and the samples were then resuspended in 250 μl of FACS staining buffer. TM Samples were analyzed on a cell analyzer (BD Biosciences).

[0165] Data Analysis

[0166] Flow cytometry data were reanalyzed using FlowJo model 9 software to set gates for specific phenotype populations. VISTA expression in different cell subsets was compared using the enumeration of geometric means. Each population was standardized for background by subtracting the isotype control value from the mean of the anti-VISTA treated samples. Prism was used to make curves and the Student's T-test was used to perform statistics when only two samples were compared or single-factor ANOVA and Bonferroni post-test were used for statistics.

[0167] result:

[0168] VISTA expression on human bone marrow and lymphoid subsets:

[0169] like Figure 2A-2E, 3A-3G, 4, 5A-5B and 6A-6C, CD14 + VISTA expression on monocytes was significantly different from all other groups (p<0.001). No significant differences were found between the other groups. Monocytes expressed the highest levels of VISTA in peripheral blood, with CD14 + CD16 - Subpopulations compared to CD14 lo CD16 + Cells had significantly higher expression. Although APCs showed moderate expression, lymphoid subsets showed low expression.

[0170] VISTA expression on human T and NK subsets:

[0171] like Figures 7A-7E , 8A-8G and 9, for NK subsets, CD56 lo Cells showed significantly higher CD56 Hi VISTA expression in NK cells. Among T cell subsets, CD8 + Memory cells expressed the highest expression, but it was not significantly higher than CD8 + Naive or CD4 + T cells.

[0172] VISTA expression on human dendritic cell subsets:

[0173] like Figures 10A-10D As shown in , 11A-11C and 12, no significant differences in VISTA expression were found; DCs and basophils exhibited low VISTA expression, among which plasmacytoid dendritic cells (pDCs) were generally higher but not significantly so.

[0174] Conclusion: These results show VISTA expression on various immune cell subsets and that VISTA is most highly expressed on monocytes, with some expressed on different T cell subsets and NK cells and little expressed on B cells.

[0175] Example 2: VISTA expression on peripheral blood cells

[0176] method:

[0177] Whole blood staining: Freshly isolated whole blood (100 μl) was stained with the antibody cocktail shown below by incubation at 4°C for 30 minutes. Red blood cells (RBCs) were lysed with RBC lysis buffer and the remaining cells were washed once with staining buffer. The cells were resuspended in 200 μl staining buffer. Data were collected using a MACSQuant flow cytometer and analyzed using FlowJo analysis software.

[0178] Peripheral blood mononuclear cell (PBMC) staining: Peripheral blood mononuclear cells were isolated from whole blood using a Ficoll gradient. Freshly isolated 1×10 6 Incubate samples at 4°C for 30 minutes and then wash once with staining buffer. Resuspend cells in 100 μl staining buffer. Use Data were collected by flow cytometry (Miltenyi Biotec) and analyzed using FlowJo analysis software.

[0179] The antibodies used were CD11b, CD33, CD177, CD16, CD15, CD14, CD20, HLADR, CD3, CD4, CD8, CD127, CD69, and FOXP3 antibodies (Biolegend, San Diego, CA). APC-bound mouse anti-human VISTA (clone GG8) was prepared by ImmuNext (Lebanon, NH).

[0180] in conclusion:

[0181] VISTA expression on healthy human peripheral blood cells

[0182] VISTA expression in whole blood and peripheral blood mononuclear cells was analyzed by multicolor flow cytometry. Fig.15A and 15B As shown in Figure 2, the highest level of VISTA expression was detected on monocytes, followed by neutrophils. CD4+ and CD8+ T cells expressed low levels of VISTA, such as Fig. 13C and 13D as shown in .

[0183] VISTA expression on peripheral blood cells in cancer patients

[0184] like Fig.14A -C, peripheral blood mononuclear cells (PBMCs) from lung cancer patients were analyzed. Fig.14A To show CD14 + Monocytes and CD15 + Representative flow cytometric analysis of bone marrow cells showing the results of myeloid suppressor cell (MDSC) analysis. The results showed that the phenotype CD15 + The cells are MDSCs derived from neutrophils. In addition, these cells are not present in healthy blood samples. Fig. 14B is a representative histogram of VISTA expression on monocytes from healthy and cancer patients, showing that higher levels of VISTA are expressed on cancer patient cells compared to healthy controls. Similarly, higher levels of VISTA were found on MDSCs from cancer patients, such as Fig. 14C as shown in .

[0185] Fig.15A Representative FACS images showing the presence of neutrophil-derived MDSCs in the blood of colon cancer patients. Fig. 15B and 15C Representative histogram showing higher levels of VISTA expression in mononuclear spheres from cancer patients compared to blood samples from healthy donors.

[0186] VISTA expression on peripheral blood cells in cynomolgus macaques

[0187] like Fig.16A and 16B As shown in , flow cytometric analysis of monkey whole blood revealed a VISTA expression pattern similar to that of human cells. + ( Fig. 16C ) and CD8 + ( Fig.16D )T cells, monocytes and neutrophils all expressed the highest levels of VISTA.

[0188] Example 3: VISTA expression at RNA and protein levels in hemoglobin malignant cell lines

[0189] Because VISTA is expressed in heme-derived malignancies, anti-VISTA antibodies may target malignant cells for destruction and block VISTA and promote anti-tumor immune responses.

[0190] The data include RNAseq analysis results for approximately 140 heme malignant cell lines (some cell lines were replicated in the analysis). Fig.17 middle.

[0191] RNAseq values ​​are listed as FPKM (fragments per kilobase of exon per million mapped fragments).

[0192] Essentially, this means counting all reads in the exon region belonging to a gene and normalizing by the length of the gene and the total number of reads per sample (to calculate sample-to-sample differences). The cutoff value is 1; above 1 is VISTA expression positive (RNA level), and below 1 is VISTA expression negative.

[0193] The results showed that multiple cell lines were positive at the RNA level, mainly acute myeloid leukemia and chronic myeloid leukemia. This is expected because VISTA is highly expressed in normal bone marrow cells and because its function is believed to be to weaken immune responses, including anti-tumor immune responses.

[0194] Example 4: Generation of monoclonal antibodies against VISTA

[0195] Phage panning

[0196] Twenty-four phage panning experiments were performed to enrich for phages active against Cyno VISTA-His. These experiments were performed using macaque VISTA protein because it showed better biotin binding than human VISTA protein. To determine the success of the phage experiment, the phage pool from the individual rounds of panning was added to a neutral chain avidin plate coated with biotin-labeled cyno VISTA-His and detected with an anti-M13 antibody bound to HRP. Individual colonies were picked from the phage selection round and Fab proteins were prepared in 96-well plates. The expressed Fab supernatant was analyzed for binding to biotin-labeled cyno VISTA-His. This operation resulted in more than 200 successful results.

[0197] The VH and VL regions from the Fab disk were amplified, submitted for DNA sequencing and exported as FASTA files. When picking the clones to be converted into MABs and tested in MAB format, the clones were selected based on sequence diversity and with limited risk of post-translational modifications and as few hydrophobic residues as possible. The VH and VL from the phage clones were sub-cloned in mammalian IgG1 / κ expression vectors and transfected into HEK293 cells. The antibodies were purified on protein A agarose fast flow affinity resin. The concentration of phage MABs was determined by quantitative ELISA using Nanodrop measurement. The antibody group was expressed at a high level. SDS-PAGE analysis showed the integrity of each expressed antibody variant.

[0198] Online maturation of phage antibodies was performed by amplifying the VH domains of a multi-strain antibody mixture from the last round of panning to be selected in a phage vector with VL diversity. This operation resulted in an enriched VH pool that had additional VL diversity after sampling. The phage was subjected to 1-2 rounds of rigorous panning in order to identify extremely high affinity binders of the VISTA ECD His protein. A single Fab ELISA was operated to determine the success of maturation. The ELISA and expression data were standardized according to a reference pure line set to 100% from the original rebirth panning experiment, and affinity mature pure lines with higher binding signals for cyno VISTA antigens were identified compared to the reference pure lines. This process produced several pure lines that showed up to 200% binding when screened at low antigen concentrations (1 nM), and the pure lines with the highest affinity were sequenced and made into MABs.

[0199] Fusion tumor generation

[0200] A group of BALB / cAnNCrl mice received an intraperitoneal (IP) injection of 50 μg Hu VISTA-Ig recombinant protein (Sino) emulsified in Complete Freund'sAdjuvant, followed by an intraperitoneal injection of 50 μg Hu VISTA-Ig recombinant protein emulsified in Incomplete Freund'sAdjuvant two weeks later. Two weeks later, the mice received an intraperitoneal injection of 50 μg cyno VISTA-Fc recombinant protein emulsified in Incomplete Freund'sAdjuvant. All mice received a final injection of PBS containing 25 μg human and 25 μg cynoVISTA at the base of the tail, and spleens were collected five days later for fusion.

[0201] Another group of BALB / cAnNCrl mice received an intraperitoneal injection of 50 μg of Hu VISTA-His recombinant protein emulsified in Chuan's complete adjuvant. Two weeks later, the mice received an intraperitoneal injection of 50 μg of HuVISTA-His recombinant protein emulsified in Chuan's incomplete adjuvant. Two weeks later, the mice received an intraperitoneal injection of 50 μg of Cyno VISTA-His recombinant protein emulsified in Chuan's incomplete adjuvant. Two weeks later, all mice received a final injection of PBS containing 25 μg HuVISTA-His and 25 μg Cyno VISTA-His, and spleens were collected three days later for fusion.

[0202] On the day of fusion, mice were euthanized by CO2 asphyxiation, spleens were removed and placed in 10mL cold phosphate buffered saline. Single cell suspensions of splenocytes were prepared by grinding spleens through a fine mesh sieve with a small pestle and rinsing with PBS at room temperature. Cells were washed once with PBS and RBC lysis was performed. In brief, cells were resuspended in 3mL RBC lysis buffer (Sigma#R7757) / spleen and placed on ice for 5 minutes. Cells were washed once again with PBS at room temperature and marked for magnetic sorting. According to the manufacturer's instructions, cells were labeled with anti-mouse Thy1.2, anti-mouse CD11b and anti-mouse IgM magnetic beads (MiltenyiBiotec#130-049-101, 130-049-601 and 130-047-301, respectively), and then sorted using the MS column with Midi MACS. Negative cell elution fractions (positive cell elution fractions discarded) were fused with FO cells. Mouse myeloma cells were fused with live spleen cells at a ratio of 1:1. Briefly, spleen and myeloma cells were mixed together, pelleted and washed once with 50 mL PBS. Aggregate pellets were resuspended for 30 seconds at 37°C with 1 mL polyethylene glycol (PEG) solution (2 g PEG (molecular weight 4000), 2 mL DMEM, 0.4 mL DMSO) per 10e8 spleen cells. The cell / fusion mixture was then immersed in a 37°C water bath for about 60 seconds under gentle stirring. 37°C DMEM was slowly added over 1 minute to terminate the fusion reaction. The fused cells were left to stand at room temperature for 5 minutes and then centrifuged at 150 × g for 5 minutes. The cells were then resuspended in medium E-HAT (Medium E, StemCell Technologies catalog number 03805) containing HAT (Sigma catalog number H0262) and inoculated in a 96-well flat-bottom polystyrene tissue culture dish (Corning #3997).

[0203] Fusion tumor supernatants were screened for antibodies specific to cyno VISTA using capture EIA. Briefly, goat anti-mouse IgG (Fc) antibodies (Jackson #115-006-071) were used in coating buffer (Thermo 28382) to coat the disc (Nunc-Maxisorp #446612) for at least 60 minutes at 4 μg / ml. The disc was blocked with 200 microliters of 0.4% (w / v) bovine serum albumin (BSA) in PBS solution per well for 30 minutes at room temperature. The disc was washed once, 50 μl of fusion tumor supernatant was added to each well and incubated at room temperature for at least 30 minutes. The disc was washed once, 50 μl of 0.1 μg / mL cyno VISTA-huIg was added to each well and incubated at room temperature for 30 minutes. The plate was washed once, and a solution of 1:40,000 streptavidin HRP (Jackson 016-030-084) in 0.4% BSA / PBS was added to the plate and incubated for 30 minutes at room temperature. The plate was washed 3 times, and then 100 μl of TMBTurbo substrate (Thermo Scientific 34022) was used per well to develop the color and incubate for about 10 minutes at room temperature. The reaction was stopped using 25 μl of 4N sulfuric acid per well and the absorbance was measured at 450 nm using an automated plate spectrophotometer. 15 primary successes were selected for sub-cloning by limiting dilution and screened in the same primary screening format.

[0204] All cyno VISTA active fusion tumor cell lines were cross-screened with human VISTA-Ig to assess cross-reactivity. Briefly, 0.1 M sodium carbonate-bicarbonate buffer (pH 9.4, Pierce 28382 BupH TM) in PBS (Invitrogen) at 4 μg / mL. The wells were blocked with 200 μl of blocking agent (0.4% BSA (Sigma) (w / v) in PBS (Invitrogen)) overnight at 4°C without washing. After removing the blocking solution, undiluted fusion tumor supernatants were incubated on the coated plates for 30 minutes at room temperature. The plates were washed once with PBST (0.02% Tween (Tween) 20 (Sigma) (w / v) in PBS) and then incubated with Hu VISTA-Ig diluted to 100 ng / ml in blocking agent for 30 minutes. The plates were washed once and probed with goat anti-human Fc-HRP (Jackson #109-036-098) diluted 1:10,000 in blocking agent for 30 minutes at room temperature. The plates were washed again and then developed using 100 μl TMB Turbo substrate (Thermo Scientific 34022) per well and incubated for approximately 10 minutes at room temperature. The reaction was stopped using 25 μl 4N sulfuric acid per well and the absorbance was measured at 450 nm using an automated plate spectrophotometer.

[0205] The V region antibody sequences of human and macaque VISTA display active fusion tumors were cloned. Fusion tumor cells were prepared with Invitrogen's SuperScript III cell direct cDNA system before reverse transcriptase (RT) reaction. In brief, the culture medium was discarded and the dish was placed on ice and resuspended in 200 μl cold PBS. Forty microliters were transferred to MicroAmp fast 96-well reaction PCR trays and the trays were placed on a cold metal tray base, sealed with plastic film and rotated at 700 rpm for 3 minutes. PBS was discarded and 10 μl resuspension buffer and 1 μl lysis enhancer were added to each well. The tray was sealed, incubated at 75 ° C for 10 minutes and stored at -80 ° C.

[0206] For RT reaction, each well contained 5 μl water, 1.6 μl 10× DNase buffer, 1.2 μl 50 mM EDTA, 2 μl Oligo(dT)20 (50 mM) and 1 μl 10 mM dNTP mix. The plate was incubated at 70°C for 5 minutes, followed by incubation on ice for 2 minutes, after which the following reagents were added to each well: 6 μl 5× RT buffer, 1 μl RNaseOUT TM (40U / μl), 1μl SuperScript TMIII RT (200U / μl) and 1 μl 0.1M DTT. Seal the plate, place on a thermocycler preheated to 50°C and incubate at 50°C for 50 minutes, followed by inactivation (incubation at 85°C for 5 minutes). Freeze the reaction on ice and store single strand cDNA at -80°C until further use.

[0207] For V region amplification, 20 μl PCR reactions were provided. Each well contained 16.2 μl water, 2.0 μl 10× PCR reaction buffer, 0.8 μl MgSO4 (50 mM), 0.4 μl 10 mM dNTPs, 0.15 μl 100 μM forward primer mix, 0.05 μl 100 μM reverse primer, 0.2 μl HiFi Tag enzyme. The cDNA prepared as described above was transferred (2 μl per well) to the PCR component mixture, the plate was sealed and the amplification reaction was run; for VH, the program was (i) 94°C for 1 minute, (ii) 94°C for 15 seconds, (iii) 55°C for 30 seconds, (iv) 68°C for 1 minute. Steps (ii-iv) were repeated for a total of 35 cycles, followed by a final extension at 68°C for 3 minutes. For VL, the program was (i) 94°C for 1 min, (ii) 94°C for 15 sec, (iii) 55°C for 30 sec, (iv) 65°C for 30 sec, (v) 68°C for 1 min. Steps (ii-v) were repeated for a total of 35 cycles, followed by a final extension at 68°C for 3 min.

[0208] Premix the forward primer and use this mixture with the reverse primer in a ratio of 3:1. Check the PCR products on an agarose gel. Prepare the reaction for infusion cloning by adding the enhancer (In-Fusion HC Cloning Kit, catalog number 639650, Clontech). Transfer five microliters of the PCR reaction to a PCR plate, followed by 2 μl of enhancer per well. Seal the plate and incubate in a thermocycler (15 minutes at 37°C and 15 minutes at 80°C). Prepare the destination vector (vDR243 or vDR301) by Esp3I digestion; (digest 1.5 μg of vector in 30 μl reaction at 37°C in 3 μl Tango buffer, 2 l Esp3I and water for 2 hours).

[0209] For infusion selection, 2 μl of enhancer-treated PCR product was mixed with 100 ng of Esp3I-digested vector and 2 μl of 5× infusion enzyme (Clontech). Infusion reactions were performed in 96-well plate format. Plates were incubated on a PCR machine at 50°C for 15 minutes and Stella competent cells were transformed by heat shock at 42°C without shaking for 40 seconds and spread on LB agar plates with the selected antibiotics and incubated overnight at 37°C. The next day, colonies were picked in 96-well deep well plates containing LB / Carbenicillin medium and grown overnight at 37°C. Frozen stocks were made from overnight cultures mixed with an equal volume of 30 w / v% glycerol. Sequencing of the V region was performed using sequencing primer SPF0052. Sequences were analyzed and one positive well was selected for each fusion tumor vH and vL, re-plated in a new plate and grown overnight in enriched medium with ampicillin. Clones were then subjected to miniprep DNA preparation for small-scale transfection in 96-well plates.

[0210] Forty-eight selected mouse fusion tumor sequences for heavy and light chains were adapted to human frameworks using an in-house software program. A human framework was selected for each of the mouse vH or vL. V region DNA sequences were obtained via reverse translation. Synthetic DNA regions corresponding to the HFA amino acid sequence were sequenced by Integrated DNA Technologies (Coralville, IA). Selections were made into pre-cut vDR149 and vDR157, human IgG1, and human κ, respectively. Prepared using the Qiagen Endo-free Maxi-prep kit. This panel of antibodies was expressed using Expi293 (100 ml) cultures.

[0211] Example 5: Protocol for in vitro human VISTA-IGT cell inhibition assay

[0212] Mouse A20 cells were stably transfected with GFP or human VISTA. They were incubated with ova peptide and DO11.10 T cells. CD25 expression of T cells was measured 24 hours after the start of incubation. A20-huVISTA cells suppressed CD25 expression of T cells, but this readout was significantly restored by incubation with VSTB95 ( Fig.18 ).

[0213] Example 6: Human framework region adaptation of anti-VISTA antibodies

[0214] Human framework adaptation of mouse fusion tumor sequences of heavy and light chains was performed by CDR grafting (Jones et al., Nature, 321: 522-525 (1986)) using an in-house software program. The program depicts the complementary determining regions (CDRs) of the V region sequences according to the Kabat definition (Wu, TT & Kabat, EA (1970). J Exp Med, 132, 211-50) and uses Blast to compare the framework regions with human germline genes. The human germline with the highest sequence identity to the mouse framework was selected as the recipient gene for human framework adaptation (HFA). In some cases, based on the experience of the aforementioned well-expressed human framework, closely related human germline genes were selected as alternatives. The DNA sequence of the human framework selected for each of the mouse vH or vL V regions was obtained via reverse translation. The synthetic DNA region corresponding to the HFA amino acid sequence was sequenced by Integrated DNA Technology (Coralville, IA). Selection was performed in human IgG1 and human κ, respectively.

[0215] Example 7: Anti-VISTA Antibody Constructs

[0216] Plasmids and sequence information for cell line-produced molecules: Plasmid constructs were generated for anti-VISTA antibodies with VSTB112 variable regions and IgG1κ constant regions (VSTB174, new numbering due to allotypic changes in the constant regions), IgG2δ constant regions (VSTB140), or IgG1 antiprotease constant regions (VSTB149).

[0217] Lonza Vectors

[0218] The pEE6.4 and pEE12.4 Chinese hamster ovary (CHO) expression vector systems (Lonza Biologics plc) were established in Biologics Research (BR) and Pharmaceutical Development & Manufacturing Sciences (PDMS) as the primary expression systems for producing therapeutic mAbs in mammalian expression cell lines. Each vector contains a human cytomegalovirus (huCMV-MIE) promoter driving expression of either the heavy chain (HC) or the light chain (LC) and contains an ampicillin resistance gene. The pEE12.4 vector also includes a gene encoding glutamate synthase (GS). Growth conditions requiring glutamate synthase activity exert selective pressure on the cells to maintain the expression vector (GS Gene Expression System Manual Version 4.0). pEE6.4 was used as a single gene vector to clone the HC gene and pEE12.4 was used as a single gene vector to clone the LC gene. Lonza dual gene plasmids were generated from these two Lonza single gene vectors.

[0219] Amino acid sequences of the variable heavy chain regions of selected VISTA mAbs

[0220] >VSTB112 heavy chain (SEQ ID NO: 37)

[0221] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS

[0222] >VSTB50 heavy chain (SEQ ID NO: 38)

[0223] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSS

[0224] >VSTB53 heavy chain (SEQ ID NO: 39)

[0225] QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDYYAMDYWGQGTLVTVSS

[0226] >VSTB95 heavy chain (SEQ ID NO:40)

[0227] EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSS

[0228] Amino acid sequence of the variable light chain region of the selected VISTA mAb

[0229] >VSTB50 light chain (SEQ ID NO:41)

[0230] DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIK

[0231] >VSTB53 light chain (SEQ ID NO:42)

[0232] DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEIK

[0233] >VSTB95 light chain (SEQ ID NO:43)

[0234] DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIK

[0235] >VSTB112 light chain (SEQ ID NO:44)

[0236] DIQMTQSPSSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK

[0237] >VSTB116 light chain (SEQ ID NO:45)

[0238] DIQMTQSPSSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQGTKVEIK

[0239] Example 8: ELISA and FACS screening of anti-VISTA antibodies

[0240] These experiments were used to determine the ability of the antibodies generated to bind to human or macaque VISTA protein in ELISA, and to determine the ability of the antibodies to bind to VISTA protein on the surface of K562 cells (a human myeloid leukemia cell line) expressing human or macaque VISTA protein using FACS screening.

[0241] method:

[0242] ELISA procedure overview: Plates were coated overnight at 4°C with 1 μg / ml SB0361 (human) or SB0361 (cyno (macaque)) proteins, which are the extracellular domains of VISTA from the respective species. Antibodies were diluted stepwise from the initial concentration of 1:4 to 1 μg / ml, a total of 4 concentrations were obtained, and incubated for 2 hours at room temperature (RT). 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%).

[0243] Overview of FACS procedure: 5 μg / ml of each test antibody was used to detect 2 × 10 5 K562-G8 (human) or K562-C7 (cynomolgus monkey) cells were stained and incubated at 4°C for 30 minutes. 5 μg / ml of goat anti-human IgG1-PE (phycoerythrin) antibody was used as the secondary detection antibody. Cells were operated on a BD Fortessa and the MFI (mean fluorescence intensity) of the live population was analyzed using FlowJo software (Tree Star, Inc., Ashlang, OR).

[0244] Data Analysis / Results: For each antibody, a subjective score (yes / no) was given as to whether the antibody bound firmly for both the ELISA and FACS analysis in each of the four assays. If the antibody gave a "no" result for binding in any of the assays, the assay was repeated to confirm that it was negative. The results are shown in Table 7 and Figures 19A-19F and 20A-20F.

[0245] Table 7.

[0246]

[0247]

[0248] Example 9: Results of screening anti-human VISTA antibodies using mixed lymphocyte reaction (MLR) and Staphylococcus enterotoxin B (SEB) activation assays

[0249] The aim of this study was to provide data supporting the identification of multifunctional α-VISTA antibodies that enhance cellular immune responses in mixed lymphocyte reaction (MLR) assays and Staphylococcal enterotoxin B activation (SEB) assays.

[0250] The mixed lymphocyte reaction (MLR) is a standard immunoassay that depends on MHC class I and class II mismatches to drive allogeneic T cell responses. Peripheral blood mononuclear cells are isolated from two mismatched individuals, cultured together, and as a result of these mismatches, proliferation and cytokine production occur.

[0251] Materials and Methods

[0252] Prepare 10% AB medium by combining 500 ml RPMI with 50 ml human AB serum, 5 ml penicillin / streptomycin (10,000 U / ml), 5 ml L-glutamic acid (100×), and 10 ml HEPES (1 M). Store the medium for no more than 14 days. Prepare 1 mCi of tritiated thymidine by diluting 0.2 ml thymidine stock solution (1 mCi / ml) with 9.8 ml RPMI.

[0253] Soluble VISTA antibodies were diluted to 20 μg / ml with 10% AB serum medium. 100 μl of the appropriate antibody solution was added to the appropriate wells of a 96-well U-bottom plate (Falcon product #353077 or equivalent). After adding various cell populations, the final concentration was 10 μg / ml.

[0254] Separation of leukocytes: Donors were at least 18 years old, generally healthy and randomly selected from the local population. Donor blood was transferred from the separation tube to a 50 ml conical flask. 15 ml Ficoll 1077 was placed under each 25 ml of blood, being careful not to mix with the blood. The cells were centrifuged at 1250 g for 25 minutes at room temperature without braking. Leukocytes were separated at the interface of Ficoll and serum and the cells were diluted with 40 ml Hanks Balanced Salt Solution (HBSS). Centrifuged at 453 g (1500 rpm) for 10 minutes at 4°C. The cells were resuspended in 50 ml HBSS and counted by transferring 500 μl to individual tubes.

[0255] Mixed lymphocyte reaction (MLR) 96-well plate setup: Determine the appropriate number of "stimulator cells" and "responder cells" required for the analysis based on the number of samples to be analyzed. 5 The reaction population was plated at 1.0×10 5 All conditions must be repeated three times. The appropriate number of "stimulator cells" were pipetted into a new conical flask and centrifuged as before. The cells were resuspended in 10 ml and irradiated with 4000 rad. The cells were centrifuged as before and 1×10 6 Resuspend in 10% AB serum medium at a concentration of 10 cells / ml and add 50 μl to appropriate wells. Isolate the desired number of responding cells by centrifugation as described previously and centrifuge at 2×10 6 Cells were resuspended in 10% AB serum medium at a concentration of 100 μl / ml and 50 μl was added to the appropriate wells. Cells were cultured for 5 days at 37°C and 5% CO2. On the fifth day, 30 μl of supernatant was removed to analyze interferon gamma (IFN-γ) production. On the fifth day, 25 μl of 40 μCi / ml tritiated thymidine solution was added to each well and cultured for 8 hours at 37°C and 5% CO2. Cells were transferred to a 96-well microscintillation plate according to the manufacturer's instructions. Microscintillation counter was used for counting according to the manufacturer's instructions. IFN-γ concentration was determined using the manufacturer's protocol by ELISA (eBioscience catalog number 88-7316-88).

[0256] Data analysis: Calculate the average counts per minute (CPM) or IFN-γ concentration of untreated wells. Calculate the average CPM or IFN-γ of each test group. Log the data group 10 Using the 12 MLR fold-scores for each compound, the average of the 12 test groups for each compound was calculated: Average score of 12 experiments = Σ[(log10 (average CPM of three replicates of the test compound))-(log 10 (average CPM of three untreated replicates))] / 12.

[0257] Acceptance Criteria: All test reagents and appropriate controls were tested for endotoxin prior to assay and should contain <0.1 EU / mg. The average CPM count for cells reacting alone was less than 700 CPM, indicating that the cells were quiescent when incubated alone. The CPM for the MLR group was at least 2-fold that of cells reacting alone, indicating that a reaction occurred and the donor was mismatched. All MLR analyses included a human IgG1 negative control protein. The results for the human IgG1 negative control were not statistically different from the untreated sample based on the use of a Studen's t-test.

[0258] Screening of anti-VISTA antibodies in MLR: Initial screening of all compounds. Before operating the MLR with anti-VISTA antibodies, determine whether the antibodies bind to cell-bound VISTA (via FACS analysis) and VISTA protein (via ELISA). Antibodies S26 (VSTB77), S30 (VSTB86), S31 (VSTB88), S32 (VSTB90) and S39 (VSTB74) failed this initial screening but are still being tested in the analysis. For the purpose of preliminary screening, all antibodies were tested at 10 μg / ml in the MLR, where the parameters measured were proliferation and IFN-γ ( Figures 21A-21D and 22A-22B).

[0259] Selection of the top six antibodies From the initial screening, six candidates were selected for further analysis: VSTB112 (S2), VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43), and VSTB60 (S47).

[0260] Dilution studies of the top six candidates in MLR: protocol adjustments. The protocol was the same as previously described with some adjustments: the antibodies were diluted to the following concentrations: 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 μg / ml.

[0261] Determination of IC 50 Value: IC of each antibody was determined using raw CPM counts and IFN-γ concentrations. 50 Value. IC is performed using the program "EZ-R stats." 50 IC values ​​were calculated using six individual responders. 50 Individual CPM counts and IFN-γ concentrations were obtained in an MLR and dose titration was performed for the preceding candidates.

[0262] Table 8: IC for CPM and IFN-γ in MLR 50 value

[0263]

[0264] **Value is log of antibody concentration 10 .

[0265] Conclusion: Preliminary screening showed that multiple VISTA-specific antibodies were able to enhance MLR cellular immune responses. Antibodies were ranked based on efficacy and variability and based on these results, VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, and VSTB60 were selected for evaluation in a dose titration experiment. In the dose titration experiment, VSTB60 induced a weaker response compared to the other five antibodies.

[0266] Staphylococcal enterotoxin B (SEB) activation assay: SEB is a bacterial superantigen that induces specific Vβ+ T cell activation. Peripheral blood mononuclear cells were isolated and incubated with SEB antigen in culture, inducing robust cytokine production. This assay was performed on the top five candidates.

[0267] Preparation of 10% AB medium, preparation of 1 mCi tritiated thymidine, preparation of soluble VISTA antibody and isolation of leukocytes were performed as described above in MLR.

[0268] SEB 96-well plate setup: Determine the appropriate number of reaction cells required for the analysis based on the number of samples to be analyzed. Plate the reaction population in a 96-well U-bottom plate at 2.0 × 10 5 All conditions were repeated three times. Cells were centrifuged and plated at 4 × 10 6 The cells were resuspended in 10% AB serum medium at a concentration of 100 μl / ml 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 experiment, SEB was obtained from Sigma Aldrich (Catalog No. S0812). The final concentration in the wells was 10 ng / ml. The cells were cultured for 3 days at 37°C and 5% CO2. On the third day, 30 μl of supernatant was removed to analyze IFN-γ production. 25 μl of 1 mCi / ml tritiated thymidine solution was added to each well and cultured for 8 hours at 37°C and 5% CO2. The cells were transferred to a 96-well microscintillation plate according to the manufacturer's instructions. A microscintillation counter was used for counting according to the manufacturer's instructions. IFN-γ concentration was determined by ELISA (eBioscience Catalog No. 88-7316-88) using the manufacturer's protocol.

[0269] Protocol: Data analysis. Calculate the counts per minute (CPM) or IFN-γ concentration per minute for each antibody at all concentrations. The acceptance criteria are performed as previously described. IC 50 value determination is performed as described. Individual CPM counts and IFN-γ concentrations are obtained in the SEB analysis, and dose titrations are performed on the previous candidates.

[0270] Table 9: IC 50 values for CPM and IFN-γ in SEB.

[0271]

[0272] **The values are the log 10 of the antibody concentration.

[0273] Conclusion: VISTA-specific antibodies enhance cytokine production and proliferation in a dose-dependent manner in the SEB analysis. The IC 50 values from the SEB study are generally similar to the results of the MLR dilution study.

[0274] Example 10: Epitope binning analysis

[0275] Method: Epitope binning is performed using the ProteOn XPR36 system (BioRad). Two sets of 6 monoclonal antibodies (mAbs) are coated on ProteOn GLC chips (BioRad, catalog number 176-5011) using the manufacturer's instructions for amine coupling chemistry (BioRad, catalog number 176-2410).

[0276] Excess competitive mAb (final concentration 250 nM) is pre-incubated with human VISTA (final concentration 25 nM) for 4 hours at room temperature, and 6 are run at a time on the chips coated with the coated mAb sets, with an association time of 4 minutes followed by a dissociation of 5 minutes. After each run, the chips are regenerated with 100 mM phosphoric acid.

[0277] Data analysis involves grouping all sensorgrams by ligand and applying an alignment wizard, which automatically performs X and Y axis alignment and artifact removal. Subsequently, Interspot correction is applied to the data.

[0278] Non-competitive mAbs are defined as having the same or >A1 signal for binding (binding only to human VISTA).

[0279] Competitive mAbs are defined as having a binding signal <<A1 signal (i.e., binding only to human VISTA).

[0280] Results: In Fig.23In the exemplary sensorgram shown, the VSTB85 antibody was coated onto a Proteon SPR chip and VISTA protein pre-incubated with the designated competitor was passed over the chip. VSTB50 is an example of a non-competitive antibody as a positive response was seen when operating the VISTA / VSTB50 complex. GG8, VSTB49 and VSTB51 complexed with VISTA do not bind to the VSTB85 coated on the chip and are thus classified as competing with VSTB85 for the same binding site on VISTA.

[0281] Table 10:

[0282]

[0283]

[0284] mAb immobilized on sensor

[0285] Y = Competitive (signal << A1 - human VISTA only)

[0286] N = Non-competitive (signal > A1 - human VISTA only)

[0287] I = Indeterminate (signal similar to A1 - human VISTA only)

[0288] Example 11: PROTEON Affinity Assay

[0289] Antibodies were captured on the ProteOn chip using an anti-IgG Fc coated surface. Binding of test antibodies to the extracellular domain (ECD) of human and cynomolgus (VISTA) with VISTA protein concentrations in the range of 0.39 nM to 100 nM was tested. The antigen was allowed to bind / associate with the antibody-coated chip for 4 minutes, followed by monitoring of dissociation for 30 minutes. The chip was regenerated twice by treatment with 100 mM phosphoric acid for 18 seconds. All experiments were conducted at 25 °C and the data was fit to a 1:1 Langmuir binding model.

[0290] Example 12: Effect of Anti-VISTA Treatment in the MB49 Murine Bladder Tumor Model

[0291] Methods:

[0292] C57BL / 6 mice were injected with MB49 tumor cells. After tumors had formed, anti-VISTA treatment was initiated. Tumor growth was then monitored 3 times / week. According to IACUC regulations, the mice were sacrificed after any tumor reached 15 mm in size.

[0293] 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 plated at 5×10 6 The cells were resuspended in RPMI at a concentration of 10 cells / ml and 50 μl was injected into each mouse.

[0294] Female C57BL / 6 mice aged 6-8 weeks were purchased from the National Cancer Institute. After arrival, they were allowed to acclimate for one day, then their right flanks were shaved and their tails were pierced. They were then injected three to five days later.

[0295] Tumor injection (intradermal): 50 μl of MB49 cell suspension (approximately 250,000 cells) were injected intradermally (id) into the shaved flank of mice.

[0296] Monitoring Tumor Growth: Tumor growth was measured using electronic calipers first across the widest dimension (L) and second at a 90° angle of the first measurement (W). Tumor volume was obtained as follows:

[0297] Volume = (L 2 *W 2 ) / 2.

[0298] When the tumor reaches about 5 mm in diameter (about 60 mm in volume) 3 ) was considered to have formed a tumor. After formation, treatment began. During the treatment, tumor growth was measured three times per week and until the experiment was terminated.

[0299] Anti-VISTA treatment: 10 mg / kg chimeric 13F3-mIgG2a monoclonal antibody was injected intraperitoneally three times a week for four weeks.

[0300] Mouse Euthanasia: According to IACUC requirements, animals were euthanized after their tumors reached 15 mm in the longest dimension.

[0301] Analysis: Data were managed using Excel and graphed using GraphPad Prism to analyze mouse tumor volumes. Statistical analysis was performed using macros in R statistical computing software.

[0302] The experimental design is presented in Fig.24 middle.

[0303] result:

[0304] Ch13F3-mIgG2a treatment of female mice resulted in complete tumor inhibition (CR) in 70% of animals and partial remission (PR) in 30% of animals (n=7) (Table 13 and Table 14). Fig.25B). In contrast, all control mIgG2a-treated mice displayed progressive tumor growth (6 / 6) ( Fig.25A ). These data show that anti-VISTA treatment can have a profound effect on tumor growth.

[0305] Table 11: Complete remission (CR) and partial remission (PR)

[0306] Female 13F3 IgG2a (n=7) CR 5 PR Until the 32nd day, 2

[0307] Human VISTA sequences are displayed in Fig.26 and 27 , modified from Wang et al., 2011, supra, the entire contents of which are incorporated herein.

[0308] Example 13: Antigenic determinant localization of anti-VISTA antibodies using hydrogen / deuterium (H / D) exchange studies

[0309] To identify the antigenic determinants for VSTB50, 60, 95 and 112 on human VISTA, solution hydrogen / deuterium exchange mass spectrometry (HDX-MS) was performed using the corresponding Fab. For H / D exchange, the procedure for analyzing Fab perturbations was similar to that previously described (Hamuro et al., J. Biomol. Techniques 14: 171-182, 2003; Horn et al., Biochemistry 45: 8488-8498, 2006), but with some modifications. Fab was prepared from IgG by papain digestion and protein A was captured using the Pierce Fab preparation kit (Thermo Scientific, catalog number 44985). The human VISTA protein sequence contains six N-linked glycosylation sites. To improve sequence coverage, the protein was deglycosylated with PNGaseF. The deglycosylated VISTA protein was incubated in a deuterated aqueous solution for a predetermined time so that deuterium was incorporated at the exchangeable hydrogen atoms. At 4 ° C, in 46 μL deuterium oxide (DO), the deuterated VISTA protein was compounded with any Fab of VSTB50, VSTB60, VSTB95 or VSTB112 for 30 seconds, 2 minutes, 10 minutes and 60 minutes. The exchange reaction was quenched by lowering the pH value and the protein was digested with pepsin. The deuterium content of the identified peptides was monitored by mass shift on LC-MS. As a reference control, the VISTA protein was treated similarly, except that it was not compounded with the Fab molecule. The region bound to the Fab is inferred to be relatively difficult to exchange and therefore contains a higher fraction of deuterium than the reference VISTA protein. About 94% of the protein can be located in a specific peptide.

[0310] Solution HDX-MS perturbation localization of VISTA using VSTB50 / VSTB60 and VSTB95 / VSTB112 are shown in Fig.28 In the upper and lower figures. Two antigenic determinants are identified. Anti-VISTA VSTB50 and VSTB60 recognize the same antigenic determinant; VSTB95 binds to another antigenic determinant region on VISTA that VSTB112 binds to. Anti-VISTA VSTB50 and 60 share the same antigenic determinant, which includes segment 103NLTLLDSGL111 (SEQ ID NO: 62) and 136 VQTGKDAPSNC146 (SEQ ID NO: 63) Fig.28 Anti-VISTA VSTB95 and 112 appear to target similar epitopes, which include segments 27 PVDKGHDVTF36 (SEQ ID NO: 75) and 54 RRPIRDLTFQDL65 (SEQ ID NO:65) Fig.28 Figure below). There are two other segments showing weak perturbations by VSTB95 and 112, including residues 39-52 and 118-134. However, the degree of reduction is not as strong as the aforementioned regions (27-36 and 54-65) in different positioning. Although a peptide 100TMR102 showing strong perturbations by VSTB95 and 112 is located on another surface of the VISTA surface, it is far away from the antigenic determinant regions 27-36 and 54-65. This perturbation may be attributed to allotopic effects. These HDX-MS results provide peptide-level antigenic determinants of anti-VISTA antibodies. There is no overlapping antigenic determinant region for these two antigenic determinants. These results are consistent with the aforementioned competitive grouping data in that they do not compete with each other.

[0311] Example 14: Determination of the structure of the human VISTA ECD:VSTB112 FAB complex by protein crystallography

[0312] In an effort to determine the structure of VISTA and delineate the antigenic determinants and paratopes that define the interaction between the VISTA extracellular domain (ECD) and the Fab fragment of the first antibody VSTB112, the complex was crystallized and the structure determined to a resolution of Efforts are made to determine the structure of ECD in human VISTA compounded with the Fab fragment of antibody VSTB112 to determine the structure of VISTA ECD itself and define the antigenic determinant / paratope for this interaction. The structure reveals that VISTA adopts IgV folding, and its chain topology is similar to TCR Vα chain. In addition to the standard disulfide bonds of B and F strands in the back and front of the bridging β sandwich, the structure reveals that ECD has two additional disulfide bonds, one tethering the CC' loop to the front sheet, and the second between A' and G' strands. Although there are crystal contacts between VISTA molecules, it is a small amount and there is no evidence of dimers of VISTA ECD based on this structure. The VSTB112 antigenic determinant is shown to include the residues (C'CFG) of the VISTA BC, CC' and FG loops and the front β sheet closest to those loops. The paratope is mainly biased towards heavy chain interactions, with CDR L3 producing the least contact.

[0313] Defining the epitope / paratope of the VISTA:VSTB112 interaction

[0314] VSTB112 Fab is buried after binding to VISTA ECD of the total surface area, of which the buried heavy chain surface accounts for VISTA forms seven hydrogen bonds and four salt bridge interactions with the VSTB112 light chain, and VISTA forms 10 hydrogen bonds and two salt bridge interactions with the VSTB112 heavy chain. VSTB112 recognizes residues proximal to the FG loop in the C', C, F, and G of the prostration strand, as well as residues in the BC, FG, and CC' loops ( Fig.29 and 30 ). Interactions with the CC' loop account for the majority of contacts with the Fab light chain that has only residues E125 and R127 in the FG loop, resulting in additional light chain interactions. Residues 119 to 127 corresponding to the VISTA FG loop account for a total of 1.3% of the surface area buried after binding to VSTB112. 38%. Notably, this loop has high polarity, comprising the following sequence -IRHHHSEHR-(SEQ ID NO:76). In addition, W103 in VSTB112 CDR H3 is well packaged against the backbone of VISTA residues H122 and H123, and VISTA H121 is the edge of the interaction with the aromatic ring of F55 in CDR H2.

[0315] Comparison of the epitope regions identified by crystallography and HDX is shown in Fig.31 middle.

[0316] Example 15: Activation of T cells and monocytes by anti-VISTA antibodies

[0317] The functional effects of anti-VISTA antibodies were evaluated in two in vitro assays, mixed leukocyte reaction (MLR) and SEB (Staphylococcal enterotoxin B). Both assays measure T cell proliferation and cytokine induction as their primary readouts, but these effects are attributed to different mechanisms. In MLR, peripheral blood mononuclear cells (PBMCs) from two different human donors are cultured together, and major histocompatibility complex (MHC) mismatches between T cells from one donor and dendritic cells from another donor cause T cell proliferation and interferon (IFNγ) production. In the SEB assay, PBMCs from a single donor are cultured together with bacterial superantigens, which directly connect MHC class II proteins on the surface of antigen presenting cells (APCs) to T cell receptors (TCRs) on T cells, thereby causing T cell activation, proliferation, and cytokine secretion. In both assays, VSTB112, the parent molecule of VSTB174, exhibited dose-dependent induction of T cell proliferation and cytokine production and was the most effective among the candidates ( Figures 21A-21D , Table 12).

[0318] Table 12. EC50 values ​​of the MLR assay readouts. VSTB112 (parent molecule of VSTB174) was the most potent molecule.

[0319]

[0320] Monocyte activation assay

[0321] The analytical data shown in Table 12 were generated using the parent molecule of VSTB174, VSTB112. To fully understand the activity of VSTB174, a monocyte activation assay was performed. The results show that incubation of VSTB174 with whole PBMCs induced upregulation of activation markers (CD80 and HLA-DR) on CD14+ monocytes, indicating the effect of antibodies that bind to immune cell subsets known to express high levels of VISTA ( Fig.32 ). Another question is whether the effect on monocyte activation in whole PBMCs can be promoted by any antibody that binds VISTA and has IgG1 Fc. Antibodies VSTB103 and VSTB63 bind to VISTA with high affinity (KD 6.36E-10 and 8.30E-10, respectively) and bind to cells expressing VISTA protein similarly to VSTB112 and VSTB111. VSTB103 and VSTB112 are in the same antigenic determinant group, while VSTB63 is in a different antigenic determinant group; neither antibody promotes monocyte activation. Taken together, these results show that one mechanism by which VSTB174 can exert its effect on T cell activation / proliferation is monocyte activation promoted by NK cells.

[0322] Preparation of culture medium

[0323] 500 ml RPMI 1640 (Corning, 10-040-CV) was combined with 50 ml human AB serum (Valley Biomedical, Lot No. 3C0405), 5 ml penicillin / streptomycin (Lonza, 17-602E) (10,000 U / ml), 5 ml L-glutamine (100×) (Gibco, 25030-081) and 10 ml HEPES (1 M) (Fisher BP299-100, Lot No. -1). The culture medium was stored at 4°C for no more than 14 days.

[0324] Preparation of soluble VISTA and control antibodies

[0325] The antibody was diluted to 2× the desired concentration with 10% AB serum medium, VSTB174: Lot No. VSTB174.003.

[0326] 100 μl of the appropriate antibody solution was added to the appropriate wells of a 96-well plate (Falcon, 353077). After adding various cell populations at 100 μl, the final concentration of each antibody was 1, 0.1 or 0.01 g / ml. The IgG1 control antibody CNTO 3930 (lot 6405, ENDO < 0.1 EU / mg) was added at a final concentration of 1 μg / ml.

[0327] Isolation of PBMCs.

[0328] Donors were at least 18 years old, generally healthy and randomly selected from the local population.

[0329] Donor blood was transferred from the separation tube to a 50 ml Erlenmeyer flask.

[0330] Place 15 ml of Ficoll 1077 (SIGMA, 10771) underneath, being careful not to mix it with the blood. Do this for every 25 ml of blood.

[0331] The cells were centrifuged at 1250 g for 25 minutes at room temperature without braking.

[0332] Leukocytes were separated at the interface of Ficoll and serum and cells were diluted with 40 ml Hank's Balanced Salt Solution (HBSS).

[0333] The cells were centrifuged at 453 g (1500 rpm) for 10 min at 4°C.

[0334] The cells were resuspended in 50 ml HBSS and counted by transferring 500 μl into individual eppendorf tubes.

[0335] Additionally, CD14+ cells were isolated by negative selection in several treatment groups using the pan-monocyte isolation kit from Miltenyi according to the manufacturer's instructions (Cat. No. 130-096-537).

[0336] In vitro culture settings

[0337] Determine the appropriate number of cells required for the analysis based on the number of samples to be analyzed. Plate the reaction population at 2.0 × 10 cells per well in a 96-well U-bottom plate. 5 For CD14 negative selection, 0.5×10 5 All conditions were performed in triplicate.

[0338] The cells were centrifuged as above and 2 × 10 6 The concentration of CD14 negative selection population was 0.5×10 6 The cells were resuspended in 10% AB serum medium at a concentration of 1 μg / ml and 100 μl was added to appropriate wells to bring the total volume in each well to 200 μl.

[0339] The cells were incubated at 37°C and 5% CO2 for 1, 2 or 3 days.

[0340] Antibody staining and flow cytometry

[0341] The 96-well U-bottom plates were centrifuged at 453 g for 5 minutes and the supernatant removed.

[0342] Wash cells with 200 μl PBS and centrifuge as in step 5.5.1.

[0343] Discard the supernatant and resuspend in 50 μl PBS containing the following antibodies:

[0344] .CD14-APC (clone HCD14) 1:250 (Biolegend catalog number 325608)

[0345] .HLA-DR-PE Cy7 (clone L243) 1:250 (Biolegend catalog number 307616)

[0346] .CD80-PE (clone 2D10) 1:250 (Biolegend catalog number 305208)

[0347] .Hu FcR Binding Inhibitor (eBioscience Cat. No. 14-9161-73)

[0348] Incubate on wet ice in the dark for 20 min.

[0349] Add 150 μl PBS and centrifuge as in step 5.5.1.

[0350] 150 1 PBS buffer was added and analyzed via FACS.

[0351] Samples were run on a Miltenyi MACSQuant 10 parameter flow cytometer and the expression of HLA-DR and CD80 on the CD14+ population was analyzed using FlowJo 9.7.5. The geometric mean of fluorescence intensity (MFI), a statistical parameter that defines the central tendency of a group of numbers, was used as the designated statistical parameter for treatment comparison.

[0352] Statistical analysis

[0353] All statistics were performed in Prism GraphPad model 6. Pairwise comparisons between groups were performed at each time point using one-way ANOVA with Tukey correction for diversity. All p values ​​tested were below 0.05 and the contrasts were considered significant. For all curves and tables, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0354] Example 16: ADCC and ADCP activity of anti-VISTA antibodies

[0355] VSTB174 has an IgG1 Fc, which can confer antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) activity. Two types of analyses were performed and showed that VSTB174 could lyse or phagocytose K562-VISTA cells ( Figure 33-34 ), but not K562 myeloma cell line blasts (data not shown). Another mechanism of action by which VSTB174 modulates the inhibitory effect of VISTA could be the lysis or phagocytosis of cells expressing high levels of VISTA, thereby removing them from the local microenvironment.

[0356] Example 17: ADCP activity of anti-VISTA antibodies

[0357] 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). These mAbs were cloned in different Fc backbones (IgG1 WT (wild type), IgG1 PR (protease resistance) and IgG2σ) and were hypothesized to have different activities for enhancing phagocytosis. The IgG1 and IgG1 PR backbones are able to bind to Fc receptors and have the potential to cause ADCP, while IgG2σ does not bind to Fc receptors and cannot modulate ADCP.

[0358] Anti-VISTA antibodies were tested in ADCP assays using K562 parent cells and K562-VISTA target cells. Figure 35-36 As shown in Figure 2, VSTB174, VSTB149, VSTB173, and VSTB145 increased hMac phagocytosis of K562-VISTA cells. VISTA antibodies VSTB140 or VSTB132 with IgG2σ Fc that do not bind to Fc receptors did not increase phagocytosis as expected. VISTA mAbs VSTB174 and VSTB173 with IgG1 Fc showed more robust phagocytosis (for EC) than VSTB149 and VSTB145 with IgG1PR Fc. 50 For values, see Tables 13 and 14).

[0359] Table 13. Anti-human VISTA mAb EC 50 value.

[0360] deal with VSTB174 VSTB149 VSTB140 EC50 0.0782 0.1142 NA

[0361] Table 14. Anti-human mAb EC 50 value.

[0362] deal with VSTB173 VSTB145 VSTB132 EC50 0.0146 0.1075 NA

[0363] VSTB174 and VSTB173 showed weak enhancement of phagocytosis of K562 mother cells at the highest concentration ( Figure 35-36 ), which can be attributed to the low expression of VISTA in K562 cells. Other anti-VISTA antibodies could not enhance the phagocytosis of K562 cells.

[0364] Negative control antibodies were tested at two different concentrations in the K562-VISTA phagocytosis assay, but they did not induce any phagocytosis. This result indicates that anti-VISTA antibody-mediated phagocytosis is specific and due to the VISTA antigen expression of K562-VISTA cells.

[0365] Example 18: ADCC activity of other anti-VISTA antibodies

[0366] The following three human anti-VISTA antibodies were tested for their ability to induce ADCC:

[0367] VSTB174 (IgG1)

[0368] VSTB149 (IgG1 PR)

[0369] VSTB174.LF (IgG1 LF (low trehalose)).

[0370] In two separate experiments, each antibody was tested at six different concentrations in triplicate on the same plate, yielding a total of six data points.

[0371] VSTB174, VSTB149, and VSTB174.LF at 10, 1, 0.1, and 0.01 μg / mL each exhibited measurable ADCC activity, while only the LF antibody exhibited measurable ADCC activity at 0.001 μg / mL; none of the antibodies exhibited ADCC at 0.0001 μg / mL. This result is expected when each of these antibodies has an IgG1 or IgG1 variant Fc. The LF antibody exhibited increased ADCC potency, which is demonstrated by the EC of the LF antibody curve compared to the conventional IgG1 antibody curve (0.02381 μg / mL). 50 The EC value of IgG1 PR antibody curve is smaller (0.002293μg / mL). 50 The value is similar to the conventional IgG1 curve (0.01846 μg / mL).

[0372] Table 15. EC values ​​of three tested anti-VISTA antibodies as determined by ADCC analysis 50 Value (μg / mL).

[0373] Anti-VISTA Antibodies <![CDATA[EC 50 (μg / mL)]]> VSTB174 (IgG1) 0.02381 VSTB149 (IgG1 PR) 0.01846 VSTB174.LF (IgG1 LF) 0.002293

[0374] At 10, 1, 0.1 and 0.01 μg / mL antibody concentrations, human IgG1, human IgG1 PR and human IgG1 LF antibodies all showed measurable ADCC-mediated killing, while only LF antibodies showed killing at 0.001 μg / mL antibody concentration. Anti-VISTA antibodies did not show killing at 0.0001 μg / mL antibody concentration.

[0375] Such as EC 50 As can be seen from the values, the LF antibody exhibited approximately 10 times more effective ADCC killing than the conventional IgG1 antibody or IgG1 PR antibody.

[0376] Example 19: Affinity of VSTB174 for human and macaque VISTA

[0377] The affinity of VSTB174 for human and cynomolgus macaque VISTA extracellular domain (ECD) was determined by surface plasmon resonance (SPR) on a ProteOn instrument. VSTB174 showed very similar KD values ​​for each protein, 1.56E-10M for human ECD and 8.66E-11M for macaque VISTA.

[0378] Example 20: VISTA antibody demonstrates efficacy in a mouse tumor model

[0379] Mouse strains, reagents, and tumor models

[0380] For in vivo studies, human VISTA knockin (VISTA-KI) mice backcrossed onto a C57BL / 6 background were used.

[0381] Anti-human VISTA antibodies were generated using the VSTB174 variable region (VSTB123) grafted onto mouse Fc IgG2a to enable testing in VISTA-KI mice.

[0382] MB49 bladder cancer was evaluated in VISTA KI mice.

[0383] In addition to published studies showing that anti-VISTA antibody therapy inhibits tumor growth in wild-type mice (Le Mercier et al., 2014), alternative hamster antibodies have been used to demonstrate anti-tumor efficacy in wild-type mice and in VISTA-KI mice treated with VSTB123 using different dosing schedules.

[0384] In vivo efficacy study in the MB49 tumor model in VISTA-KI mice

[0385] MB49 efficacy studies were conducted in female VISTA-KI mice to test VSTB123 at several doses ranging from 1-10 mg / kg. Mice were injected intradermally with 250,000 MB49 tumor cells on day 0. On day 6, Fig.37 Dosing was initiated as indicated in (10 mg / kg isotype control mIgG2a or indicated doses of VSTB123; 10 mice / group).

[0386] like Fig.37 As shown in Figure 2, VSTB123 was more effective at higher and lower doses. Doses of 10 mg / kg and 7.5 mg / kg were equivalent, while tumors grew more rapidly in mice given 5 or 1 mg / kg.

[0387] Example 21: Detection of VISTA expression in human tumors using anti-VISTA antibodies

[0388] Figure 1 shows VISTA expression in AML tumor cells - and Fig.17 The RNA sequence expression data in support the idea that VISTA is expressed by AML cells and that anti-VISTA drugs are effective by targeting these cells for immunomodulation or antibody-mediated killing.

[0389] Data evaluating VISTA expression in lung cancer were obtained from lung tumor samples from surgical resections. Cells were dissociated and characterized for the expression of VISTA and multiple other markers. Results showed that 13 / 13 lung tumors (squamous carcinoma or adenocarcinoma) contained CD14+VISTA+ myeloid cells ( Fig.38 ).

[0390] Example 22: Detection of VISTA expression in lung tumors using anti-VISTA antibodies

[0391] Immunohistochemistry was performed using cloned GG8, an anti-human VISTA mouse IgG1. This mAb was used to study staining of VISTA in non-small cell lung cancer (NSCLC) FFPE tumor sections.

[0392] FFPE tumor sections were processed using standard antigen retrieval methods prior to staining. GG8 mouse anti-human VISTA antibody was used at a dilution of 1:500. GG8 binding was detected using rabbit anti-mouse polyclonal antibody followed by anti-rabbit polymer HRP. Tumor sections were then scored following counterstaining with hematoxylin.

[0393] VISTA expression in lung cancer is mainly restricted to immune infiltration ( Fig.39 Examples shown in ) and high levels of VISTA-positive cells were present in multiple lung cancer samples.

[0394] Example 23: Structure of the extracellular domain (ECD) of human VISTA in complex with the FAB fragment of VSTB174

[0395] VISTA antigen variants were generated and purified for crystallography. Recombinant VSTB174 Fab tagged with his was expressed internally and purified. Crystals were generated and used to collect higher resolution data of the VISTA ECD:VSTB174 Fab complex using synchrotron radiation, and the structure was determined using a combination of homology modeling and electron density analysis ( Fig.29 (Picture above)).

[0396] The structure of the VISTA ECD:VSTB174 Fab complex was determined by X-ray crystallography at 1.3 Å resolution. The first structure of VISTA ECD was obtained and the VSTB174 antigenic determinant and paratope were depicted. VISTA ECD adopts an IgV fold, which is topologically similar to CTLA-4 ECD, but has a unique G' strand extending in the front sheet of the β sandwich. A' and G' are additionally chemically tethered via a disulfide bridge formed between residue C12 in the A' strand and C146 in the G' strand. Six cysteines were found to participate in three intramolecular disulfide bonds, and based on crystal contacts, there is no evidence of dimeric VISTA.

[0397] VSTB174 recognizes residues in the front strand C', C, F and G proximal to the FG loop as well as residues in the BC, FG and CC' loops.

[0398] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0399] While the invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. Sequence Listing <110> Janssen Pharmaceuticals; Linda Snyder; Gordon Powers; <120> Anti-VISTA antibodies and fragments <130> 0148.1142-003 <140> PI-210246 / 1F <150> 61 / 920,695 <151> 2013-12-24 <150> 62 / 085,086 <151> 2014-11-26 <160> 79 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Homo sapiens <400> 1 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 2 <211> 8 <212> PRT <213> Homo sapiens <400> 2 Ile Asn Pro Tyr Thr Gly Glu Pro 1 5 <210> 3 <211> 12 <212> PRT <213> Homo sapiens <400> 3 Ala Arg Glu Gly Tyr Gly Asn Tyr Ile Phe Pro Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Glu Ser Val Asp Thr Tyr Ala Asn Ser Leu 1 5 10 <210> 5 <211> 3 <212> PRT <213> Homo sapiens <400> 5 Arg Ala Ser 1 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Gln Gln Thr Asn Glu Asp Pro Arg Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Homo sapiens <400> 7 Gly Tyr Thr Phe Thr His Tyr Thr 1 5 <210> 8 <211> 8 <212> PRT <213> Homo sapiens <400> 8 Ile Ile Pro Ser Ser Gly Tyr Ser 1 5 <210> 9 <211> 16 <212> PRT <213> Homo sapiens <400> 9 Ala Arg Gly Ala Tyr Asp Asp Tyr Tyr Asp Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <210> 10 <211> 11 <212> PRT <213> Homo sapiens <400> 10 Gln Thr Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 11 <211> 3 <212> PRT <213> Homo sapiens <400> 11 Lys Val Ser 1 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Phe Gln Ala Ser His Val Pro Trp Thr 1 5 <210> 13 <211> 8 <212> PRT <213> Homo sapiens <400> 13 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 14 <211> 8 <212> PRT <213> Homo sapiens <400> 14 Ile Asn Thr Tyr Thr Gly Glu Ser 1 5 <210> 15 <211> 12 <212> PRT <213> Homo sapiens <400> 15 Ala Arg Asp Tyr Tyr Gly Ile Tyr Val Ser Ala Tyr 1 5 10 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <400> 16 Glu Ser Val Asp Asn Tyr Ala Asn Ser Phe 1 5 10 <210> 17 <211> 3 <212> PRT <213> Homo sapiens <400> 17 Arg Ala Ser 1 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Gln Gln Ser His Glu Asp Pro Tyr Thr 1 5 <210> 19 <211> 8 <212> PRT <213> Homo sapiens <400> 19 Gly Phe Thr Phe Arg Asn Tyr Gly 1 5 <210> 20 <211> 8 <212> PRT <213> Homo sapiens <400> 20 Ile Ile Ser Gly Gly Ser Tyr Thr 1 5 <210> 21 <211> 15 <212> PRT <213> Homo sapiens <400> 21 Ala Arg Ile Tyr Asp His Asp Gly Asp Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <210> 22 <211> 11 <212> PRT <213> Homo sapiens <400> 22 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 23 <211> 3 <212> PRT <213> Homo sapiens <400> 23 Lys Val Ser 1 <210> 24 <211> 9 <212> PRT <213> Homo sapiens <400> 24 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 25 <211> 8 <212> PRT <213> Homo sapiens <400> 25 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Homo sapiens <400> 26 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 27 <211> 13 <212> PRT <213> Homo sapiens <400> 27 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr 1 5 10 <210> 28 <211> 6 <212> PRT <213> Homo sapiens <400> 28 Gln Ser Ile Asp Thr Arg 1 5 <210> 29 <211> 3 <212> PRT <213> Homo sapiens <400> 29 Ser Ala Ser 1 <210> 30 <211> 9 <212> PRT <213> Homo sapiens <400> 30 Gln Gln Ser Ala Tyr Asn Pro Ile Thr 1 5 <210> 31 <211> 8 <212> PRT <213> Homo sapiens <400> 31 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 32 <211> 8 <212> PRT <213> Homo sapiens <400> 32 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 33 <211> 13 <212> PRT <213> Homo sapiens <400> 33 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr 1 5 10 <210> 34 <211> 6 <212> PRT <213> Homo sapiens <400> 34 Gln Ser Ile Asn Thr Asn 1 5 <210> 35 <211> 3 <212> PRT <213> Homo sapiens <400> 35 Ala Ala Ser 1 <210> 36 <211> 9 <212> PRT <213> Homo sapiens <400> 36 Gln Gln Ala Arg Asp Thr Pro Ile Thr 1 5 <210> 37 <211> 120 <212> PRT <213> Homo sapiens <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 119 <212> PRT <213> Homo sapiens <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Gly Asn Tyr Ile Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 39 <211> 123 <212> PRT <213> Homo sapiens <400> 39 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ile Pro Ser Ser Gly Tyr Ser Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Asp Asp Tyr Tyr Asp Tyr Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 122 <212> PRT <213> Homo sapiens <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ile Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asp His Asp Gly Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 41 <211> 111 <212> PRT <213> Homo sapiens <400> 41 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Tyr 20 25 30 Ala Asn Ser Leu Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 42 <211> 112 <212> PRT <213> Homo sapiens <400> 42 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 43 <211> 112 <212> PRT <213> Homo sapiens <400> 43 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 44 <211> 107 <212> PRT <213> Homo sapiens <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Arg 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Tyr Asn Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 107 <212> PRT <213> Homo sapiens <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Thr Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Arg Asp Thr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 295 <212> PRT <213> Homo sapiens <400> 46 Met Gly Val Pro Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser 1 5 10 15 Leu Leu Phe Ala Leu Phe Leu Ala Ala Ser Leu Gly Pro Val Ala Ala 20 25 30 Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45 Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 50 55 60 Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 85 90 95 Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp 100 105 110 Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn 115 120 125 Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr 130 135 140 Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg Val 145 150 155 160 His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser 165 170 175 Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr 180 185 190 Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu 195 200 205 Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn 210 215 220 Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile 225 230 235 240 Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln Gly Ile Pro Glu 245 250 255 Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser 260 265 270 Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr Pro Leu Ser Pro 275 280 285 Pro Gly Pro Gly Asp Val Phe 290 295 <210> 47 <211> 449 <212> PRT <213> Homo sapiens <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Gly Asn Tyr Ile Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 48 <211> 218 <212> PRT <213> Homo sapiens <400> 48 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Tyr 20 25 30 Ala Asn Ser Leu Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 49 <211> 453 <212> PRT <213> Homo sapiens <400> 49 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ile Pro Ser Ser Gly Tyr Ser Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Asp Asp Tyr Tyr Asp Tyr Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 50 <211> 219 <212> PRT <213> Homo sapiens <400> 50 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 51 <211> 449 <212> PRT <213> Homo sapiens <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Tyr Gly Ile Tyr Val Ser Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 52 <211> 218 <212> PRT <213> Homo sapiens <400> 52 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Ala Asn Ser Phe Met His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 53 <211> 452 <212> PRT <213> Homo sapiens <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ile Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asp His Asp Gly Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Methionine, Histidine, Glutamic acid, Alanine, Leucine, Histidine, Asparagine, Histidine, Tyrosine, Threonine, Glutamine, Lysine, Serine, Leucine, Serine, Leucine 435 440 445 Serine, Proline, Glycine, Lysine 450 <210> 54 <211> 219 <212> PRT <213> Homo sapiens <400> 54 Aspartic acid, Isoleucine, Valine, Methionine, Threonine, Glutamine, Serine, Proline, Leucine, Serine, Leucine, Proline, Valine, Threonine, Proline, Glycine 1 5 10 15 Glutamic acid, Proline, Alanine, Serine, Isoleucine, Serine, Cysteine, Arginine, Serine, Serine, Glutamine, Serine, Isoleucine, Valine, Histidine, Serine 20 25 30 Asparagine, Glycine, Asparagine, Threonine, Tyrosine, Leucine, Glutamic acid, Tryptophan, Tyrosine, Leucine, Glutamine, Lysine, Proline, Glycine, Glutamine, Serine 35 40 45 Proline, Glutamine, Leucine, Leucine, Isoleucine, Tyrosine, Lysine, Valine, Serine, Asparagine, Arginine, Phenylalanine, Serine, Glycine, Valine, Proline 50 55 60 Aspartic acid, Arginine, Phenylalanine, Serine, Glycine, Serine, Glycine, Serine, Glycine, Threonine, Aspartic acid, Phenylalanine, Threonine, Leucine, Lysine, Isoleucine 65 70 75 80 Serine, Arginine, Valine, Glutamic acid, Alanine, Glutamic acid, Aspartic acid, Valine, Glycine, Valine, Tyrosine, Tyrosine, Cysteine, Phenylalanine, Glutamine, Glycine 85 90 95 Serine, Histidine, Valine, Proline, Tryptophan, Threonine, Phenylalanine, Glycine, Glutamine, Glycine, Threonine, Lysine, Leucine, Glutamic acid, Isoleucine, Lysine 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 55 <211> 450 <212> PRT <213> Homo sapiens <400> 55 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 56 <211> 214 <212> PRT <213> Homo sapiens <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Arg 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Tyr Asn Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 57 <211> 450 <212> PRT <213> Homo sapiens <400> 57 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 58 <211> 214 <212> PRT <213> Homo sapiens <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Thr Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Arg Asp Thr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 59 <211> 446 <212> PRT <213> Homo sapiens <400> 59 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215 220 Glu Cys Pro Pro Cys Pro Ala Pro Pro Ala Ala Ala Ser Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Ala Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 60 <211> 449 <212> PRT <213> Homo sapiens <400> 60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 225 230 235 240 Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro Ile Ala Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 61 <211> 450 <212> PRT <213> Homo sapiens <400> 61 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Gly Trp Ser Tyr Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 62 <211> 9 <212> PRT <213> Homo sapiens <400> 62 Asn Leu Thr Leu Leu Asp Ser Gly Leu 1 5 <210> 63 <211> 11 <212> PRT <213> Homo sapiens <400> 63 Val Gln Thr Gly Lys Asp Ala Pro Ser Asn Cys 1 5 10 <210> 64 <211> 13 <212> PRT <213> Homo sapiens <400> 64 Leu Leu Gly Pro Val Asp Lys Gly His Asp Val Thr Phe 1 5 10 <210> 65 <211> 12 <212> PRT <213> Homo sapiens <400> 65 Arg Arg Pro Ile Arg Asp Leu Thr Phe Gln Asp Leu 1 5 10 <210> 66 <211> 309 <212> PRT <213> Mus musculus <400> 66 Met Gly Val Pro Ala Val Pro Glu Ala Ser Ser Pro Arg Trp Gly Thr 1 5 10 15 Leu Leu Leu Ala Ile Phe Leu Ala Ala Ser Arg Gly Leu Val Ala Ala 20 25 30 Phe Lys Val Thr Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45 Asn Ala Thr Leu Thr Cys Arg Ile Leu Gly Pro Val Ser Lys Gly His 50 55 60 Asp Val Thr Ile Tyr Lys Thr Trp Tyr Leu Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Met Cys Lys Glu His Arg Pro Ile Arg Asn Phe Thr Leu Gln His 85 90 95 Leu Gln His His Gly Ser His Leu Lys Ala Asn Ala Ser His Asp Gln 100 105 110 Pro Gln Lys His Gly Leu Glu Leu Ala Ser Asp His His Gly Asn Phe 115 120 125 Ser Ile Thr Leu Arg Asn Val Thr Pro Arg Asp Ser Gly Leu Tyr Cys 130 135 140 Cys Leu Val Ile Glu Leu Lys Asn Met Met Pro Glu Gln Arg Phe Tyr 145 150 155 160 Gly Ser Met Glu Leu Gln Val Gln Ala Gly Lys Gly Ser Gly Ser Thr 165 170 175 Cys Met Ala Ser Asn Glu Gln Asp Ser Asp Ser Ile Thr Ala Ala Ala 180 185 190 Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu Pro Leu Ile 195 200 205 Leu Leu Leu Val Tyr Lys Gln Arg Gln Val Ala Ser His Arg Arg Ala 210 215 220 Gln Glu Leu Val Arg Met Asp Ser Ser Asn Thr Gln Gly Ile Glu Asn 225 230 235 240 Pro Gly Phe Glu Thr Thr Pro Pro Phe Gln Gly Met Pro Glu Ala Lys 245 250 255 Thr Arg Pro Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser Glu Ser 260 265 270 Gly Arg Tyr Leu Leu Ser Asp Pro Ser Thr Pro Leu Ser Pro Pro Gly 275 280 285 Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp Ser Pro 290 295 300 Asn Ser Glu Ala Ile 305 <210> 67 <211> 311 <212> PRT <213> Homo sapiens <400> 67 Met Gly Val Pro Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser 1 5 10 15 Leu Leu Phe Ala Leu Phe Leu Ala Ala Ser Leu Gly Pro Val Ala Ala 20 25 30 Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45 Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 50 55 60 Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 85 90 95 Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp 100 105 110 Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn 115 120 125 Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr 130 135 140 Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg Val 145 150 155 160 His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser 165 170 175 Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr 180 185 190 Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu 195 200 205 Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn 210 215 220 Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile 225 230 235 240 Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln Gly Ile Pro Glu 245 250 255 Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser 260 265 270 Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr Pro Leu Ser Pro 275 280 285 Pro Gly Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp 290 295 300 Ser Pro Asn Phe Glu Val Ile 305 310 <210> 68 <211> 312 <212> PRT <213> Red kangaroo (Macropus rufus) <400> 68 Met Asn Val Pro Thr Ser Val Leu Glu Ser Gly Gly Arg Arg Trp Gly 1 5 10 15 Pro Leu Leu Leu Ala Phe Phe Leu Ala Ala Ser Arg Gly Leu Val Ala 20 25 30 Ala Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly 35 40 45 Glu Asn Ile Thr Leu Ala Cys Gln Leu Leu Gly Pro Val Pro Lys Gly 50 55 60 His Asp Val Ser Phe Tyr Lys Thr Trp Phe Arg Ser Ser Arg Gly Glu 65 70 75 80 Val Gln Val Cys Ser Glu His Arg Pro Ile Arg Asn Val Thr Leu Gln 85 90 95 Asn Leu His Pro Tyr His Gly Gly His Gln Ala Ser Asn Thr Ser His 100 105 110 Asn Leu Leu Gln Ser His Gly Leu Glu Thr Ala Ser Asp His His Gly 115 120 125 Asn Phe Ser Ile Thr Met Arg Asn Leu Thr Val Gln Asp Gly Gly Leu 130 135 140 Tyr Cys Cys Leu Val Val Glu Met Arg His Arg His Ser Glu His Arg 145 150 155 160 Val His Ala Ala Asn Glu Leu Gln Val Gln Lys Gly Lys Asp Ala Pro 165 170 175 Ser Lys Cys Ile Thr Tyr Pro Ser Ser Pro Glu Glu Ser Asp Asn Ile 180 185 190 Thr Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys 195 200 205 Leu Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Val Ala Ser 210 215 220 His Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Ser Pro Gln Gly 225 230 235 240 Ile Glu Asn Pro Gly Phe Glu Ala Pro Pro Ser Ser Gln Gly Leu Pro 245 250 255 Glu Ala Lys Val Arg Pro Pro Leu Ser Tyr Met Ala Gln Arg Gln Pro 260 265 270 Ser Glu Ser Gly Arg His Leu Leu Ser Glu Pro Asn Thr Pro Leu Ser 275 280 285 Pro Pro Gly Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro 290 295 300 Asp Ser Pro Asn Ser Glu Phe Asn 305 310 <210> 69 <211> 310 <212> PRT <213> Common dolphin (Delphinus delphis) <400> 69 Met Gly Val Pro Pro Val Pro Glu Ala Gly Ser Trp Arg Arg Gly Pro 1 5 10 15 Val Leu Leu Ala Phe Phe Leu Ala Ala Ser Arg Gly Leu Val Ala Ala 20 25 30 Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45 Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Leu Ala Lys Gly His 50 55 60 Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Ala Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 85 90 95 Leu His Leu His His Gly Gly His Gln Ala Asn Ser Ser Gln Asp Leu 100 105 110 Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn Phe 115 120 125 Thr Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Gly Gly Leu Tyr Cys 130 135 140 Cys Leu Val Val Glu Ile Arg His Arg His Ser Glu Gln Arg Leu Tyr 145 150 155 160 Gly Ala Met Glu Leu Gln Val Gln Arg Gly Glu Glu Ala Pro Ser Lys 165 170 175 Cys Thr Val Tyr Pro Pro Ser Ser Lys Glu Ser Glu Ser Ile Thr Ala 180 185 190 Ala Ala Leu Ala Thr Ser Ala Cys Ile Val Gly Ile Leu Cys Leu Pro 195 200 205 Leu Ile Leu Ile Leu Val Trp Lys Gln Arg Gln Val Ala Ser Asn Arg 210 215 220 Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Thr Gln Gly Ile Glu 225 230 235 240 Asn Pro Gly Phe Glu Thr Ser Pro Pro Ser His Gly Met Pro Glu Thr 245 250 255 Lys Pro Arg Gln Pro Leu Thr Tyr Met Ala Arg Arg Gln Pro Ser Glu 260 265 270 Ser Gly Arg His Leu Leu Ser Glu Pro Asn Thr Pro Leu Ser Pro Pro 275 280 285 Gly Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp Ser 290 295 300 Pro Asn Ser Glu Ala Ile 305 310 <210> 70 <211> 288 <212> PRT <213> Original rooster (Gallus gallus) <400> 70 Gly Gly Thr Ala Ala Phe Leu Val Thr Val Pro Tyr Thr Leu Cys Ile 1 5 10 15 Cys Pro Glu Gly Gln Asn Val Thr Leu Ser Cys Arg Val Ser Gly Pro 20 25 30 Pro Ala Asp His His Asp Leu Ile Phe Lys Thr Trp Tyr Phe Ser Asn 35 40 45 Asn Gly Asp Gln Ser Cys Ser Glu Lys Arg His Val Arg Asn Leu Thr 50 55 60 Glu Lys Glu Leu Arg His Asp Pro Gly Arg His His Ser Thr Ala Ala 65 70 75 80 Asn Ser Thr Ala Arg Ser Pro His Gly Ser Leu Ala Ser His His Gly 85 90 95 Val Glu Phe Val Pro Asp His His Gly Ala Phe His Ile Val Val Met 100 105 110 Asn Leu Thr Leu Gln Asp Ser Gly Asn Tyr Cys Cys Tyr Ala Met Glu 115 120 125 Thr Arg Arg Asp His Gly Lys Ala His Thr Leu His Ile Ala His Gly 130 135 140 Phe Val Glu Leu Gln Ile Gln Arg Gly Arg Gly Ser Leu Gln Asn Cys 145 150 155 160 Thr Phe His Thr Ala Thr Ser Lys Asp Ile Thr Ala Ala Ala Leu Ala 165 170 175 Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu Pro Leu Ile Leu Leu 180 185 190 Leu Ile Tyr Lys Gln Arg Gln Ala Val Ser His Arg Arg Ala His Glu 195 200 205 Leu Val Arg Met Glu Ser Ser Ala Gln Gly Ile Glu Asn Pro Val Phe 210 215 220 Glu Ala Leu Pro Ala Gly Ser Thr Glu Gln Arg Pro Arg Pro Gln Leu 225 230 235 240 Ser Tyr Leu Gly Gly Arg Gln Leu Ser Glu Ser Gly Arg His Leu Leu 245 250 255 Ser Glu Pro Asn Thr Pro Leu Ser Pro Pro Ala Pro Gly Glu Cys Phe 260 265 270 Phe Pro Thr Leu Asp Pro Val Pro Asp Ser Pro Asn Ser Leu Lys Ala 275 280 285 <210> 71 <211> 261 <212> PRT <213> Xenopus laevis <400> 71 Asp Ala Ile Thr Ala Phe Ser Val Ser Ala Leu Tyr Ser His Ile Thr 1 5 10 15 Cys Pro Glu Gly Gln Asn Val Asn Leu Thr Cys Thr Val Ser Gly His 20 25 30 Val Ala Asp Lys His Asp Val Leu Phe Ser Leu Trp His Phe Ser Lys 35 40 45 Asp Lys Asn Ser Asn Cys Leu Glu Arg Arg His Ile Gln Asn Thr Thr 50 55 60 Glu Arg Asp His Leu His Lys Glu His Leu Ser His Ser Met Met His 65 70 75 80 Asn Gly Ala Phe Gln Ile Thr Leu Thr Asn Val Ser Gln Gln Asp Ser 85 90 95 Gly Gly Tyr Cys Cys Tyr Val Ile Glu Ala Ser Lys Lys His His Thr 100 105 110 Arg His Tyr Ser Tyr Ile Glu Phe Gln Val Lys Thr Asp Asp Leu Asn 115 120 125 Leu Tyr Thr Cys Met Phe His Ser Pro Thr Glu Gly Asp Asn Ser Ser 130 135 140 Thr Ala Ala Ala Leu Ala Ile Val Ser Cys Val Ile Gly Ile Leu Cys 145 150 155 160 Met Pro Leu Ile Leu Phe Leu Val Tyr Lys Gln Arg Arg Ala Ile Ser 165 170 175 His Arg Arg Ser Tyr His Phe Val Phe Ile Asp Phe Ser Glu Ala Gln 180 185 190 Gly Ile Glu Asn Pro Val Phe Asp Asp Pro Pro Pro Ala Asn Val Val 195 200 205 Glu Gln Arg Pro Arg Leu Ala Phe Met Ala Ser Arg Gln Gln Ser Glu 210 215 220 Ser Asp Arg His Leu Leu Ser Glu Pro Asn Thr Pro Leu Ser Pro Ser 225 230 235 240 Cys Pro Asn Glu Cys Phe Phe Pro Ser Leu Pro Val Pro Asp Ser Pro 245 250 255 Asp Pro Gly Asn Val 260 <210> 72 <211> 315 <212> PRT <213> Taeniopygia guttata <400> 72 Gly His Pro Ala Thr Met Gly Thr Ala Ser Pro Arg Pro Gly Leu Leu 1 5 10 15 Leu Ala Ala Leu Cys Leu Leu Ala Ser His Gly Gly Ala Asp Ala Phe 20 25 30 Leu Ile Ser Thr Pro Tyr Ser Leu Cys Val Cys Pro Glu Gly Gln Asn 35 40 45 Val Thr Leu Ser Cys Arg Ile Ser Gly Ala Leu Ala Glu Arg His Asp 50 55 60 Leu Leu Tyr Lys Thr Trp Tyr Phe Ser Ser Thr Gly Asp Gln Ser Cys 65 70 75 80 Ser Asp Lys Arg His Ile Arg Asn Val Thr Asp Lys Glu Leu Arg His 85 90 95 Asp Leu Gly Arg His His Glu Leu Pro Gly Asn Ala Ser Gln Lys Pro 100 105 110 Pro Phe Gly Trp Gln Ser Gly His His Gly Val Glu Leu Val Leu Asp 115 120 125 His His Gly Ala Phe His Leu Val Val Met Asn Leu Thr Leu Gln Asp 130 135 140 Ser Gly Asn Tyr Cys Cys Tyr Ala Val Glu Val Arg Arg Glu Gly His 145 150 155 160 Ser Lys Pro His Thr Val Gln Ala Ala His Gly Phe Val Glu Leu Gln 165 170 175 Ile Gln Arg Gly Glu Pro Cys Ser His Ala Arg Ala Gln Ser Gln Arg 180 185 190 Ala Ala Asp Asp Ile Thr Ala Ala Val Leu Ala Thr Gly Ala Cys Ile 195 200 205 Val Gly Ile Leu Cys Leu Pro Leu Ile Leu Leu Leu Ile Tyr Lys Gln 210 215 220 Arg Gln Ala Ala Ser Ser Arg Arg Ala His Glu Leu Val Arg Met Asp 225 230 235 240 Ser Gly Ala Gln Gly Ile Glu Asn Pro Val Phe Glu Ala Val Pro Ser 245 250 255 Ala Gly Ala Glu Pro Arg Pro Arg Ala Gln Leu Ser Tyr Val Ala Ser 260 265 270 Arg Leu Pro Ser Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr 275 280 285 Pro Leu Ser Pro Pro Gly Pro Gly Asp Cys Phe Phe Pro Thr Leu Asp 290 295 300 Pro Val Pro Asp Ser Pro Asn Ser Leu Lys Ala 305 310 315 <210> 73 <211> 275 <212> PRT <213> Danio rerio <400> 73 Met Asp Val Phe Arg Ala Val Leu Leu Cys Phe His Val Phe Thr Ala 1 5 10 15 Ile Gln Ala Ser Gly Asp His His Ser Leu Arg Val Ser Val Pro His 20 25 30 Arg Thr Tyr Glu Cys Pro Glu Gly Ala Asp Val Ile Leu Lys Cys Val 35 40 45 Pro Ser Gly Thr Lys Ala Tyr Pro Gln Asp Thr Phe Trp Thr Thr Trp 50 55 60 Leu Tyr Thr Pro Arg Ser Gln Asp His Cys Gln Lys Gly Ala His Pro 65 70 75 80 Arg Lys Ala Asn His Thr Asn Arg Ser Leu Gly Val Val Tyr Ser Ser 85 90 95 Gly Asp Lys Val Phe Ser Val Ser Leu Lys Asn Val Lys His Thr Asp 100 105 110 Gln Gly Lys Tyr Cys Cys Trp Leu Leu Asp Leu His Gly Arg His Lys 115 120 125 Glu Gln Glu Ala His Asp Phe Met Tyr Leu Ser Val Met Pro Thr Pro 130 135 140 Lys Asp Ala His Asn Gly Ser Leu Lys Cys Leu Glu Tyr Ser His Thr 145 150 155 160 Ala Ser Asp Asp Ser Val Ala Glu Gly Leu Ala Ile Ala Ala Cys Val 165 170 175 Ala Phe Val Leu Cys Leu Pro Leu Ile Leu Met Leu Val Tyr Arg Gln 180 185 190 Arg Gln Thr Val Glu Arg His Arg Arg Ala His Glu Leu Val Arg Met 195 200 205 Asp Ser Glu Ala Gln Gly His Glu Asn Pro Val Phe Leu Gly Asp Ser 210 215 220 Pro Glu Pro Lys Met Arg Thr Val Ser Gln Ile Met Met Arg Gln Pro 225 230 235 240 Ser Glu Thr Gly His His Leu Leu Ser Glu Pro Gly Thr Pro Phe Ser 245 250 255 Pro Asn Ile Gln Gly Glu Leu Phe Phe Ser Ala Gln Gly Leu Pro Glu 260 265 270 Ser Asn Ile 275 <210> 74 <211> 293 <212> PRT <213> Tiger puffer (Takifugu rubripes) <400> 74 Leu Glu Lys Phe Thr Ser Ala His His Thr Lys Gln Thr Leu Glu Lys 1 5 10 15 Gly Leu Asn Leu Leu Cys Leu Thr Lys Ser Asn Ala His His Gly His 20 25 30 Pro Ala Met Ser Val Ser Ala Ser His Leu Tyr Tyr Thr Cys Pro Glu 35 40 45 Gly Ala Asn Ala Thr Leu Val Cys Asn Gln Arg Gly Gly Ala Leu His 50 55 60 Pro Asn Asp Ser Leu Trp Arg Leu Trp Phe Phe Thr Pro His Lys Asp 65 70 75 80 Gln His Cys Thr Lys His Gly Pro Arg Asn Val Thr Phe Lys His Ser 85 90 95 Lys Leu Ser Ser Gly Leu His Phe Gly Ala Thr Gln Glu Asn Phe Trp 100 105 110 Val Gln Leu Gln Asn Val Thr His Ala Asp Gln Gly Arg Tyr Cys Cys 115 120 125 Ala Ala Leu Glu Ile Glu Ser Ile His His Glu Ala Val Gln Arg Thr 130 135 140 His Ser His Met Phe Leu Asn Ile Ile Pro Arg Gly Thr Gly Ser Pro 145 150 155 160 Asn Cys Thr Val Ser Ala Pro Ser Ala Pro Glu Gly Asn Ala Thr Leu 165 170 175 Cys Thr Val Pro Val Ala Leu Ala Met Gly Ala Cys Ile Leu Ala Leu 180 185 190 Leu Ser Leu Pro Leu Ile Leu Leu Leu Val Tyr Arg Gln Arg Gln Ser 195 200 205 Ala Gln Ser Arg Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Glu 210 215 220 Ala His Gly His Glu Asn Pro Val Phe Leu Gly Gly Ser Pro Gln Ile 225 230 235 240 Lys Asn Arg Thr Val Ser Gln Ile Met Ala Arg Gln Ser Ser Glu Thr 245 250 255 Gly Arg His Leu Leu Ser Glu Pro Gly Thr Pro Leu Ser Pro Pro Ala 260 265 270 His Gly Asp Val Phe Phe Pro Ala Glu Asp Thr Ile Phe Glu Thr Pro 275 280 285 Glu Leu Arg Gln Val 290 <210> 75 <211> 168 <212> PRT <213> Homo sapiens <400> 75 Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 1 5 10 15 Asp Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 20 25 30 Asp Val Thr Phe Tyr Lys Thr Asn Tyr Arg Ser Ser Arg Gly Glu Val 35 40 45 Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asp Leu Thr Pro Gln Asp 50 55 60 Leu Leu Leu His His Gly Gly His Gln Ala Ala Asp Thr Ser His Asp 65 70 75 80 Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn 85 90 95 Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr 100 105 110 Cys Cys Leu Val Val Glu Ile Arg Met Met Met Ser Glu Met Arg Val 115 120 125 Met Gly Ala Asn Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser 130 135 140 Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Glu Ser Glu Asn Ile Thr 145 150 155 160 Ala Ala His His His His His His 165 <210> 76 <211> 9 <212> PRT <213> Homo sapiens <400> 76 Ile Arg His His His Ser Glu His Arg 1 5 <210> 77 <211> 169 <212> PRT <213> Homo sapiens <400> 77 Ala Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly 1 5 10 15 Gln Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly 20 25 30 His Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu 35 40 45 Val Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Gln Leu Thr Phe Gln 50 55 60 Asp Leu His Leu His His Gly Gly His Gln Ala Ala Gln Thr Ser His 65 70 75 80 Asp Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly 85 90 95 Asn Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu 100 105 110 Tyr Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg 115 120 125 Val His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro 130 135 140 Ser Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Glu Ser Glu Gln Ile 145 150 155 160 Thr Ala Ala His His His His His His 165 <210> 78 <211> 241 <212> PRT <213> Homo sapiens <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Arg 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Tyr Asn Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Gln Gln Val Gln Leu Val Gln Ser 115 120 125 Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys 130 135 140 Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe 165 170 175 Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr 180 185 190 Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 195 200 205 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ser Tyr Gly Trp 210 215 220 Ser Tyr Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ser <210> 79 <211> 169 <212> PRT <213> Homo sapiens <400> 79 Ala Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly 1 5 10 15 Gln Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly 20 25 30 His Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu 35 40 45 Val Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Gln Leu Thr Phe Gln 50 55 60 Asp Leu His Leu His His Gly Gly His Gln Ala Ala Gln Thr Ser His 65 70 75 80 Asp Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly 85 90 95 Asn Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu 100 105 110 Tyr Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg 115 120 125 Val His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro 130 135 140 Ser Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Glu Ser Glu Gln Ile 145 150 155 160 Thr Ala Ala His His His His His His 165

Claims

1. An isolated antibody or antibody fragment comprising a variable light chain polypeptide and a variable heavy chain polypeptide, wherein the antibody or antibody fragment binds to a V-domain Ig inhibitory factor of T cell activation (VISTA), wherein: (i) the heavy chain of the antibody or antibody fragment comprises a variable heavy chain (V H ) polypeptide, the variable heavy chain (V H ) The polypeptide comprises a V consisting of the amino acid sequence of SEQ ID NO: 19 H CDR1, V consisting of the amino acid sequence of SEQ ID NO: 20 H CDR2 and V consisting of the amino acid sequence of SEQ ID NO: 21 H CDR3, and (ii) the light chain of the antibody or antibody fragment comprises a variable light chain (V L ) polypeptide, the variable light chain (V L ) The polypeptide comprises a V consisting of the amino acid sequence of SEQ ID NO: 22 L CDR1, V consisting of the amino acid sequence of SEQ ID NO: 23 L CDR2 and V consisting of the amino acid sequence of SEQ ID NO: 24 L CDR3.

2. The antibody or antibody fragment according to claim 1, wherein the binding of the antibody or antibody fragment to VISTA promotes an immune response selected from the following: activation of monocytes; induction of T cell proliferation; induction of cytokine secretion; antibody-dependent cell-mediated cytotoxicity of cells expressing VISTA; or antibody-dependent cellular phagocytosis of cells expressing VISTA.

3. The antibody or antibody fragment of claim 1, wherein the antibody fragment is selected from the group consisting of: Fab, F(ab')2 or scFv antibody fragment. The antibody or antibody fragment according to claim 1 , comprising an antibody constant region.

5. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the antibody is a humanized antibody.

7. The antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody comprises a human constant region.

8. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the antibody has a concentration of at least 1×10 -8 L / mol affinity binds to VISTA.

9. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the antibody or antibody fragment has a concentration of at least 1×10 -7 L / mol affinity binds to VISTA.

10. The antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody or antibody fragment comprises a variable heavy chain polypeptide of SEQ ID NO: 40 and a variable light chain polypeptide of SEQ ID NO:

43.

11. The antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody or antibody fragment comprises a human heavy chain constant region.

12. The antibody or antibody fragment of claim 11, wherein the human heavy chain constant region comprises a human IgG1 heavy chain constant region.

13. The antibody or antibody fragment of claim 12, wherein the human heavy chain constant region comprises the human IgG1 heavy chain constant region contained in SEQ ID NO: 61 or SEQ ID NO:

60.

14. The antibody or antibody fragment of claim 11, wherein the human heavy chain constant region comprises a human IgG1 heavy chain constant region comprising a modification that increases protease resistance of the antibody compared to a human heavy chain constant region lacking such modification.

15. The antibody or antibody fragment of any one of claims 1 to 4, comprising a human light chain constant region.

16. The antibody or antibody fragment of claim 15, wherein the human light chain constant region comprises a human light chain constant region comprising the amino acid sequence of SEQ ID NO:

56.

17. The antibody or antibody fragment according to any one of claims 1 to 4, wherein (i) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 53, and (ii) the light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

54.

18. The antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody or antibody fragment is expressed in a recombinant cell.

19. The antibody or antibody fragment of claim 10, wherein the antibody or antibody fragment is expressed in a recombinant cell.

20. The antibody or antibody fragment of claim 17, wherein the antibody or antibody fragment is expressed in a recombinant cell.

21. The antibody or antibody fragment of claim 18, wherein the cell lacks trehalosylase.

22. The antibody or antibody fragment of claim 19, wherein the cell lacks trehalosylase.

23. The antibody or antibody fragment of claim 20, wherein the cell lacks trehalosylase.

24. The antibody or antibody fragment of claim 18, wherein the cell is a CHO cell.

25. The antibody or antibody fragment of claim 19, wherein the cell is a CHO cell.

26. The antibody or antibody fragment of claim 20, wherein the cell is a CHO cell.

27. A composition comprising the antibody or antibody fragment according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

28. A composition comprising the antibody or antibody fragment of claim 10 and a pharmaceutically acceptable carrier.

29. A composition comprising the antibody or antibody fragment of claim 17 and a pharmaceutically acceptable carrier.

30. One or more nucleic acids encoding the antibody or antibody fragment of any one of claims 1 to 4 for expression.

31. One or more nucleic acids encoding the antibody or antibody fragment of claim 10 for expression.

32. One or more nucleic acids encoding the antibody or antibody fragment of claim 17 for expression.

33. An expression vector comprising one or more nucleic acids encoding the antibody or antibody fragment of any one of claims 1 to 4 for expression.

34. An expression vector comprising one or more nucleic acids encoding the antibody or antibody fragment of claim 10 for expression.

35. An expression vector comprising one or more nucleic acids encoding the antibody or antibody fragment of claim 17 for expression.

36. A recombinant cell expressing one or more nucleic acids of claim 30.

37. A recombinant cell expressing one or more nucleic acids of claim 31.

38. A recombinant cell expressing one or more nucleic acids of claim 32.

39. The recombinant cell of claim 36, which is a CHO cell.

40. The recombinant cell of claim 37, which is a CHO cell.

41. The recombinant cell of claim 38, which is a CHO cell.

42. A method for expressing the antibody or antibody fragment of any one of claims 1 to 4, the method comprising culturing a cell comprising a nucleic acid encoding the antibody or antibody fragment under conditions that promote expression of the antibody or antibody fragment.

43. A method for expressing the antibody or antibody fragment of claim 10, the method comprising culturing a cell comprising a nucleic acid encoding the antibody or antibody fragment under conditions that promote expression of the antibody or antibody fragment.

44. A method of expressing the antibody or antibody fragment of claim 17, the method comprising culturing a cell comprising a nucleic acid encoding the antibody or antibody fragment under conditions that promote expression of the antibody or antibody fragment.

45. One or more pharmaceutical compositions comprising (i) the antibody or antibody fragment according to any one of claims 1 to 4, and (ii) Cancer vaccines.

46. ​​One or more pharmaceutical compositions comprising (i) the antibody or antibody fragment according to claim 10, and (ii) Cancer vaccines.

47. One or more pharmaceutical compositions comprising (i) the antibody or antibody fragment according to claim 17, and (ii) Cancer vaccines.

48. An application of an antibody or antibody fragment as described in any one of claims 1 to 4 for the preparation of a pharmaceutical for treating cancer associated with VISTA-mediated immunosuppression or immune cells or cancer cells expressing VISTA, wherein the cancer comprises myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

49. A use of an antibody or antibody fragment as described in claim 10, for preparing a medicament for treating cancer associated with VISTA-mediated immunosuppression or immune cells or cancer cells expressing VISTA, wherein the cancer comprises myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

50. Use of an antibody or antibody fragment as described in claim 17, for preparing a medicament for treating cancer associated with VISTA-mediated immunosuppression or immune cells or cancer cells expressing VISTA, wherein the cancer comprises myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

51. The use of claim 48, wherein the cancer comprises leukemia, lymphoma, myelodysplastic syndrome or myeloma.

52. The use of claim 49, wherein the cancer comprises leukemia, lymphoma, myelodysplastic syndrome or myeloma.

53. The use of claim 50, wherein the cancer comprises leukemia, lymphoma, myelodysplastic syndrome or myeloma.

54. The use according to claim 48, wherein the leukemia comprises lymphocytic leukemia, myeloid leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T-cell leukemia.

55. The use according to claim 49, wherein the leukemia comprises lymphocytic leukemia, myeloid leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T-cell leukemia.

56. The use according to claim 50, wherein the leukemia comprises lymphocytic leukemia, myeloid leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T-cell leukemia.

57. The use of claim 48, wherein the leukemia comprises acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), or chronic myeloid leukemia (CML).

58. The use of claim 49, wherein the leukemia comprises acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), or chronic myeloid leukemia (CML).

59. The use of claim 50, wherein the leukemia comprises acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), or chronic myeloid leukemia (CML).

60. The use of claim 48, wherein the cancer comprises lung cancer.

61. The use of claim 49, wherein the cancer comprises lung cancer.

62. The use of claim 50, wherein the cancer comprises lung cancer.

63. The use of claim 48, wherein the cancer comprises bladder cancer.

64. The use of claim 49, wherein the cancer comprises bladder cancer.

65. The use of claim 50, wherein the cancer comprises bladder cancer.

66. Use of an antibody or antibody fragment as described in any one of claims 1 to 4 for the preparation of a pharmaceutical product for enhancing an anti-tumor immune response, wherein the anti-tumor immune response is against a cancer selected from myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

67. A use of the antibody or antibody fragment as described in claim 10 for preparing a medicament for enhancing an anti-tumor immune response, wherein the anti-tumor immune response is against a cancer selected from myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

68. Use of the antibody or antibody fragment of claim 17 for the preparation of a medicament for enhancing an anti-tumor immune response, wherein the anti-tumor immune response is against a cancer selected from myeloma, leukemia, lymphoma, myelodysplastic syndrome, lung cancer, colon cancer or bladder cancer.

69. The use according to claim 66, wherein the medicament is suitable for parenteral or enteral administration.

70. The use according to claim 67, wherein the medicament is suitable for parenteral or enteral administration.

71. The use according to claim 68, wherein the medicament is suitable for parenteral or enteral administration.

72. The use of claim 66, wherein the medicament is suitable for intravenous, subcutaneous or oral administration.

73. The use according to claim 67, wherein the medicament is suitable for intravenous, subcutaneous or oral administration.

74. The use according to claim 68, wherein the medicament is suitable for intravenous, subcutaneous or oral administration.

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