TWI836472B - Anti-vista antibodies and fragments - Google Patents

Abstract

The present invention relates to novel antibodies and fragments that bind to a V-domain Ig Suppressor of T cell Activation (VISTA), and methods of making and using same. Methods of use include methods of treatment of cancer, including leukemias, lymphomas, solid tumors and melanomas.

Description

Anti-VISTA antibodies and fragments

The present invention relates to novel antibodies and fragments that bind to V-domain Ig inhibitor of T cell activation (VISTA), and methods for their preparation and use.

In the tumor microenvironment, immune checkpoint negative regulators expressed by cancer cells or immune cells can suppress the host's immune response against the tumor. To effectively fight cancer, it is necessary to block tumor-mediated suppression of the host's immune response. Therefore, there is a need for new and effective therapeutic agents that inhibit immune checkpoint negative regulators in the tumor microenvironment, wherein the immune checkpoint negative regulators inhibit the anti-tumor immune response.

The present invention relates to antibodies and antibody fragments comprising an antigen-binding region that binds to V-domain Ig inhibitor of T cell activation (VISTA). VISTA is a checkpoint regulator that adversely suppresses the immune response. See Wang et al. , "VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses," J. Exp. Med ., 208(3) 577-92 (2011). It is manifested in normal human neutrophils, monocytes and T cell subsets. Furthermore, cynomolgus monkey cells expressed VISTA in a pattern similar to normal human cells. VISTA is also expressed in peripheral blood cells of cancer patients.

The antibody or antibody fragment binds to VISTA to modulate or promote an immune response. The antibody fragment may include, for example, Fab, F(ab') ₂ or scFv antibody fragments. The antibody or antibody fragment may include an antibody constant region. The antibody or antibody fragment may bind to VISTA expressed on 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.

The antibody or antibody fragment may comprise one or more heavy chain complementation 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 specific examples, the antibody or fragment thereof is selected from the group consisting of VSTB112, VSTB95, VSTB116, VSTB50, VSTB53, and VSTB60. In one specific example, the antibody or fragment comprises 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 includes 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 includes 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.

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

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

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

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 to the antigenic determinant of VISTA, such as the amino acid sequence SEQ ID NO: 9. In some embodiments, the antibody or antibody fragment binds to the antigenic determinant of VISTA with an affinity of at least 1× ^10-7 liters/mole (e.g., at least 1× ^10-8 liters/mole, such as at least 1× ^10-9 liters/mole).

In some embodiments, modulating the immune response includes increasing CD45+ leukocytes, CD4+ T cells, and/or CD8+ T cells. In some embodiments, modulating an immune response includes increasing (eg, T cell) cytokine (eg, IFNγ, IL 10, TNFα, IL-17) production, increasing T cell responses, and/or modulating Foxp3 expression.

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

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

In some embodiments, the antibody or antibody fragment binds to VISTA to modulate or promote an immunogenic response to cancer. In some embodiments, the cancer is leukemia, lymphoma, myelodysplastic syndrome and/or myeloma. In some embodiments, the 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 granulocytic lymphocytic leukemia, or adult T-cell leukemia. In some embodiments, the lymphoma is a histiocytic lymphoma, and in some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a solid tumor, such as melanoma or bladder cancer. In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)). Some treatment methods further include administering a vaccine (e.g., 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 described herein.

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)).

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.

The invention also includes methods of making antibodies, fragments and compositions, such as methods of making the antibodies or fragments described herein, which comprise culturing host cells under conditions for making the antibodies. The method may further comprise isolating the antibody. The 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., operably linked to a promoter), and expression vectors transformed with such expression vectors host cell.

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.

The invention also provides an isolated antibody or an antibody fragment thereof, which comprises an antigen-binding region that binds to V domain Ig inhibitory factor of T cell activation (VISTA), wherein the antibody comprises an antibody VH domain, and the antibody VH domain contains SEQ ID VH CDR1 with the amino acid sequence of NO:25, VH CDR2 with the amino acid sequence of SEQ ID NO:26, and VH CDR3 with the amino acid sequence of SEQ ID NO:27, and the antibody further comprises an antibody VL domain , the antibody VL domain includes VL CDR1 with the amino acid sequence of SEQ ID NO:28, VL CDR2 with the amino acid sequence of SEQ ID NO:29, and VL CDR3 with the amino acid sequence of SEQ ID NO:30 . In some embodiments, an antibody or antibody fragment contains one or more humanized or human structural regions. In certain embodiments, 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 that found in SEQ ID NO:56 ). The heavy chain constant region is preferably an IgG1 heavy chain constant region (eg, the IgG1 heavy chain constant region found in SEQ ID NO: 61). In certain embodiments, the IgGl heavy chain constant region is modified to increase the protease resistance of the antibody. An example of an IgG1 heavy chain constant region modified to increase protease resistance is the IgG1 heavy chain constant region found 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 The light chain. In certain embodiments, the antibody or antibody fragment is expressed in cells lacking trehalosylase (eg, Chinese hamster ovary (CHO) cells lacking trehalosylase).

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.

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

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

In addition, the present invention relates to a method of enhancing an immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or an antibody fragment thereof that binds to 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.

In another embodiment, the present invention provides a method of eliciting a biological 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 V-domain Ig inhibitory factor of T cell activation (VISTA) , which contains the antigen-binding region that binds to VISTA, thereby promoting the immune response to cancer. Examples of biological responses include activation of monocytes; induction of T cell proliferation and cytokine secretion; antibody-dependent cell-mediated cytotoxicity (ADCC) of VISTA-expressing cells; and antibody-dependent phagocytosis of VISTA-expressing cells ( ADCP).

Illustrative embodiments of the present invention are described below.

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 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 ⁺ and CD8 ⁺ T cell proliferation and cytokine production independently of PD-1 via cognate receptors. In a passive EAE (experimental autoimmune encephalomyelitis) disease model, VISTA-specific monoclonal antibodies enhance T cell-dependent immune responses and exacerbate disease. Overexpression of VISTA on tumor cells impairs protective anti-tumor immunity in tumor-bearing hosts. Studies of human VISTA have identified its inhibitory function on human T cells (Lines et al., Cancer Research 74:1924, 2014). Studies from Flies et al. also identified VISTA (referred to as PD-1H) as a potent immunosuppressive molecule (Flies et al., 2011). VISTA is further described in U.S. Published Application US 20130177557 A1 and U.S. Patent Nos. 7,919,585 and 8,236,304, which are incorporated herein by reference in their entirety.

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

Antibodies and fragments of the invention

The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, human antibodies and anti-Id antibodies as well as antibodies produced by any known technology such as ( But not limited to) its fragments, regions or derivatives provided by enzymatic cleavage, peptide synthesis or recombinant technology). The anti-VISTA antibodies of the invention are capable of binding to the portion of VISTA that modulates, modulates or enhances the immune response. In some embodiments, the antibodies competitively inhibit one or more of the anti-VISTA antibodies described herein. Methods of 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 one antibody blocks the binding of another antibody to the target. Typically, competitive binding assays involve the use of purified target antigen (eg, PD-1) bound to a solid substrate or cells, an unlabeled test binding molecule, and a labeled reference binding molecule. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cell in the presence of a test binding molecule. Typically the test binding molecule is present in excess. Typically, when the competitive binding molecule is present in excess, it will inhibit the specific binding of the reference binding molecule to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75 % or above 75%. In some embodiments, competitive inhibition is determined using a competitive inhibition ELISA assay.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of animals immunized with the antigen. Monoclonal antibodies contain a substantially homogeneous population of antibodies to a specific antigen that contain substantially similar epitope 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., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); and Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory (1988); Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), where the owner's content appears in full text Incorporated herein by reference. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD, and any subclasses thereof. Fusionomas producing monoclonal antibodies of the present invention can be cultured in vitro, in situ or in vivo.

The present invention also encompasses digestion fragments, designated portions and variants thereof, including antibody mimetics or antibody portions that mimic the structure and/or function of an antibody 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 invention encompasses antibody fragments, or portions thereof, that are capable of binding 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 reagglutination), Fv or scFv (e.g. by molecular biology techniques) fragments. Antibody fragments of the invention also include those described in Aaron L. Nelson, mAbs 2:1, 77-83 (January/February 2010), the content of which is incorporated by reference in its entirety.

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 known techniques, or can be prepared as contiguous proteins using genetic engineering techniques.

In a specific example, the amino acid sequence of an immunoglobulin chain or a 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) with 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.

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

Antibodies or Variants of the Invention A given variant may comprise any number of contiguous amino acid residues from an antibody of the 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 the 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. Furthermore, 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.

Those skilled in the art will appreciate 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 that of a natural (non-synthetic), endogenous or related and known antibody. Methods for analyzing and quantifying enzyme activity and substrate specificity measurements are well known to those skilled in the art.

Substantially similar means that the compound has at least 85% (e.g., at least 95%) identity to a natural (non-synthetic), endogenous or related and known antibody and has at least 85% (e.g., at least 95%) of its activity.

The term "human antibody" as used herein refers to an antibody in which substantially all parts of the protein (e.g., CDRs, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinge (VL, VH)) Substantially non-immunogenic in humans with only minor sequence alterations or variations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees and the like), rodents (mice, rats and the like) and other mammals refer to such species, subgenus, genus, subgenus Family, family-specific antibodies. Additionally, chimeric antibodies may include any combination of the above. Such alterations or variations, as appropriate and preferably retain or reduce immunogenicity in humans or other species relative to unmodified antibodies. Therefore, human antibodies are different from chimeric or humanized antibodies. It is noted that human antibodies can be produced from non-human animals or prokaryotic or eukaryotic cells capable of functionally expressing rearranged human immunoglobulin (eg, heavy chain and/or light chain) genes. In addition, when the human antibody is a single chain antibody, it may contain linking peptides that are not present in native human antibodies. For example, an Fv may comprise a linker peptide, such as two to about eight glycine or other amino acid residues, linking the variable region of the heavy chain to the variable region of the light chain. Such linker peptides are believed to be of human origin.

Bispecific, heterospecific, heterobinding or similar antibodies, which are monoclonal, preferably human or humanized antibodies with binding specificities for at least two different antigens, may also be used. In the context of 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, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). See also WO 93/08829; U.S. Patent 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, No. 5,959,083, No. No. 5,932,448, No. 5,833,985, No. 5,821,333, No. 5,807,706, No. 5,643,759, No. 5,601,819, No. 5,582,996, No. 5,496,549, No. 4,676,980, WO 91/00360, WO 92/0 0373, EP 03089; Traunecker et al. , EMBO J. 10:3655 (1991); Suresh et al., Methods in Enzymology 121:210 (1986), each of which is incorporated by reference in its entirety.

In one specific example, the invention relates to a bispecific antibody that targets VISTA and a second target protein, such as an immune checkpoint protein. Exemplary bispecific antibodies include bispecific antibodies targeting VISTA and PD-L1 and bispecific antibodies targeting VISTA and PD-L2.

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

Other specific or general mammalian antibodies can be raised similarly. Immunogenic antigen preparation and monoclonal antibody preparation can be performed using any suitable technique.

For example, the hybridoma is prepared by fusing a suitable immortalized cell line (e.g., a myeloma cell line, such as (but not limited to) Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A or its analogs, or hybrid myeloma, its fusion product or any cell or fusion cell derived therefrom, or any other suitable cell line known in the art, see, for example, www.atcc.org) and antibody-producing cells. Antibody-producing cells may include isolated or cloned spleen, peripheral blood, lymph, tonsil or other immune cells (e.g., B cells) or any other cells expressing heavy chain or light chain constant or variable or framework 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, supra, and Colligan, Immunology, Chapter 2, supra, which are incorporated herein by reference in their entirety.

Antibody-producing cells can also be obtained from the peripheral blood or preferably the spleen or lymph nodes of a human or other suitable animal that has been immunized with the relevant antigen. Any other suitable host cell may also be used to express heterogeneous or endogenous nucleic acids encoding the antibodies of the invention, designated fragments or variants thereof. Fusion cells (fusionomas) or recombinant cells can be isolated 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 the desired specificity can be selected by suitable assays, such as enzyme-linked immunosorbent assay (ELISA).

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 the like, presentation libraries; purchased, for example, 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, e.g., 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 614 989; WO95/16027; WO88/06630; WO90/3809 ; U.S. Patent No. 4,704,692; PCT/US91/02989; WO89/06283; EP 371 998; 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) or relies on methods of immunizing transgenic animals (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 techniques (U.S. Patent 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, ed. Borrebaeck, Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods of engineering or humanizing non-human or human antibodies can also be used and are well known in the art. Generally, humanized or engineered antibodies have one or more amino acid residues derived 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 "import" variable, constant or other domains of known human sequences. Known human Ig sequences are disclosed, eg, 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.

As is known in the art, such introduced sequences can be used to reduce immunogenicity or reduce, increase or alter binding, affinity, avidity, specificity, half-life or any other suitable characteristic. In general, some or all of the non-human or human CDR sequences are maintained, while some or all of the non-human sequences of the framework and/or constant regions are replaced with human or other amino acids. Antibodies can also be humanized, as appropriate, using three-dimensional immunoglobulin models known to those skilled in the art, while retaining high affinity for antigen and other favorable biological properties. Computer programs that illustrate and present possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Detection of these presentations allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected from common and imported sequences and combined so that the desired antibody characteristic (such as increased affinity for the target antigen) is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of 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. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1992). (1993); U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567, each of which is incorporated herein by reference in its entirety, including the references cited therein.

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

Transgenic animals that produce repertoires of human antibodies that bind to human antigens can be prepared by known methods (such as, but not limited to, U.S. Patent Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126 issued to Lonberg et al. No. 5,625,825, 5,633,425, 5,661,016 and 5,789,650; 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 151 B1, Kucherlapate et al. EP 0710 719 A1, Surani et al. US Patent No. 5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. et al. EP 0438 474 B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol. 6(4)579 -591 (1994), Green et al., Nature Genetics 7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic Acids Research 20(23):6287- 6295 (1992), Tuaillon et al., Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93 (1995), and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), each of which is incorporated herein by reference in its entirety). Generally, such mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is or is functionally rearranged. The endogenous immunoglobulin locus in such mice can be disrupted or deleted to remove the animal's ability to produce antibodies encoded by the endogenous gene.

Screening for antibodies or fragments that specifically bind to similar proteins can be conveniently achieved using peptide display libraries. This method involves screening a large number of peptides for individual members with the desired function or structure. 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, typically 5-100 amino acids long, and typically about 8 to 25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves presenting peptide sequences on the surface of phages or cells. Each phage or cell contains a nucleotide sequence encoding the specific peptide sequence presented. Such methods are described in PCT Patent Publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generating libraries of peptides have 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. Patent 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. Patent 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 antibody No. 5,427,908, No. 5,580,717 and No. 5,885,793 to 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.

Antibodies of the invention can also be prepared using at least one nucleic acid encoding an anti-VISTA antibody, resulting in transgenic animals (such as goats, cows, sheep and the like) that produce such antibodies in their 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 by reference in its entirety. in this article.

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 therein. References; 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 by reference in its entirety.

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 bind to human VISTA with a _KD equal to or lower than about ^10-7 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). In some embodiments, the antibody or antibody fragment can bind to human VISTA with an affinity of 1× ^10-7 liter/mole (e.g., at least 1× ^10-8 liter/mole, such as at least 1× ^10-9 liter/mole).

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 the 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 antibody and antigen and standardized buffer.

nucleic acid molecules

Using the information provided herein, such as nucleotide sequences encoding at least 70-100% of the contiguous amino acids of at least one of the specified fragments, variants thereof, or common sequences, or a deposited vector comprising at least one of these 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, wherein the anti-VISTA antibody comprises 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.

The nucleic acid molecules of the 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). DNA can be triple-stranded, double-stranded or single-stranded or any combination thereof. Any part of at least one strand of DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also known as the antisense strand.

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 a coding sequence of an anti-VISTA antibody or fragment (e.g., a fragment comprising a variable region); and nucleic acid molecules comprising a nucleotide sequence different from the 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 generate 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.

As indicated herein, a nucleic acid molecule of the invention comprising a nucleic acid encoding an anti-VISTA antibody may include, but is not limited to, a nucleic acid molecule encoding an amino acid sequence of an antibody fragment; a coding sequence for an entire antibody or a portion thereof; an antibody, fragment or portion the coding sequence as well as other sequences, such as at least one signal leader sequence or the coding sequence of the fusion peptide, such as at least one intron, in the presence or absence of the other coding sequences described above, and other non-coding sequences, including (but not limited to) non- Encodes 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); encodes other amines Other coding sequences for amino acids, such as coding sequences that provide other functions. Thus, a sequence encoding an antibody can 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.

The human gene encoding the constant (C) region of the antibodies, fragments and regions of the present invention can be derived from a human fetal liver library by known methods. The human C region gene can be derived from any human cell, including human cells that express and produce human immunoglobulins. The human _CH 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 the H chain isotype is responsible for the various effector functions of the antibody, the selection of the _CH region will be guided by the desired effector function (such as complement fixation) or activity in antibody-dependent cellular cytotoxicity (ADCC).

Composition

The pharmaceutical compositions disclosed herein are prepared according to standard procedures and administered in doses selected to treat (e.g., to alleviate, prevent, or eliminate the condition being treated, or to 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, N.Y., 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. Patent Nos. 5,990,092; 5,039,660; 4,452,775; 3,854,480, the entire teachings of which are incorporated herein by reference.

To prepare pharmaceutical compositions from the anti-VISTA antibodies and/or fragments of the present invention, pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. For example, the compounds of the present invention may be in powder form for reconstitution upon delivery. A solid carrier can be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials. In powder form, the carrier is a finely divided solid which forms a mixture with the finely divided active ingredient.

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.

Liquid form preparations include solutions, suspensions, retention enemas and emulsions, such as water or aqueous propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in aqueous polyethylene glycol solutions.

Pharmaceutical compositions may be in unit dosage form. In such form, the composition is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation containing individual amounts of the unit dose. The dosage may vary depending on the requirements of the patient, the severity of the condition being treated, the compound used, and the route of administration. Determination of the appropriate dosage for a particular situation is within the skill of the art.

In addition, if necessary, the pharmaceutical composition may contain other compatible agents, such as drugs, therapeutic agents or preventive agents. Therapeutic or preventive agents include (but are not limited to) peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. Examples of classes of such agents (e.g., anticancer agents) include, but are not limited to, cytotoxins, angiogenesis inhibitors, immunomodulators, tumor immune agents, and agents used to relieve pain or counteract the toxic effects of one or more therapeutic agents. Agents (e.g., use of bisphosphonates that reduce the hypercalcemic effects of glucocorticoids).

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 fibronectin prolysin); anti-angiogenic antithrombin III; angiozyme. Bisphosphonates include, but are not limited to, alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate and zoledronate.

Immunomodulatory agents 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 antibody Rituxan (IDEC)), anti-CD52 antibody (e.g., CAMPATH 1H (Ilex)), anti-CD11a antibody (e.g., Xanelim (Genentech)); anti- Cytokines or anti-cytokine receptor antibodies and antagonists, such as anti-IL-2 receptor antibodies (Zenapax (Protein Design Laboratories)), anti-IL-6 receptor antibodies (e.g., MRA (Chugai)) and anti-IL-12 antibody (CNTO1275 (Janssen)), anti-TNFα antibody (Remicade (Janssen)) or TNF receptor antagonist (Enbrel (Immunex)), anti-IL-6 antibody (BE8 (Diaclone)) and siltuximab (CNTO32 (Centocor)) and antibodies that immunospecifically bind to tumor-associated antigens (such as trastuzimab (Genentech)).

Tumor immunotherapies suitable for use in 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.

The compositions are preferably prepared in dosage unit form containing a therapeutically effective amount of the antibody or fragment. Examples of dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules may contain, besides the active ingredient, customary carriers such as binding agents, for example gum arabic, gelatin, polyvinylpyrrolidone, sorbitol or tragacanth; fillers, for example phosphoric acid Calcium, glycine, lactose, cornstarch, sorbitol or sucrose; lubricants such as magnesium stearate, polyethylene glycol, silica or talc; disintegrants such as potato starch; flavoring or coloring agents ; or an acceptable wetting agent. 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, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include gum arabic, almond oil, ethanol, partially separated coconut oil, gelatin, glucose syrup, glycerol, hydrogenated edible fats, lecithin, methylcellulose, methylparaben or p-hydroxybenzoate Propyl benzoate, propylene glycol, sorbitol or sorbic acid.

Other general details regarding methods of making and using the compounds and compositions described herein are well known in the art. See, for example, U.S. Patent No. 7,820,169, the entire contents of which are incorporated herein.

Treatment

A person skilled in the art (e.g., a clinician) can determine the appropriate dose and route of administration of a particular antibody, fragment or composition for administration to an individual by considering the selected agent, pharmaceutical formulation and route of administration, various patient factors and other considerations. The dose preferably does not cause or produces minimal or no adverse side effects. In a standard multiple dosing regimen, a pharmacological agent can be administered at a dose schedule designed to maintain a predetermined or optimal plasma concentration in the individual being treated. The antibodies, fragments and compositions can be added in any appropriate dosage range or therapeutically effective amount, for example 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, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 In one embodiment, the composition, antibody or fragment is administered at a dose of 0.1-15 mg/kg per administration.

The antibody or fragment may be administered once, at least once, twice, at least twice, three times, or at least three times daily. 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 per month, at least once per month, twice per month, at least twice per month, three times per month, or at least three times per month. The antibody or antibody fragment can 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 times or at least six times per year.

Anti-VISTA antibodies, fragments and compositions may be administered, for example, via parenteral or enteral methods, including, but not limited to, intravenous, subcutaneous, oral, transrectal, intramuscular, intraperitoneal, transmucosal, transdermal, Intrathecal, nasal, or topical administration. One of ordinary skill will recognize that the following dosage forms may contain a compound or a corresponding pharmaceutically acceptable salt of a compound of the invention as an active ingredient. In some specific examples, the dosage form may contain a compound or a corresponding pharmaceutically acceptable salt of the compound as the active ingredient.

The anti-VISTA antibodies of the invention can be administered as part of a combination therapy (eg, together with each other or with one or more other therapeutic agents). The compounds of the present invention may be administered before, after, or concurrently with one or more other therapeutic agents. In some embodiments, compounds of the invention and other therapeutic agents can be co-administered simultaneously (eg, simultaneously) in separate formulations or combined formulations. Alternatively, the agents may be administered sequentially as separate compositions within an appropriate time frame determined by the skilled clinician (eg, a time sufficient to allow overlap in the drug effects of the therapy). The compounds of the invention and one or more other therapeutic agents may be administered in a single dose or in multiple doses in an order and schedule suitable to achieve the desired therapeutic effect.

The present invention also provides a method of regulating or treating at least one malignant disease in cells, tissues, organs, animals or patients. In some embodiments, the compounds and compositions of the present invention are used to treat or prevent cancer. Cancer can include any malignant or benign tumor of any organ or body system. Examples include (but are not limited to) the following: breast cancer, gastrointestinal cancer, gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, urogenital cancer, germ cell cancer, gynecological cancer, head and neck cancer, blood/blood cancer, musculoskeletal cancer , nerve cancer, respiratory/chest 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, cervix cancer, ovarian cancer and thyroid cancer. Other cancers may include leukemia, melanoma and lymphoma and any of the cancers described herein. In some embodiments, solid tumors are infiltrated with bone marrow and/or T cells. In some embodiments, the cancer is leukemia, lymphoma, myelodysplastic syndrome, and/or myeloma. In some embodiments, the cancer can be any kind or type of leukemia, including lymphocytic leukemia or myelogenous leukemia, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia, T-cell prelymphocytic leukemia, large granular lymphocytic leukemia, or adult T-cell leukemia. In some embodiments, the lymphoma is histiocytic lymphoma, follicular lymphoma, or Hodgkin lymphoma, and in some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a solid tumor, such as melanoma or bladder cancer. In one embodiment, the cancer is lung cancer, such as non-small cell lung cancer (NSCLC).

The present invention also provides a method for 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 sarcoma), colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/malignant hypercalcemia, solid tumors, adenocarcinomas, sarcomas, malignant melanomas, hemangiomas, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like. In some embodiments, the solid tumor is infiltrated by bone marrow and/or T cells. In a specific embodiment, the solid tumor is lung cancer, such as non-small cell lung cancer (NSCLC).

In some embodiments, the compounds and therapies described herein are co-administered with a vaccine (e.g., 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.

In some embodiments, the compounds and therapies described herein are co-administered with agents and/or bisphosphonates used in chemotherapy, hormonal therapy, and biological therapy. In some embodiments, the agents used in 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).

In other embodiments, anti-VISTA compounds and therapies described herein are co-administered with one or more immune checkpoint antibodies, such as nivolumab, perilizumab, tremelimumab, Pembrolizumab, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-TIM-3 antibody, anti-LAG-3v, anti-OX40 antibody and anti-GITR antibody.

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).

The anti-VISTA compounds and compositions of the present invention may be administered to a subject in need thereof to prevent (including preventing the recurrence of cancer) or treat (e.g., control or ameliorate 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) may 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; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride 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-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; mechlorethamine hydrochloride hydrochloride); megestrol acetate; melengestrol acetate; melphalan; menogaril; phenoxypurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; ormaplatin; paclitaxel; pegaspargase; porfromycin; prednimustine; procarbazine hydrochloride hydrochloride); puromycin; rogletimide; safingol hydrochloride; semustine; simtrazene; spalfosate sodium; sparsomycin; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tegafur; teloxantrone hydrochloride hydrochloride); temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate; trimetrexate glucuronate; triptorelin; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate); vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Targeted therapy includes, but is not limited to, tyrosine kinase inhibitors (e.g., imatinib, sorafenib, and vemurafenib). The present invention also encompasses the administration of an anti-VISTA compound 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 references such as the Physician's Desk Reference (57th Edition, 2003).

The anti-VISTA antibodies described herein are also suitable for use, for example, in the treatment of chronic infectious diseases such as, inter alia, HIV, HBV, HCV and HSV.

Various properties and sequence information for selecting anti-VISTA antibodies of the present invention are provided in Tables 1A, 1B and 2 herein.

Table 1A: CDR sequences of selected fully human or humanized anti-human VISTA antibodies mAb ID VH family Heavy chain cdr1 (Imgt) Heavy chain cdr2 (Imgt) Heavy chain cdr3 (Imgt) Light chain cdr1 (Imgt) Light chain cdr2 (Imgt) Light chain cdr3 (Imgt) VSTB50 B GYTFTNYG (SEQ ID NO:1) INPYTGEP (SEQ ID NO:2) AREGYGNYIFPY (SEQ ID NO:3) ESVDTYANSL (SEQ ID NO:4) RAS (SEQ ID NO:5) QQTNEDPRT (SEQ ID NO:6) VSTB53 GYTFTHYT (SEQ ID NO:7) IIPSSGYS (SEQ ID NO:8) ARGAYDDYYDYYAMDY (SEQ ID NO:9) QTIVHSNGNTY (SEQ ID NO:10) KVS (SEQ ID NO:11) FQASHVPWT（SEQ ID NO:12） VSTB60 B GYTFTNYG (SEQ ID NO:13) INTYTGES (SEQ ID NO:14) ARDYYGIYVSAY (SEQ ID NO:15) ESVDNYANSF (SEQ ID NO:16) RAS (SEQ ID NO:17) QQSHEDPYT (SEQ ID NO:18) VSTB95 GFTFRNYG (SEQ ID NO:19) IISGGSYT (SEQ ID NO:20) ARIYDHDGDYYAMDY (SEQ ID NO:21) QSIVHSNGNTY (SEQ ID NO:22) KVS (SEQ ID NO:23) FQGSHVPWT (SEQ ID NO:24) VSTB112 D GGTFSSYA (SEQ ID NO:25) IIPIFGTA (SEQ ID NO:26) ARSSYGWSYEFDY (SEQ ID NO:27) QSIDTR (SEQ ID NO:28) SAS (SEQ ID NO:29) QQSAYNPIT (SEQ ID NO:30) VSTB116 D GGTFSSYA (SEQ ID NO:31) IIPIFGTA (SEQ ID NO:32) ARSSYGWSYEFDY (SEQ ID NO:33) QSINTN (SEQ ID NO:34) AAS (SEQ ID NO:35) QQARDTPIT（SEQ ID NO:36）

Table 1B: Heavy chain and light chain sequences of selected fully human or humanized anti-human VISTA antibodies Protein ID Heavy chain AA CDS Light chain AA CDS VSTB50 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:47) DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:48) VSTB53 Question NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:49) DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:50) VSTB60 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMTWVRQAPGQGLEWMGWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCARDYYGIYVSAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51) DIVMTQTPLSLSVTPGQPASISCRASESVDNYANSFMHWYLQKPGQSPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSHEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:52) VSTB95 EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:53) DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC (SEQ ID NO:54) VSTB112 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:55) DIQMTQSPSSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO:56) VSTB116 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57) DIQMTQSPSSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO:58) VSTB140* QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:59) DIQMTQSPSSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:56) VSTB149* ^Δ QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAP PVA GP D VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN A ALPAPI A KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:60) DIQMTQSPSSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:56) VSTB174* QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:61) DIQMTQSPSSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO:56) *Constant region sequences in VSTB140, VSTB149 and VSTB174 are underlined. Amino acid residues in the heavy chain of ^ΔVSTB149 that confer protease resistance are shown in bold.

Table 2: Dissociation constants ( _KD ) of selected anti-VISTA antibodies Sample KD (M ) ka1 (1/Ms ) kd1 (1/s ) S1 1.71E-10 1.69E+06 2.89E-04 1.09E-10 1.11E+06 1.21E-04 S40 5.07E-10 1.46E+05 7.40E-05 6.96E-10 1.39E+05 9.69E-05 S41 6.32E-10 4.82E+05 3.05E-04 3.10E-10 7.08E+05 2.19E-04 S42 1.04E-10 1.05E+06 1.09E-04 2.65E-10 5.13E+05 1.36E-04 S43 2.64E-11 1.25E+06 3.30E-05 5.28E-11 1.18E+06 6.22E-05 S44 2.53E-11 1.23E+06 3.12E-05 6.40E-11 9.93E+05 6.36E-05 S45 2.35E-11 1.58E+06 3.72E-05 2.58E-11 1.46E+06 3.77E-05 S46 1.06E-10 1.56E+06 1.66E-04 2.96E-10 1.50E+06 4.44E-04 S47 3.56E-10 5.14E+05 1.83E-04 2.52E-10 5.69E+05 1.43E-04 S33 8.30E-10 1.23E+06 1.02E-03 1.22E-09 8.96E+05 1.10E-03 S34 1.08E-09 5.95E+05 6.43E-04 2.80E-09 5.20E+05 1.46E-03 S35 8.06E-11 2.08E+06 1.68E-04 1.35E-10 1.78E+06 2.41E-04 S36 6.29E-11 1.77E+06 1.12E-04 2.90E-11 1.58E+06 4.58E-05 S37 2.23E-09 5.10E+05 1.14E-03 4.43E-09 3.94E+05 1.75E-03 S38 2.26E-09 5.18E+05 1.17E-03 2.03E-09 5.37E+05 1.09E-03 S39 5.62E-10 3.97E+05 2.23E-04 3.47E-10 4.15E+05 1.44E-04 S25 1.31E-09 6.21E+05 8.12E-04 1.10E-09 5.65E+05 6.24E-04 S26 No binding 3.53E-09 2.38E+05 8.41E-04 S27 1.13E-09 8.86E+05 9.97E-04 1.61E-09 7.12E+05 1.15E-03 S48 3.12E-10 1.24E+06 3.87E-04 1.21E-09 8.78E+05 1.06E-03 S28 2.03E-09 1.08E+06 2.19E-03 2.03E-09 9.30E+05 1.88E-03 S29 3.78E-11 1.42E+06 5.38E-05 8.90E-11 9.06E+05 8.06E-05 S30 No binding No binding S31 Weak binding Weak binding S32 Weak binding Weak binding S15 9.34E-11 6.46E+05 6.04E-05 5.13E-10 3.50E+05 1.80E-04 S16 1.26E-10 5.54E+05 6.99E-05 1.92E-10 4.43E+05 8.53E-05 S17 7.68E-10 9.88E+05 7.59E-04 4.10E-10 7.09E+05 2.91E-04 S18 2.28E-09 4.90E+05 1.12E-03 1.05E-09 3.13E+05 3.29E-04 S19 1.54E-09 1.02E+06 1.58E-03 2.86E-10 7.03E+05 2.01E-04 S20 1.48E-09 6.67E+05 9.85E-04 4.57E-10 6.36E+05 2.91E-04 S21 3.18E-09 3.16E+05 1.00E-03 1.34E-09 2.70E+05 3.60E-04 S22 2.98E-09 1.09E+06 3.25E-03 1.27E-09 1.25E+06 1.59E-03 S6 6.36E-10 5.28E+05 3.36E-04 3.02E-10 5.98E+05 1.80E-04 S7 6.75E-10 1.31E+06 8.87E-04 3.27E-10 1.15E+06 3.75E-04 S8 1.15E-10 1.89E+06 2.18E-04 5.97E-11 1.25E+06 7.48E-05 S9 1.67E-10 1.87E+06 3.11E-04 9.31E-11 1.27E+06 1.18E-04 S10 8.90E-11 1.55E+06 1.38E-04 4.30E-11 1.22E+06 5.27E-05 S12 4.94E-10 1.57E+06 7.76E-04 2.39E-10 1.19E+06 2.86E-04 S13 1.02E-10 1.42E+06 1.44E-04 6.46E-11 9.55E+05 6.17E-05 S14 2.02E-10 1.26E+06 2.55E-04 7.55E-11 1.12E+06 8.43E-05 S1 2.06E-10 1.60E+06 3.29E-04 8.35E-11 1.21E+06 1.01E-04 S2 1.56E-10 9.74E+05 1.52E-04 8.66E-11 7.25E+05 6.28E-05 S3 4.33E-11 9.07E+05 3.93E-05 4.89E-11 7.41E+05 3.63E-05 S4 1.52E-10 8.98E+05 1.36E-04 7.54E-11 6.93E+05 5.23E-05 S49 1.45E-10 1.01E+06 1.46E-04 1.04E-10 7.28E+05 7.60E-05 S5 2.13E-10 1.25E+06 2.67E-04 1.37E-10 8.51E+05 1.17E-04 Embodiment

Example 1: Analysis of VISTA expression on human hematopoietic cells

method:

Preparation and staining of fresh human PBMC for VISTA performance

VISTA expression 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 line MOPC-21, 5 μg/ml) was used as an isotype control.

Donor material

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

Sample preparation

Dilute blood 1:1 with sterile PBS. Layer 22 ml of diluted umbilical cord blood over 20 ml sterile Ficoll-Hypaque (GE Healthcare catalog #17-144003) in a 50 ml conical tube. Centrifuge tube at 1800 rpm for 20 minutes at room temperature. Collect mononuclear cells at the interface after centrifugation using a 1 ml pipette and combine in two 50 ml conical tubes. Add sterile PBS to each tube to make up the volume to 50 ml and centrifuge cells at 300 g for 10 minutes at 4°C. Discard supernatant. Resuspend cells in 50 ml sterile PBS and spin tube at 300 g for 10 minutes at 4°C. Discard supernatant. Cells were pooled and resuspended in 50 ml sterile PBS before counting.

Staining protocol: A compensation control was performed using a frozen vial containing 5×10 ⁷ PBMCs and used as a comparison for staining.

Use the following reagents and/or consumables:

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 pure GA-1 from ImmunoNext lot 080612B (used at 5 μg/ml); biotinylated mIgG1,K isotype control (pure MOPC-21), Biolegend Cat. No. 400104, lot B116649 (used at 5 μg/ml); μg/ml); anti-human antibodies (see the table below for staining); near-infrared live/dead cell stain (Invitrogen, catalog number L10119); and anti-streptavidin reagents, including STP-APC (BD Biosciences catalog number 554067, lot number 04251) (used at a dilution of 1:200 in FACS buffer), STP-PE (Biolegend catalog number 405203, lot number B139688) (used at a dilution of 1:200 in FACS buffer), STP-PE Cy7 (to demonstrate nonspecific binding in isotype control samples), and STP-Q605 (Invitrogen catalog number Q10101MP, lot number 53449A) (used at a dilution of 1:200 in FACS buffer).

Cell surface staining protocol

Before staining, 1×10 ⁶ cells were transferred to 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.

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

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. Discard the supernatant.

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

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.

Table 3: Lineage staining Target Fluorescent agent antigen mouse rat human Same type Pure line supplier catalog number Batch number Titer (μl/10 ⁶ cells) FITC/AF488 CD19 X mIgG1 HIB19 Biolegend 302206 B123019 2 PE CD11b X mIgG1,K ICRF44 BD Bio. 555388 45134 2 PerCP-Cy5.5 HLA-DR X mIgG2a,K G46-6 BD Bio. 560652 25161 0.5 PE Cy7 CD16 X mIgG1,K 3G8 BD Bio. 557744 87825 0.2 APC Cy7 NIR Live/Dead X AF700 CD56 X mIgG1,K B159 BD Bio. 557919 19470 1 APC/AF647 VISTA-Bio X PB/V450 CD3 X mIgG1,K UCHT1 BD Bio. 558117 90926 0.5 Q605 CD14 X mIgG2a,K tK4 Invitrogen Q10013 1049158 0.2

Table 4: T cell staining Target Fluorescent agent antigen Mouse Rat Human Same type Pure Suppliers Catalog Number Batch No. Titer (μl/10 ⁶ cells) FITC/AF488 CD4 X mIgG1, K RPA-T4 BD Bio. 555346 38460 2 PE VISTA-Bio X PerCP-Cy5.5 CD8 X mIgG1, K RPA-T8 BD Bio. 560662 1037 0.5 PE Cy7 CD56 X mIgG1, K B159 BD Bio. 557747 47968 0.5 APC Cy7 NIR X AF700 CD45RO X mIgG2a, K UCHL1 Biolegend 304218 B143062 1 APC/AF647 TCR X mIgG, K B1 Biolegend 331212 B126473 2 PB/V450 CD45RA X mIgG2b, K HI100 BD Bio. 560363 90928 0.5 Q655 CD3 X mIgG2a S4.1 Invitrogen Q10012 982352 0.5

Table 5: DC staining Target Fluorescent agent antigen mouse rat human Same type Pure line supplier catalog number Batch number Titer (μl/10 ⁶ cells) FITC/AF488 Lin1 X Mix Mix BD Bio. 340546 2152758 5 PE CD11c X mIgG1,K BD Bio. 555392 45123 2 PerCP-Cy5.5 HLA-DR X mIgG2a,K G46-6 BD Bio. 560652 25161 0.5 APC Cy7 NIR X APC/AF647 CD83 X mIgG1,K HB15e BD Bio. 551073 57688 2 BV421 CD123 X mIgG1,K 6H6 Biolegend 306017 B148193 0.5 Q605 VISTA-Bio X

Table 6: Bone marrow staining Target Fluorescent agent antigen Mouse Rat Human Same type Pure Suppliers Catalog Number Batch No. Titer (μl/10 ⁶ cells) FITC/AF488 CD33 X mIgG1 HM3-4 Biolegend 303304 B100963 3 PE CD11b X mIgG1, K ICRF44 BD Bio. 555388 45134 2 APC Cy7 NIR X APC/AF647 VISTA-Bio X Q605 CD45 X mIgG1, K HI30 Invitrogen Q10051 880470 1

After surface staining, cells were washed twice with FACS staining buffer and centrifuged at 1300 rpm for 5 min at 4°C as previously described. Samples were resuspended in 50 μl of FACS staining buffer containing appropriately fluorescently labeled streptavidin. Incubate samples for 30 minutes at 4°C. Cells were washed with 150 μl FACS staining buffer and centrifuged at 1300 rpm for 5 min at 4°C. Repeat this wash step and then resuspend the sample in 250 μl FACS staining buffer. Samples were analyzed on the same day on a BD LSRFortessa™ Cell Analyzer (BD Biosciences).

Data Analysis

Flow cytometry data were reanalyzed using FlowJo model 9 software to gating specific phenotypic groups. Enumeration of geometric means was used to compare VISTA performance in different cell subpopulations. Each population was normalized to background by subtracting the isotype control value from the mean of autoantibody VISTA-treated samples. Use Prism to create curves and use student's T-test for statistics or one-way ANOVA and Bonferroni post-test statistics when comparing only two samples.

result:

VISTA expression in human bone marrow and lymphocyte subsets:

As shown in Figures 2A-2E, 3A-3G, 4, 5A-5B, and 6A-6C, VISTA expression on CD14 ⁺ 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 the CD14 ^{+ CD16-} subset having significantly higher expression compared to ^{CD14loCD16 +} cells. Although APCs showed moderate expression, lymphoid subsets showed low expression.

VISTA performance on human T and NK subsets:

As shown in Figures 7A-7E, 8A-8G and 9, for NK subsets, CD56 ^lo cells showed significantly higher VISTA expression than CD56 ^Hi NK cells. Among T cell subsets, CD8 ⁺ memory cells showed the highest expression, but it was not significantly higher than CD8 ⁺ naive or CD4 ⁺ T cells.

VISTA performance on human dendritic cell subsets:

As shown in Figures 10A-10D, 11A-11C, and 12, no significant differences in VISTA expression were found; DCs and philospheres exhibited low VISTA expression, while plasmacytoid dendritic cells (pDCs) generally had higher but not significant levels.

Conclusion: These results show VISTA expression on various immune cell subsets with VISTA being most highly expressed on monocytes, some on different T cell subsets and NK cells and almost not on B cells.

Example 2: VISTA expression on peripheral blood cells

method:

Whole blood staining: Stain freshly isolated whole blood (100 μl) with the antibody mixture shown below by incubating for 30 minutes at 4°C. Red blood cells (RBCs) were lysed with RBC lysis buffer and the remaining cells were washed once with staining buffer. Resuspend cells in 200 μl staining buffer. Data were collected using a MACSQuant flow cytometer and analyzed using FlowJo analysis software.

Peripheral blood mononuclear cells (PBMC) staining: Peripheral blood mononuclear cells were isolated from whole blood using a Ficoll gradient. Stain freshly isolated 1× ¹⁰ PBMC with 100 μl of antibody mixture in staining buffer. Samples were incubated for 30 minutes at 4°C, followed by one wash with staining buffer. Resuspend cells in 100 μl staining buffer. Data were collected using a MACSQuant® flow cytometer (Miltenyi Biotec) and analyzed using FlowJo analysis software.

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-conjugated mouse anti-human VISTA (pure GG8) was prepared by ImmuNext (Lebanon, NH).

Conclusion:

VISTA expression on healthy human peripheral blood cells

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

VISTA expression on peripheral blood cells of cancer patients

As shown in Figures 14A-C, peripheral blood mononuclear cells (PBMCs) from lung cancer patients were analyzed. Figure 14A is a representative flow cytometry analysis of bone marrow plot showing the analysis results of CD14 ⁺ monocytes and CD15 ⁺ bone marrow-derived suppressor cells (MDSC). The results indicate that the phenotypic CD15 ⁺ cells are neutrophil-derived MDSCs. Additionally, these cells are not present in healthy blood samples. Figure 14B is a representative histogram of VISTA expression on single nuclei from healthy and cancer patients, demonstrating higher levels of VISTA expression on cancer patient cells compared to healthy controls. Similarly, higher levels of VISTA were found on MDSC of cancer patients, as shown in Figure 14C.

Figure 15A is a representative FACS graph showing the presence of neutrophil-derived MDSCs in the blood of patients with colorectal cancer. Figures 15B and 15C are representative histograms showing higher levels of VISTA expression on mononuclear spheres of cancer patients compared to healthy donor blood samples.

VISTA expression on peripheral blood cells of cynomolgus macaques

As shown in Figures 16A and 16B, flow cytometric analysis of monkey whole blood revealed a VISTA expression pattern similar to that of human cells. Both monocytes and neutrophils expressed the highest levels of VISTA compared to CD4 ⁺ (Figure 16C) and CD8 ⁺ (Figure 16D) T cells.

Example 3: VISTA performance at RNA level and protein level in heme malignant cell lines

Because VISTA manifests in heme malignancies, anti-VISTA antibodies may target malignant cells to destroy and block VISTA and promote anti-tumor immune responses.

The data include RNAseq analysis results for approximately 140 heme malignant cell lines (some cell lines were replicated in the analysis). The data are presented in Figure 17.

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

Essentially, this means that the counts belong to all reads in the exonic regions of the gene, and are normalized by the length of the gene and the total number of reads per sample (to account for inter-sample variability). The cutoff value is 1; above 1 is VISTA positive (RNA level), below 1 is VISTA negative.

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 thought to be to weaken immune responses, including anti-tumor immune responses.

Example 4: Generation of monoclonal antibodies against VISTA

Phage panning

Twenty-four phage panning experiments were performed to enrich for phages active against Cyno VISTA-His. Macaque VISTA protein was used for these experiments because it exhibits better biotin binding compared to human VISTA protein. To confirm that the phage experiment was successful, phage pools from individual rounds of panning were added to neutravidin plates coated with biotin-labeled cyno VISTA-His and probed with HRP-conjugated anti-M13 antibodies. Individual colonies were picked from the phage selection round and Fab proteins were prepared in 96-well plates. The expressed Fab supernatants were analyzed for binding to biotin-labeled cyno VISTA-His. This operation got over 200 successful results.

The VH and VL regions from the Fab plates were expanded, submitted for DNA sequencing and exported as FASTA files. When selecting clones to be converted to MAbs and tested in MAb format, they were chosen based on sequence diversity and having limited risk of post-translational modifications and as few hydrophobic residues as possible.

VH and VL from phage pure lines were subcloned in mammalian IgG1/κ expression vector and transfected into HEK293 cells. Antibodies were purified on Protein A Sepharose Fast Flow Affinity Resin. Phage MAB concentration was determined by quantitative ELISA using Nanodrop measurements. The antibody panel performed at a high level. SDS-PAGE analysis demonstrates the integrity of each expressed antibody variant.

Online maturation of phage antibodies is performed by amplifying the VH domains from the multi-strain antibody mixture from the final round of panning for colonization in phage vectors with VL diversity. This operation results in an enriched VH pool that is sampled with additional VL diversity. The phages were subjected to 1-2 rounds of stringent panning in order to identify extremely high affinity binders of the VISTA ECD His protein. Perform individual strain Fab ELISA to determine maturation success. ELISA and performance data were normalized against a reference clone set to 100% from the original regeneration panning experiment, and affinity mature clones with higher binding signals to the cyno VISTA antigen compared to the reference clone were identified. This process generated several clones that exhibited up to 200% binding when screened at low antigen concentrations (1 nM). The clone with the highest affinity was sequenced and MAB generated.

fusion tumor generation

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

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

On the day of fusion, mice were euthanized by _CO2 asphyxiation, and spleens were removed and placed in 10 mL of cold phosphate-buffered saline. Single cell suspensions of spleen cells were prepared by grinding the spleen through a fine mesh sieve with a small pestle and rinsing with PBS at room temperature. Cells were washed once with PBS and RBC lysed. Briefly, cells were resuspended in 3 mL RBC lysis buffer (Sigma #R7757)/spleen and placed on ice for 5 min. Cells were washed once more with PBS at room temperature and labeled for magnetic sorting. Following the manufacturer's instructions, cells were labeled with anti-mouse Thy1.2, anti-mouse CD11b, and anti-mouse IgM magnetic beads (Miltenyi Biotec # 130-049-101, 130-049-601, and 130-047-301, respectively), followed by Sorting using MS column with Midi MACS. Fuse the negative cell fraction (positive cell fraction discarded) with FO cells. Murine myeloma cells were fused with viable splenocytes at a 1:1 ratio. Briefly, spleen and myeloma cells were mixed together, pelleted and washed once with 50 mL PBS. Resuspend the aggregated pellet with 1 mL of polyethylene glycol (PEG) solution (2 g PEG (molecular weight 4000), 2 mL DMEM, 0.4 mL DMSO) per 10e8 splenocytes for 30 seconds at 37°C. The cell/fusion mixture was then immersed in a 37°C water bath for approximately 60 seconds with gentle stirring. The fusion reaction was stopped by slowly adding 37°C DMEM over 1 minute. The fused cells were allowed to stand at room temperature for 5 minutes, followed by centrifugation at 150×g for 5 minutes. Cells were then resuspended in Medium E-HAT (MediumE, StemCell Technologies Catalog No. 03805) containing HAT (Sigma Catalog No. H0262) and plated in 96-well flat-bottomed polystyrene tissue culture dishes (Corning #3997).

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

All cyno VISTA active fusion tumor cell lines were cross-screened using human VISTA-Ig to assess cross-reactivity. Briefly, goat anti-ms Fc (Jackson #115-006-071) in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.4, Pierce 28382BupH™) was coated on a plate (Nunc- Maxisorp #446612), overnight at 4°C. Without washing, the wells were blocked with 200 μl of blocking agent (0.4% BSA (Sigma) (w/v) in PBS (Invitrogen)) overnight at 4°C. After removing the blocking solution, incubate undiluted fusion tumor supernatant on the coated plate for 30 minutes at room temperature. The plates were washed once with PBST (0.02% 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. Dishes 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 plate was washed again and then developed using 100 μl of TMB Turbo substrate (Thermo Scientific 34022) per well and incubated at room temperature for approximately 10 minutes. The reaction was stopped using 25 μl of 4 N sulfuric acid per well and the absorbance was measured at 450 nm using an automatic disk spectrophotometer.

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

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

For V region amplification, provide 20 μl of PCR reaction. Each well contains 16.2 μl water, 2.0 μl 10× PCR reaction buffer, 0.8 μl MgSO ₄ (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. Transfer the cDNA prepared as described above (2 μl per well) to the PCR component mix, seal the plate and run the amplification reaction; for VH, the program is (i) 94°C for 1 minute, (ii) 94°C for 15 seconds, ( iii) 55℃ for 30 seconds, (iv) 68℃ for 1 minute. Repeat steps (ii-iv) for a total of 35 cycles, followed by a final extension of 3 minutes at 68°C. For VL, the program is (i) 94°C for 1 minute, (ii) 94°C for 15 seconds, (iii) 55°C for 30 seconds, (iv) 65°C for 30 seconds, (v) 68°C for 1 minute. Repeat steps (ii-v) for a total of 35 cycles, followed by a final extension of 3 minutes at 68°C.

Premix the forward primer and use this mixture to the reverse primer in a ratio of 3:1. Check PCR products on agarose gel. Reagents were prepared for infusion selection by adding enhancers (In-Fusion HC Selection Kit, Cat. No. 639650, Clontech). Five microliters of the PCR reaction was transferred to the PCR plate, followed by 2 μl of enhancer per well. Seal the dish and incubate in a thermal cycler (15 minutes at 37°C and 15 minutes at 80°C). Prepare the vector of destination (vDR243 or vDR301) by digestion with Esp3I; (digest 1.5 μg of vector in 3 μl of Tango buffer, 2 l of Esp3I, and water in a 30 μl reaction for 2 hours at 37°C).

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). Perform infusion reactions in a 96-well plate format. Incubate the plate on a PCR machine for 15 minutes at 50°C and transform Stella competent cells by heat shock at 42°C for 40 seconds without shaking and spread with the antibiotic of choice Place on LB agar plate and incubate 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 prepared from overnight cultures mixed with an equal volume of 30 w/v% glycerol. Sequencing primer SPF0052 was used to sequence the V region. Sequences were analyzed, and one positive well per fusion tumor vH and vL was selected, rearranged in a new plate and grown overnight in enriched medium with ampicillin. The pure lines were then subjected to miniprep DNA preparation for small-scale transfection into 96-well plates.

Forty-eight selected mouse fusion tumor sequences of heavy and light chains were adapted to human constructs using in-house software programs. Select a human construct for each of mouse vH or vL. The V region DNA sequence 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 into precut vDR149 and vDR157, human IgG1 and human kappa respectively. Prepare using Qiagen Endo-free Maxi-prep kit. Expi293 (100 ml) cultures were used to express this antibody panel.

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

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

Example 6: Human structural region adaptation of anti-VISTA antibodies

Human architectural 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 delineates the complementarity determining regions (CDRs) of 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 architectural regions with human germline genes. The human germline gene with the highest sequence identity to the mouse construct was selected as the recipient gene for human construct adaptation (HFA). In some cases, closely related human germline genes are selected as replacements based on previous experience with well-performing human architectures. DNA sequences selected for the human construct for each of the mouse vH or vL V regions were 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 into human IgG1 and human kappa respectively.

Example 7: Anti-VISTA Antibody Construct

Plasmids and sequence information for cell line-produced molecules: Plasmid constructs were generated for anti-VISTA antibodies with VSTB112 variable regions and either an IgG1κ constant region (VSTB174, new numbering due to allotypic variation in the constant region), an IgG2δ constant region (VSTB140), or an IgG1 protease-resistant constant region (VSTB149).

Lonza Vectors

The pEE6.4 and pEE12.4 Chinese Hamster Ovary (CHO) expression vector systems (Lonza Biologics Public Co., Ltd.) were established in Biologics Research (BR) and Pharmaceutical Development & Manufacturing Sciences (PDMS) as methods for producing therapeutic mAbs in mammalian expression cell lines. Main performance system. Each vector contains the human cytomegalovirus (huCMV-MIE) promoter driving heavy chain (HC) or light chain (LC) expression and contains an ampicillin resistance gene. The pEE12.4 vector also includes the gene encoding glutamine synthetase (GS). Growth conditions requiring glutamine synthetase activity exert selective pressure on cells to maintain expression vectors (GS Gene Expression System Manual Version 4.0). The HC gene was selected using pEE6.4 as a single gene vector and the LC gene was selected using pEE12.4 as a single gene vector. From these two Lonza single-gene vectors, Lonza dual-gene plasmids were generated.

selected VISTA mAb The amino acid sequence of the variable heavy chain region

＞VSTB112 heavy chain (SEQ ID NO:37)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS

> VSTB50 rechain (SEQ ID NO:38)

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSS

＞VSTB53 heavy chain (SEQ ID NO:39)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDYYAMDYWGQGTLVTVSS

> VSTB95 rechain (SEQ ID NO:40)

EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSS

selected VISTA mAb The amino acid sequence of the variable light chain region

＞VSTB50 light chain (SEQ ID NO:41)

DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIK

＞VSTB53 light chain (SEQ ID NO:42)

DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEIK

＞VSTB95 light chain (SEQ ID NO:43)

DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIK

＞VSTB112 light chain (SEQ ID NO:44)

DIQMTQSPSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK

＞VSTB116 light chain (SEQ ID NO:45)

DIQMTQSPSSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQGTKVEIK

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

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.

method:

Overview of ELISA procedure: Coat plates overnight at 4°C with 1 μg/ml SB0361 (human) or SB0361 (cyno (cyno)) proteins, which are the extracellular domains of VISTA from the respective species. The antibody was gradually diluted from the initial concentration 1:4 to 1 μg/ml to obtain a total of 4 concentrations and incubated at room temperature (RT) for 2 hours. Probe with mouse anti-human IgG1-HRP (horseradish peroxidase) and incubate for 1 hour at room temperature. Use PBS-Tween (0.05%) for all washes.

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

Data Analysis/Results: For each antibody, a subjective score (yes/no) was given as to whether the antibody bound stably for each of the 4 assays, both for ELISA and FACS analysis. If an antibody gave a "no" result for binding in any assay, the assay was repeated to confirm that it was negative. The results are shown in Table 7 below and in Figures 19A-19F and 20A-20F.

Table 7. INX Number Hu ELISA Cyno ELISA Hu FACS Cyno FACS 1 Y Y Y Y 2 Y Y Y Y 3 Y Y Y Y 4 Y Y Y Y 5 Y Y Y Y 6 Y Y Y Y 7 Y Y Y Y 8 Y Y Y Y 9 Y Y Y Y 10 Y Y Y Y 11 N N N N 12 Y Y Y Y 14 Y Y Y Y 16 Y Y Y Y 17 Y Y Y Y 18 Y Y Y Y 19 Y Y Y Y 20 Y Y Y Y twenty one Y Y Y Y twenty two Y Y Y Y twenty three N N N N twenty four N N N N 25 Y Y Y Y 26 N Y N Y 28 Y Y Y Y 30 N N N N 31 N N N N 32 N N N N 33 Y Y Y Y 34 Y Y Y Y 35 Y Y Y Y 36 Y Y Y Y 37 Y Y Y Y 38 Y Y Y Y 39 Y Y N N 40 Y Y Y Y 41 Y Y Y Y 42 Y Y Y Y 43 Y Y Y Y 44 Y Y Y Y 45 Y Y Y Y 46 Y Y Y Y 47 Y Y Y Y 48 Y Y Y Y 49 Y Y Y Y

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

The purpose 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.

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, incubated together, and proliferation and cytokine production occur as a result of these mismatches.

Materials and Methods

Prepare 10% by combining 500 ml RPMI with 50 ml human AB serum, 5 ml penicillin/streptomycin (10,000 U/ml), 5 ml L-glutamine (100×) and 10 ml HEPES (1 M) AB medium. Medium should be stored for no more than 14 days. Prepare 1 mCi of tritiated thymidine by diluting 0.2 ml of thymidine stock solution (1 mCi/ml) with 9.8 ml of RPMI.

Dilute the soluble VISTA antibody to 20 μg/ml in 10% AB serum medium. Add 100 μl of the appropriate antibody solution to the appropriate wells of a 96-well U-chassis (Falcon Product #353077 or equivalent). After adding various cell populations, the final concentration was 10 μg/ml.

Isolated white blood cells: Donors are at least 18 years old, generally healthy and randomly selected from the local population. Transfer donor blood from the separation tube to a 50 ml Erlenmeyer flask. Place 15 ml of Ficoll 1077 under every 25 ml of blood, taking care not to mix with the blood. Centrifuge cells at 1250 g for 25 min at room temperature without braking. Leukocytes were separated at the interface of Ficoll and serum and the cells were diluted with 40 ml of Hanks Balances Salt Solution (HBSS). Centrifuge at 453 g (1500 rpm) for 10 min at 4 °C. Cells were resuspended in 50 ml HBSS and counted by transferring 500 μl into individual tubes.

Mixed Lymphocyte Reaction (MLR) 96-well Plate Setup: Determine the appropriate number of "stimulator cells" and "responder cells" needed for the assay based on the number of samples to be analyzed. Seed the stimulator population at 0.5 × 10 ⁵ cells per well of a 96-well U-bottom plate and the responder population at 1.0 × 10 ⁵ cells per well. All conditions must be repeated three times. Pipette the appropriate number of "stimulator cells" into a new Erlenmeyer flask and centrifuge as previously described. Resuspend the cells in 10 ml and irradiate with 4000 rad. Cells were centrifuged as previously described, resuspended in 10% AB serum medium at a concentration of 1 × 10 ⁶ / ml and 50 μl was added to the appropriate wells. The desired number of reactant cells were isolated, centrifuged as previously described, resuspended in 10% AB serum medium at a concentration of 2 × 10 ⁶ / ml and 50 μl was added to the appropriate wells. Cells were incubated at 37°C and 5% CO ₂ for 5 days. On the fifth day, 30 μl of supernatant was removed for analysis of interferon gamma (IFN-γ) production. On the fifth day, 25 μl of 40 μCi/ml tritiated thymidine solution was added to each well and incubated at 37°C and 5% CO ₂ for 8 hours. Cells were transferred to 96-well microflashes according to the manufacturer's instructions. Cells were counted using a microflashes counter according to the manufacturer's instructions. IFN-γ concentrations were determined by ELISA (eBioscience catalog number 88-7316-88) using the manufacturer's protocol.

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

Acceptance Criteria: All test reagents and appropriate controls were tested for endotoxin prior to assay and should contain < 0.1 EU/mg. The mean CPM count for cells reacting alone was less than 700 CPM, indicating that the cells were quiescent when incubated alone. The CPM of the MLR group was at least 2-fold greater than the CPM 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.

Screening of anti-VISTA antibodies in MLR: Initial screening of all compounds. Before operating the MLR with anti-VISTA antibodies, confirm antibody binding 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 were still tested in the analysis. For preliminary screening purposes, all antibodies were tested in MLR at 10 μg/ml, where the parameters measured were proliferation and IFN-γ (Figures 21A-21D and 22A-22B).

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).

Dilution study of the first six candidates in the MLR: protocol adjustments. The protocol was the same as previously described, but with some adjustments: the antibody was diluted to the following concentrations: 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 μg/ml.

Determine IC ₅₀ value: Use raw CPM counts and IFN-γ concentration to determine the IC ₅₀ value of each antibody. The IC ₅₀ value is calculated by using the program "EZ-R stats." _IC50 values were determined using six individual responders. Individual CPM counts and IFN-γ concentrations were obtained in the MLR, and dose titrations were performed on the preceding candidates.

Table 8: IC ₅₀ values for CPM and IFN-γ in MLR VSTB112 ( S2 ) VSTB116 ( S5 ) VSTB95 ( S16 ) VSTB50 ( S41 ) VSTB53 ( S43 ) VSTB60 ( S47 ) CPM -0.67 -0.78 -0.54 -0.12 -0.33 0.02 γ -0.42 -0.16 0.22 0.06 0.27 0.4

** Values are log ₁₀ of antibody concentration.

Conclusion: Initial screening indicated 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 dose titration experiments. In the dose titration experiments, VSTB60 induced a weaker response compared to the other five antibodies.

Staphylococcal enterotoxin B (SEB) activation assay: SEB is a bacterial superantigen that induces activation of specific Vβ+ T cells. Peripheral blood mononuclear cells are isolated and incubated with SEB antigen in culture to induce stable cytokine production. Perform this analysis on the top five candidates.

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.

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. Seed the reaction population at 2.0 × 10 ⁵ cells per well of a 96-well U-bottom plate. All conditions must be performed in triplicate. Centrifuge cells as previously described and resuspend in 10% AB serum medium at a concentration of 4 × 10 ⁶ cells/ml and add 50 μl to the appropriate wells. Add 50 μl of 10% AB serum medium containing SEB antigen at a concentration of 40 ng/ml. In the described experiment, SEB was obtained from Sigma Aldrich (Catalog No. S0812). The final concentration in the wells was 10 ng/ml. Incubate cells at 37°C and 5% CO ₂ for 3 days. On the third day, remove 30 μl of supernatant to analyze IFN-γ production. Add 25 μl of 1 mCi/ml tritiated thymidine solution to each well and incubate for 8 hours at 37°C and 5% _CO2 . Transfer cells to 96-well microscintillation plates according to the manufacturer's instructions. Count using a microscintillation counter according to the manufacturer's instructions. Determine IFN-γ concentration by ELISA (eBioscience catalog number 88-7316-88) using the manufacturer's protocol.

Project: Data analysis. Average counts per minute (CPM) or IFN-γ concentration was calculated for each antibody at all concentrations. Acceptance criteria were performed as previously described. _IC50 value determination was performed as described. Individual CPM counts and IFN-γ concentrations were obtained in the SEB assay, and dose titrations were performed on the preceding candidates.

Table 9: _IC50 values for CPM and IFN-γ in SEB. VSTB112 (S2) VSTB116 (S5) VSTB95 (S16) VSTB50 (S41) VSTB53 (S43) VSTB60 (S47) CPM -1.16 -1.44 -1.12 -0.74 -1.06 Not in progress γ -1.24 -0.35 0.05 1.69 -1.05 Not in progress

**Values are log ₁₀ of antibody concentration.

Conclusions: VISTA-specific antibodies increased cytokine production and proliferation in a dose-dependent manner in SEB assays. _IC50 values from SEB studies are generally similar to results from MLR dilution studies.

Example 10: Epitope binning analysis

Methods: Epitope grouping was performed using the ProteOn XPR36 system (BioRad). ProteOn GLC wafers (BioRad, catalog number 176-5011) were coated with two sets of six monoclonal antibodies (mAbs) using the manufacturer's instructions for amine coupling chemistry (BioRad, catalog number 176-2410).

Excess competitive mAb (250 nM final concentration) was preincubated with human VISTA (25 nM final concentration) for 4 hours at room temperature and run 6 at a time on wafers coated with the coated mAb set, where the association time for 4 minutes, followed by 5 minutes of dissociation. After each operation, the wafer was regenerated with 100 mM phosphoric acid.

Data analysis involves grouping all sensor maps by ligand and applying alignment guides, which automatically perform X- and Y-axis alignment and artifact removal. Interspot correction was then applied to the data.

Non-competitive mAbs were defined as those with binding signals equal to or greater than A1 signal (binding only to human VISTA).

Competitive mAbs were defined as those with a binding signal << A1 signal (i.e., binding only to human VISTA).

Results: In the exemplary sensor profile shown in Figure 23, the VSTB85 antibody was coated on a Proteon SPR chip and VISTA protein pre-incubated with a designated competitor was passed over the chip. VSTB50 is an example of a non-competitive antibody because a positive reaction was seen when the VISTA/VSTB50 complex was run. GG8, VSTB49, and VSTB51 complexed with VISTA did not bind to VSTB85 coated on the chip and were therefore classified as competing with VSTB85 for the same binding site on VISTA.

Table 10: Sample set #1 : coupled to sensor Sample set #2 : coupled to sensor L1 L2 L3 L4 L5 L6 L1 L2 L3 L4 L5 L6 Sample Group GG8 B85 B95 B104 B112 B113 B50 B53 B66 B67 IE8 B116 GG8 1 Y Y Y Y Y Y N Y N Y Y Y VSTB100.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB101.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB102.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB103.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB104.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB105.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB106.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB107.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB108.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB109.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB110.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB111.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB112.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB113.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB114.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB115.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB116.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB49.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB51.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB53.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB59.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB65.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB67.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB70.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB81.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB92.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB95.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB97.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB98.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB99.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB50.001 2 N N N N N N Y N Y N N N VSTB54.001 2 N N N N N N Y N Y N N N VSTB56.001 2 N N N N N N Y N Y N N N VSTB60.001 2 N N N N N N Y N Y N N N VSTB63.001 2 N N N N N N Y N Y N N N VSTB66.001 2 N N N N N N Y N Y N N N VSTB73.001 2 N N N N N N Y N Y N N N VSTB76.001 2 N N N N N N Y N Y N N N VSTB78.001 2 N N N N N N Y N Y N N N VSTB84.001 2 N N N N N N Y N Y N N N VSTB85.001 3 Y Y Y Y Y Y N Y N Y I Y VSTB74.001 4 N N N N N N N N N N N N IE8 5 Y I Y Y Y Y N Y N Y Y Y mAb immobilized on the sensor Y = Competition (Signal << A1-only human VISTA) N = No competition (signal > A1-human VISTA only) I = Uncertain (signal similar to A1 - human VISTA only)

Example 11: PROTEON affinity determination

Antibodies were captured on ProteOn chips using an anti-IgG Fc coated surface. Antibodies were tested for binding to human and macaque (VISTA) extracellular domains (ECD) at concentrations ranging from 0.39 nM to 100 nM of VISTA protein. Antigens were allowed to bind/associate to the antibody coated chips for 4 minutes followed by monitoring dissociation for 30 minutes. The chips were regenerated twice by treatment with 100 mM phosphoric acid for 18 seconds. All experiments were run at 25°C and data were fit to a 1:1 Langmuir binding model.

Example 12: Effect of anti-VISTA treatment in MB49 mouse bladder tumor model

method:

C57BL/6 mice were injected with MB49 tumor cells. After tumor formation, anti-VISTA treatment was initiated. Tumor growth was subsequently monitored 3 times/week. In accordance with IACUC regulations, mice were sacrificed after tumors reached 15 mm in any size.

For each experiment, frozen vials of MB49 cells were thawed and grown in RPMI 1640 (+L-Glut) with 10% serum and penicillin/streptomycin antibiotics. After three days of culture, cells were collected using StemPro Accutase and resuspended in RPMI at a concentration of 5×10 ⁶ cells/ml, and 50 μl was injected per mouse.

Female C57BL/6 mice aged 6-8 weeks were purchased from the National Cancer Institute. After arriving, let it get used to it for a day, then shave the right side of its belly and tattoo its tail with a mark. They are then injected three to five days later.

Tumor injection (intradermal): 50 μl MB49 cell suspension (approximately 250,000 cells) was injected intradermally (i.d.) into the shaved flank of mice.

Monitor tumor growth: Use an electronic caliper to measure tumor growth first across the widest dimension (L) and second at the 90° angle of the first measurement (W). Tumor volume was obtained as follows:

Volume = (L ² _* W ² )/2 .

Tumors were considered established when they reached approximately 5 mm in diameter (approximately 60 mm ³ in volume). After establishment, treatment was initiated. During treatment, tumor growth was measured three times per week and until the end of the experiment.

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

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

Data management was performed using Excel and graphs were drawn using GraphPad Prism to analyze mouse tumor volume. Statistical analysis was performed using macros of R statistical computing software.

The experimental design is shown in Figure 24.

result:

Ch13F3-mIgG2a treatment of female mice resulted in complete tumor suppression (CR) in 70% of animals and partial response (PR) in 30% of animals (n=7) (Table 11 and Figure 25B). In contrast, all control mIgG2a treated mice exhibited progressive tumor growth (6/6) (Figure 25A). These data demonstrate that anti-VISTA treatment can have a dramatic effect on tumor growth.

Table 11: Complete response (CR) vs. partial response (PR) Female 13F3 IgG2a (n=7) CR 5 PR Until day 32, 2

The human VISTA sequence is shown in Figures 26 and 27, modified from Wang et al., 2011, supra, the entire contents of which are incorporated herein.

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

To identify the epitopes on human VISTA for VSTB50, 60, 95, and 112, solution hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed using the corresponding Fabs. For H/D exchange, the procedure used to analyze Fab perturbations was similar to that described previously (Hamuro et al., J. Biomol. Techniques 14:171-182, 2003; Horn et al., Biochemistry 45:8488-8498, 2006), but with some modifications. Fabs were prepared from IgG by papain digestion and captured with Protein A using the Pierce Fab Preparation Kit (Thermo Scientific, Catalog No. 44985). The human VISTA protein sequence contains six N-linked glycosylation sites. To improve sequence coverage, the protein was deglycosylated using PNGaseF. Deglycosylated VISTA protein was incubated in a deuterated aqueous solution for a predetermined time to allow deuterium to be incorporated at exchangeable hydrogen atoms. Deuterated VISTA protein was complexed with any Fab of VSTB50, VSTB60, VSTB95 or VSTB112 in 46 μL of deuterium oxide (D ₂ O) for 30 seconds, 2 minutes, 10 minutes and 60 minutes at 4°C. The exchange reaction was quenched by lowering the pH 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, VISTA protein was treated similarly, except that it was not complexed with Fab molecules. The regions bound to Fab are inferred to be relatively difficult to exchange and therefore contain a higher fraction of deuterium than those sites of the reference VISTA protein. About 94% of the protein can be located at a specific peptide.

Solution HDX-MS perturbation localization of VISTA using VSTB50/VSTB60 and VSTB95/VSTB112 is shown in the upper and lower images of Figure 28, respectively. Identify two epitopes. Anti-VISTA VSTB50 and VSTB60 recognize the same epitope; VSTB95 binds to another epitope region on VISTA that VSTB112 binds to. Anti-VISTA VSTB50 and 60 share the same epitope, which includes the segments ₁₀₃ NLTLLDSGL111 (SEQ ID NO:62) and ₁₃₆ VQTGKDAPSNC146 (SEQ ID NO:63) (Figure 28, upper panel). Anti-VISTA VSTB95 and 112 appear to target similar epitopes, including the segments ₂₇ PVDKGHDVTF36 (SEQ ID NO:75) and ₅₄ RRPIRNLTFQDL65 (SEQ ID NO:65) (Figure 28, bottom panel). There are two other segments showing weak perturbation of VSTB95 and 112, including residues 39-52 and 118-134. However, the reduction is not as strong as in the previously mentioned areas (27-36 and 54-65) in different positionings. Although one peptide showing strong perturbation by VSTB95 and 112, 100TMR102, was located on another surface of the VISTA surface, it was far away from epitope regions 27-36 and 54-65. This perturbation may be attributed to aliasing effects. These HDX-MS results provide peptide-level epitopes of anti-VISTA antibodies. There are no overlapping epitope regions for these two epitopes. These results are consistent with the competitive grouping data described above in that they do not compete with each other.

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

In an effort to determine the structure of VISTA and delineate the epitopes and complements 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 1.85Å resolution. The structure of the ECD in human VISTA in complex with the Fab fragment of the antibody VSTB112 was determined to determine the structure of the VISTA ECD itself and define the epitopes/complements for this interaction. The structure reveals that VISTA adopts an IgV fold with a chain topology similar to the TCR Vα chain. In addition to the canonical disulfide bonds bridging the back and front B and F strands of the β-sheet, the structure reveals that the ECD has two additional disulfide bonds, one tethering the CC' loop to the front sheet and the second between the A' and G' strands. Although there are crystal contacts between VISTA molecules, they are few and there is no evidence of dimers of the VISTA ECD based on this structure. The VSTB112 epitope was shown to include portions of the VISTA BC, CC' and FG loops and the residues of the front β-sheet closest to those loops (C'CFG). The complementary positions are primarily biased towards heavy chain interactions, with CDR L3 making the fewest contacts.

Defining the epitope/complementary site of the VISTA:VSTB112 interaction

VSTB112 Fab buries 1024.3 Å2 surface area after binding to VISTA ECD, of which the buried heavy chain surface accounts for 715.3 Å2 of the total. Seven hydrogen bonds and 4 salt bridge interactions are formed between VISTA and the VSTB112 light chain, and 10 hydrogen bonds and 2 salt bridge interactions are formed between VISTA and the VSTB112 heavy chain. VSTB112 recognizes residues proximal to the FG loop in prolamellar strands C', C, F and G and residues in BC, FG and CC' loops (Figures 29 and 30). Interactions with the CC' loop accounted for the majority of contacts with the Fab light chain having residues E125 and R127 only in the FG loop, creating additional light chain interactions. Residues 119 to 127 corresponding to the VISTA FG loop account for 38% of the total 1034.8 Å2 buried surface area upon binding to VSTB112. Notably, this loop is highly polar and contains the following sequence -IRHHSEHR- (SEQ ID NO:76). In addition, W103 in VSTB112 CDR H3 is well packaged for the main chain of VISTA residues H122 and H123, and VISTA H121 is the edge of interaction with the aromatic ring of F55 in CDR H2.

A comparison of the epitope regions identified by crystallography and HDX is shown in FIG31 .

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

The functional role of anti-VISTA antibodies was 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 (PBMC) from two different human donors are cultured together, and the major histocompatibility complex (major histocompatibility complex) between T cells from one donor and dendritic cells from the other donor MHC) mismatch causes T cell proliferation and interferon (IFNγ) production. In the SEB assay, PBMC from a single donor are incubated with bacterial superantigens that directly link 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 analyses, VSTB112, the parent molecule of VSTB174, exhibited dose-dependent induction of T cell proliferation and cytokine production and was the most potent among the candidates (Figures 21A-21D, Table 12).

Table 12. EC50 values of the MLR assay readouts. VSTB112 (the parent molecule of VSTB174) was the most potent molecule. Candidates EC ₅₀ proliferation (μg/ml) EC ₅₀ IFNγ production (μg/ml) VSTB112 0.21 0.38 VSTB116 0.17 0.69 VSTB95 0.29 1.67 VSTB50 0.77 1.14 VSTB53 0.47 1.88 VSTB60 1.04 2.48

Monocyte activation assay

The analytical data shown in Table 12 were generated using VSTB112, the parent molecule of VSTB174. To fully understand the activity of VSTB174, a single-core sphere activation assay was performed. Results demonstrate that incubation of VSTB174 with whole PBMC induces upregulation of activation markers (CD80 and HLA-DR) on CD14+ monocytes, demonstrating the role of antibodies that bind to a subset of immune cells known to exhibit high levels of VISTA (Figure 32) . Another question is whether the effect on monocyte activation in whole PBMCs can be promoted by any antibody that binds VISTA and has an IgGl Fc. Antibodies VSTB103 and VSTB63 bind to VISTA with high affinity (KD 6.36E-10 and 8.30E-10, respectively) and similarly to VSTB112 and VSTB111 bind to cells expressing VISTA protein. VSTB103 and VSTB112 are in the same epitope group, while VSTB63 is in a different epitope group; neither antibody promotes monocyte activation. Taken together, these results demonstrate that one mechanism by which VSTB174 may exert its effect on T cell activation/proliferation is through monocyte activation promoted by NK cells.

Prepare medium

Mix 500 ml RPMI 1640 (Corning, 10-040-CV) with 50 ml human AB serum (Valley Biomedical, lot number 3C0405), 5 ml penicillin/streptomycin (Lonza, 17-602E) (10,000 U/ml), Combination of 5 ml L-glutamic acid (100×) (Gibco, 25030-081) and 10 ml HEPES (1 M) (Fisher BP299-100, lot number-1). Store the culture medium at 4°C for no more than 14 days.

Preparation of soluble VISTA and control antibodies

Dilute the antibody to 2× the desired concentration in 10% AB serum medium, VSTB174: lot number VSTB174.003.

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

Isolate PBMC.

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

Transfer the donor blood from the separation tube to a 50 ml Erlenmeyer flask.

Place 15 ml of Ficoll 1077 (SIGMA, 10771) underneath, taking care not to mix with the blood. Do this for every 25 ml of blood.

Centrifuge cells at 1250 g for 25 min 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).

Centrifuge the cells at 453 g (1500 rpm) for 10 min at 4 °C.

Cells were resuspended in 50 ml HBSS and counted by transferring 500 μl into individual eppendorf tubes.

Additionally, CD14+ cells were isolated by negative selection in several treatment groups using a pan-monocyte isolation kit from Miltenyi according to the manufacturer's instructions (catalog number 130-096-537).

In vitro culture settings

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

Cells were centrifuged as above, resuspended in 10% AB serum medium at a concentration of 2 × ¹⁰ /ml for the whole PBMC population and 0.5 × ¹⁰ /ml for the CD14 negative selection population and added to the appropriate wells. Add 100 μl to bring the total volume in each well to 200 l.

_Incubate cells at 37°C and 5% CO for 1, 2, or 3 days.

Antibody staining and flow cytometry

Centrifuge the 96-well U-plate at 453 g for 5 minutes and remove the supernatant.

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

Discard the supernatant and resuspend in 50 μl PBS containing the following antibodies: ․ CD14-APC (pure line HCD14) 1:250 (Biolegend catalog number 325608) ․ HLA-DR-PE Cy7 (pure line L243) 1:250 (Biolegend catalog number 307616) ․ CD80-PE (pure line 2D10) 1:250 (Biolegend catalog number 305208) ․ Hu FcR binding inhibitor (eBioscience catalog number 14-9161-73)

Incubate on wet ice for 20 minutes in the dark.

Add 150 μl PBS and centrifuge as in step 5.5.1.

150 1 PBS buffer was added and analyzed via FACS.

Samples were run on a Miltenyi MACSQuant 10 parametric flow cytometer and the expression of HLA-DR and CD80 on the CD14+ population was analyzed using FlowJo 9.7.5. The mean geometric fluorescence intensity (MFI), a statistical parameter that defines the central trend of a set of numbers, was used as the specified statistical parameter for process comparisons.

Statistical analysis

All statistics were performed in Prism GraphPad Model 6. Pairwise comparisons were performed between groups at each time point using one-way ANOVA with Tukey correction for diversity. The p-value for all tests was below 0.05 and the contrast was considered significant. For all curves and tables, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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

VSTB174 has an IgG1 Fc that confers antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) activities. Two types of analyzes were performed and demonstrated that VSTB174 may lyse or phagocytose K562-VISTA cells (Figures 33-34), but not K562 myeloma cell line blasts (data not shown). Another mechanism by which VSTB174 mediates the inhibitory effects of VISTA could be lysis or phagocytosis of cells expressing high levels of VISTA, thereby removing them from the local microenvironment.

Example 17: ADCP activity of anti-VISTA antibodies

An in vitro phagocytosis assay was used to study the enhancement of macrophage-mediated phagocytosis of cells expressing VISTA ectopically by anti-human VISTA mAbs (VSTB173 and VSTB174). These mAbs were selected in different Fc backbones (IgG1 WT (wild type), IgG1 PR (protease resistant) and IgG2σ) and were hypothesized to have different activities in increasing phagocytosis. The IgG1 and IgG1 PR backbones can bind to Fc receptors and have the potential to cause ADCP, while IgG2σ does not bind to Fc receptors and cannot regulate ADCP.

Anti-VISTA antibodies were tested in ADCP assays using K562 blast cells and K562-VISTA target cells. As shown in Figures 35-36, VSTB174, VSTB149, VSTB173 and VSTB145 enhance hMac phagocytosis of K562-VISTA cells. VISTA antibodies VSTB140 or VSTB132 with an IgG2σFc that does not bind Fc receptors did not enhance phagocytosis as expected. VISTA mAbs VSTB174 and VSTB173 with IgG1 Fc demonstrated more robust phagocytosis compared to VSTB149 and VSTB145 with IgG1 PR Fc (for _EC50 values, see Tables 13 and 14).

Table 13. Anti-human VISTA mAb _EC50 values. handle VSTB174 VSTB149 VSTB140 EC50 0.0782 0.1142 NA

Table 14. Anti-human mAb _EC50 values. handle VSTB173 VSTB145 VSTB132 EC50 0.0146 0.1075 NA

VSTB174 and VSTB173 were shown to weakly enhance phagocytosis of K562 mother cells at the highest concentration (Figures 35-36), which could be attributed to the low VISTA performance of K562 cells. Other anti-VISTA antibodies were unable to enhance phagocytosis of K562 cells.

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 VISTA antigen expression by K562-VISTA cells.

Example 18: ADCC activity of other anti-VISTA antibodies

To test their ability to induce ADCC, the following three human anti-VISTA antibodies were tested: VSTB174 (IgG1) VSTB149 (IgG1 PR) VSTB174.LF (IgG1 LF (low trehalose)).

In two separate experiments, each antibody was tested in the same plate at six different concentrations, repeated three times, to obtain a total of six data points.

VSTB174, VSTB149, and VSTB174.LF each exhibited measurable ADCC activity at 10, 1, 0.1, and 0.01 μg/mL, 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 as evidenced by a smaller EC ₅₀ value for the LF antibody curve (0.002293 μg/mL) compared to the conventional IgG1 antibody curve (0.02381 μg/mL). The EC ₅₀ value for the IgG1 PR antibody curve was similar to the conventional IgG1 curve (0.01846 μg/mL).

Table 15. _EC50 values (μg/mL) of three tested anti-VISTA antibodies as determined by ADCC analysis. Anti-VISTA Antibodies _EC50 (μg/mL) VSTB174 (IgG1) 0.02381 VSTB149 (IgG1 PR) 0.01846 VSTB174.LF (IgG1 LF) 0.002293

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 the LF antibody showed killing at 0.001 μg/mL antibody concentration. Anti-VISTA antibodies showed no killing at 0.0001 μg/mL antibody concentration.

As can be seen in the _EC50 values, the LF antibody exhibited approximately 10-fold more potent ADCC killing compared to conventional IgG1 antibody or IgG1 PR antibody.

Example 19: Affinity of VSTB174 for human and macaque VISTA

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

Example 20: VISTA antibody demonstrated efficacy in a mouse tumor model

Mouse strains, reagents, and tumor models

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

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

Assessment of MB49 bladder cancer in VISTA KI mice.

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

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

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, dosing was initiated as shown in FIG37 (10 mg/kg isotype control mIgG2a or specified doses of VSTB123; 10 mice/group).

As shown in Figure 37, VSTB123 was more effective at higher versus lower doses. Doses of 10 mg/kg and 7.5 mg/kg were equivalent, but tumors grew more rapidly in mice given 5 or 1 mg/kg.

Example 21: Detection of VISTA expression in human tumors using anti-VISTA antibodies Figure 1 shows VISTA expression in AML tumor cells - this and the RNA-seq expression data in Figure 17 support the idea that VISTA is expressed by AML cells and that anti-VISTA drugs are used for immunomodulation or antibody-mediated killing through these cells. Cells are targeted and effective.

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

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

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

FFPE tumor sections were processed using standard antigen retrieval methods before staining. GG8 mouse anti-human VISTA antibody was used at a 1:500 dilution. GG8 binding was detected using rabbit anti-mouse polyclonal antibody and anti-rabbit polymer HRP in sequence. This was followed by contrast staining with hematoxylin, and tumor sections were subsequently scored.

VISTA expression in lung cancer is primarily limited by immune infiltration (example shown in Figure 39) and high levels of VISTA-positive cells are present in multiple lung cancer samples.

Example 23: Structure of the extracellular domain (ECD) of human VISTA complexed with the FAB fragment of VSTB174

VISTA antigen variants were generated and purified for crystallography. His-tagged recombinant VSTB174 Fab 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 (Figure 29 (top)).

The structure of the VISTA ECD:VSTB174 Fab complex was determined by X-ray crystallography with a resolution of 1.85Å. The first structure of VISTA ECD was obtained and the VSTB174 epitope and paratope were delineated. VISTA ECD adopts an IgV fold that is topologically similar to CTLA-4 ECD but has a unique G' strand that extends in front of the beta sandwich. A' and G' are additionally chemically tethered via a disulfide bridge formed between residues 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 was no evidence of dimeric VISTA.

VSTB174 recognizes residues in the prolamellar strands C', C, F and G proximal to the FG loop and residues in the BC, FG and CC' loops.

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

While this invention has been specifically shown and described with reference to illustrative embodiments thereof, those skilled in the art will understand that various modifications may be made therein without departing from the scope of the invention as encompassed by the appended claims. Various changes in details.

without

The patent or application file contains at least one drawing in color. Upon application and payment of the necessary fee, the Patent Office will provide a copy of this patent or patent application publication with color drawings.

[Figure 1A-1C]: Curve showing the performance of VISTA on TF1 AML cells. Demonstrating the expression of VISTA protein by flow cytometry in the TF-1 AML cell line.

[Figure 2A-2E]: Curves of staining and gating strategies for identifying human bone marrow and lymphoid subpopulations.

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

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

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

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

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

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

[Figure 9]: Curve showing the performance of VISTA on human T and NK cell subsets from multiple healthy normal donors.

[Figure 10A-10D]: Curves showing staining and gating strategies for identifying the expression of VISTA on human dendritic cell subpopulations.

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

[Figure 12]: Curve showing the performance of VISTA on human dendritic cell subsets and basophilic spheres from multiple healthy normal donors.

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

[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 was obtained using multicolor flow cytometry analysis: representative FACS plots from one individual are shown (Figure 14A). Peripheral blood mononuclear cells were separated by Ficoll and analyzed for VISTA expression on mononuclear cells (CD14+ CD11b+ CD33+ HLADR+ CD15-) (Figure 14B) and bone marrow-derived suppressor cells (CD14-CD11b+ CD33-HLADR-CD15+ CD16+) (Figure 14C).

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

[Figure 16A-16D]: Characteristics of VISTA expression on peripheral blood cells of cynomolgus macaques obtained using multicolor flow cytometry analysis: analysis of mononuclear spheres (SSC ^lo CD11b ^hi CD14 ^hi) from whole blood of 4 different monkeys VISTA performance on HLA-DR ^hi CD16 ^-ve CD19 ^-ve ) (Figure 16A) and neutrophil CD11b ^hi CD14 ^lo HLA-DR ^-ve CD16 ^-ve CD19 ^-ve (Figure 16B). Peripheral blood mononuclear cells from three monkeys were separated using Ficoll gradients to analyze CD4+ T cells (TCRα/β ^+ve CD4 ^+ve ) (Figure 16C) and CD8+ T cells (TCRα/β ^+ve CD8 ^+ve ) (Figure 16D ).

[Figure 17]: Shows the absolute performance value of VISTA RNA in heme cell lines.

[Figure 18]: Mouse A20 cells were stably transfected with GFP or human VISTA. This was incubated with ova peptide and DO11.10 T cells. The 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.

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

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

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

[Figure 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).

[Figure 23]: Sensorgram obtained using anti-VISTA antibody VSTB85 and VISTA protein coated on a Proteon SPR wafer with designated competitors (competitors listed in Table 16) passing over the wafer.

[Figure 24]: Experimental design of MB49 mouse bladder tumor model.

[Figures 25A-25B]: MB49 tumor growth in female C57BL/6 mice. Curves illustrate tumor growth in individual mice treated with anti-mouse VISTA antibody (Figure 25B) or control IgG (Figure 25A).

[Figure 26]: Amino acid sequence of human VISTA (SEQ ID NO:46).

[Figure 27]: Multiple sequence alignment of VISTA orthologs.

[Figure 28]: Human VISTA region bound by VSTB50 and VSTB60 antibodies (top) or VSTB95 and VSTB112 antibodies (bottom) as determined by HDX.

[Figure 29]: VISTA epitope binding to VSTB112. (Top) VISTA shown in sketch, with strands labeled. Residues with at least one atom within 5 Å of VSTB112 in the complex are colored blue. Blue and orange balls highlight chain breaks, and cyan and green balls mark the N- and C-termini of the VISTA structure, respectively. (Bottom) Sequence of the VISTA construct used in structure determination. Circles under the sequence are used to indicate residues that make only main chain contacts with VSTB112, triangles indicate side chain contacts, and squares indicate side chain contacts that create hydrogen bonding or salt bridge interactions, as calculated by PISA. Shapes are colored to indicate the CDR with the greatest number of atoms in contact with a given residue, where the CDR color is defined in Figure 59. Secondary structural elements are defined by the formula MOE, where yellow arrows represent beta strands and red rectangles indicate alpha helices.

[Figure 30]: VSTB112 complementation. (Top) VISTA antigen is displayed in an illustrative manner, and VSTB112 within 5 angstroms (Å) of VISTA is displayed in the surface with colors used to indicate CDR identities specified in the sequence below. Contact framework residues adjacent to the CDRs are similar to the corresponding CDRs of the VSTB112 Fv region (Bottom) Sequence coloring. The colored background specifies the CDRs according to the Kabat definition. As calculated by PISA, circles under the sequence are used to indicate residues that make only main chain contacts with VISTA, triangles indicate side chain contacts, and squares indicate side chain contacts that produce hydrogen bond or salt bridge interactions.

[Figure 31]: Comparison of epitope regions identified by crystallography and hydrogen-deuterium exchange (HDX). Sequence of the VISTA construct used in structure determination. Circles under the sequence are used to indicate residues that make only backbone contacts with VSTB112, triangles indicate side chain contacts, and squares indicate side chain contacts that create hydrogen bonding or salt bridge interactions, as calculated by PISA.

[Figure 32]: Activation of CD14+ mononuclear spheres in whole PBMCs by VSTB174 (derived from VSTB112). In each part of the 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 showing representative results).

[Figure 33]: A curve showing the ADCC activity of VSTB174 against K562-VISTA cells.

[Figure 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.

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

[Figure 36]: Curves showing phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAbs against 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.

[Figure 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 ³ when dosing started on day 6 post-implantation. VSTB123 is VSTB112 Fab grafted onto a mouse Fc backbone and binds to human VISTA in VISTA-KI mice.

[Figure 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 the distal lung tissue and peripheral blood of the patients.

[Figure 39]: IHC staining of lung cancer VISTA using GG8.

         
 <![CDATA[ <110> Janssen Pharmaceutica NV]]>
 <![CDATA[ <120> Anti-VISTA antibodies and fragments]]>
 <![CDATA[ <150> US 62/085,086 ]]>
 <![CDATA[ <151> 2014-11-26 ]]>
           <![CDATA[ <150> PCT/IB2014/002868 ]]>
           <![CDATA[ <151> 2014-12-22 ]]>
 <![CDATA[ <150> US 62/101,352 ]]>
 <![CDATA[ <151> 2015-01-08 ]]>
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          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
           <![CDATA[ <210> 41]]>
 <![CDATA[ <211> 111]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
           <![CDATA[ <210> 42]]>
 <![CDATA[ <211> 112]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 44]]>
 <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 107]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 295]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 47]]>
 <![CDATA[ <211> 449]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
 <![CDATA[ <210> 48]]>
 <![CDATA[ <211> 218]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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 
 <![CDATA[ <210> 49]]>
 <![CDATA[ <211> 453]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
           <![CDATA[ <210> 50]]>
 <![CDATA[ <211> 219]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 449]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
 <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 218]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
           <![CDATA[ <210> 53]]>
 <![CDATA[ <211> 452]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 
Ser Pro Gly Lys
450 
 <![CDATA[ <210> 54]]>
 <![CDATA[ <211> 219]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 54]]>
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 
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 
 <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 450]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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 
 <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 214]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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 
 <![CDATA[ <210> 57]]>
 <![CDATA[ <211> 450]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 214]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
           <![CDATA[ <210> 59]]>
 <![CDATA[ <211> 446]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 449]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
 <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 450]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 9]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 62]]>
          Asn Leu Thr Leu Leu Asp Ser Gly Leu
           1 5
           <![CDATA[ <210> 63]]>
 <![CDATA[ <211> 11]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <400> 63]]>
Val Gln Thr Gly Lys Asp Ala Pro Ser Asn Cys
1 5 10 
 <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 64]]>
          Leu Leu Gly Pro Val Asp Lys Gly His Asp Val Thr Phe
           1 5 10
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <400> 65]]>
          Arg Arg Pro Ile Arg Asp Leu Thr Phe Gln Asp Leu
           1 5 10
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 309]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Mus musculus]]>
           <![CDATA[ <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
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 311]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 312]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Red Kangaroo]]>
 <![CDATA[ <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
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 310]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Real Dolphin]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 288]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Jungle fowl]]>
           <![CDATA[ <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 
 <![CDATA[ <210> 71]]>
 <![CDATA[ <211> 261]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> African Xenopus]]>
           <![CDATA[ <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
           <![CDATA[ <210> 72]]>
 <![CDATA[ <211> 315]]>
 <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pearl Bird]]>
 <![CDATA[ <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
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 275]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Zebrafish]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 293]]>
           <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Red-finned dolphin]]>
 <![CDATA[ <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 
 <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 168]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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
                          165
           <![CDATA[ <210> 76]]>
 <![CDATA[ <211> 9]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 76]]>
Ile Arg His His His Ser Glu His Arg
1 5 
 <![CDATA[ <210> 77]]>
 <![CDATA[ <211> 169]]>
 <![CDATA[ <212> PRT]]>
 <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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 
 <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 241]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <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
 <![CDATA[ <210> 79]]>
 <![CDATA[ <211> 169]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
 <![CDATA[ <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 (66)

An isolated antibody or antibody fragment thereof comprising an antigen-binding region that binds to the V domain Ig inhibitor of human T cell activation (VISTA), wherein the antibody or antibody fragment comprises: a heavy chain variable (VH) domain, It includes VH CDR1 having the amino acid sequence of SEQ ID NO: 7; VH CDR2 having the amino acid sequence of SEQ ID NO: 8 and VH CDR3 having the amino acid sequence of SEQ ID NO: 9; and further comprising A light chain variable (VL) domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 10; a VL CDR2 having the amino acid sequence of SEQ ID NO: 11; and an amine having the amino acid sequence of SEQ ID NO: 12 The amino acid sequence of VL CDR3. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 39; and/or (ii) a VL domain comprising SEQ ID NO: 39; Amino acid sequence of ID NO: 42. The antibody or antibody fragment of claim 1, wherein the antibody fragment is a Fab, F(ab') ₂ or scFv antibody fragment. The antibody or antibody fragment of claim 2, wherein the antibody fragment is a Fab, F(ab') ₂ or scFv antibody fragment. The antibody or antibody fragment of claim 1 comprises a human or non-human antibody constant region. The antibody or antibody fragment of claim 2 comprises a human or non-human antibody constant region. The antibody or antibody fragment of claim 5 comprises a human IgG1 antibody constant region. The antibody or antibody fragment of claim 6, which comprises a human IgG1 antibody constant region. The antibody or antibody fragment of claim 1, wherein the VISTA bound by the isolated antibody or antibody fragment is human VISTA or cyno VISTA. As claimed in claim 2, the antibody or antibody fragment, wherein the VISTA bound by the separated antibody or antibody fragment is human VISTA or cynomolgus macaque VISTA. The antibody or antibody fragment of claim 9, wherein the human VISTA or cynomolgus VISTA is expressed on hematopoietic cells, on tumor cells or in the tumor microenvironment (TME). An antibody or antibody fragment as claimed in claim 10, wherein the human VISTA or cynomolgus macaque VISTA is expressed on hematopoietic cells, on tumor cells or in a tumor microenvironment (TME). The antibody or antibody fragment of claim 11, wherein the hematopoietic cells are myeloid cells and/or lymphocytes, monocytes, neutrophils, T cells, natural killer (NK) cells or natural killer T (NKT) cells. The antibody or antibody fragment of claim 12, wherein the hematopoietic cells are myeloid cells and/or lymphocytes, monocytes, neutrophils, T cells, natural killer (NK) cells or natural killer T (NKT) cells. The antibody or antibody fragment of claim 1, further comprising at least one heavy chain and at least one light chain. The antibody or antibody fragment of claim 2 further comprises at least one heavy chain and at least one light chain. The antibody or antibody fragment of claim 2, wherein the antibody comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and/or (ii) a light chain comprising SEQ ID NO: 50 amino acid sequences. The antibody or antibody fragment of claim 1, wherein the antibody is a monoclonal antibody; is a humanized antibody; contains one or more human or humanized framework regions; or contains a human constant region. The antibody or antibody fragment of claim 2, wherein the antibody is a monoclonal antibody; is a humanized antibody; contains one or more human or humanized framework regions; or contains a human constant region. The antibody or antibody fragment of claim 1, wherein the antibody binds to VISTA with an affinity of at least 1×10 ^-9 M, at least 1×10 ^-8 M, or at least 1×10 ^-7 M. For example, the antibody or antibody fragment of claim 1 inhibits or blocks the immunosuppressive response caused by VISTA and/or triggers an anti-tumor response. For example, the antibody or antibody fragment of claim 2 inhibits or blocks the immunosuppressive response caused by VISTA and/or triggers an anti-tumor response. The antibody or antibody fragment of claim 21, wherein: the suppression or blocking of the immunosuppressive response caused by VISTA results in one or more of the following: an increase in CD45 ⁺ leukocytes, an increase in CD4 ⁺ T cells, and an increase in CD8 ⁺ T cells An increase in cells or a decrease in immune cells expressing VISTA; or the suppression or blockage of the immunosuppressive response caused by VISTA results in enhanced cytokine production, enhanced T-cell responses and/or modulation of Foxp3 expression. The antibody or antibody fragment of claim 22, wherein: the inhibition or blocking of the immunosuppressive response caused by VISTA results in one or more of the following: an increase in CD45 ⁺ leukocytes, an increase in CD4 ⁺ T cells, an increase in CD8 ⁺ T cells, or a decrease in immune cells expressing VISTA; or the inhibition or blocking of the immunosuppressive response caused by VISTA results in enhanced cytokine production, enhanced T-cell response and/or regulation of Foxp3 expression. The antibody or antibody fragment as claimed in claim 1 comprises the whole antibody. Such as the antibody or antibody fragment of claim 2, which includes a whole antibody. An antibody or antibody fragment as claimed in claim 1, wherein the antibody or antibody fragment is expressed in cells lacking a fucosylation enzyme. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is expressed in CHO cells. The antibody or antibody fragment of claim 2, wherein the antibody or antibody fragment is expressed in CHO cells. A composition comprising the antibody or antibody fragment of any one of claims 1-29 and a pharmaceutically acceptable carrier, diluent or excipient. A composition comprising: (a) a VISTA antagonist comprising an antibody or antibody fragment thereof according to any one of claims 1-29; and (b) a vaccine. A pharmaceutical composition comprising: (a) a VISTA antagonist, which includes an antibody or an antibody fragment thereof according to any one of claims 1-29; and (b) a vaccine; wherein said antibody or antibody fragment is identical to VISTA Binding modulates or enhances immune responses suppressed by VISTA. A composition as claimed in claim 31, wherein the vaccine is a viral vector vaccine, a bacterial vaccine, a DNA vaccine, an RNA vaccine, a peptide vaccine or a protein vaccine. The pharmaceutical composition of claim 32, wherein the vaccine is a viral vector vaccine, bacterial vaccine, DNA vaccine, RNA vaccine, peptide vaccine or protein vaccine. A use of an antibody or antibody fragment as claimed in any one of claims 1 to 29 for the manufacture of a medicament for treating or preventing cancer in an individual in need thereof. A use of a composition as claimed in claim 30 for the manufacture of a medicament for treating or preventing cancer in an individual in need thereof. Use of a composition according to claim 31 for the manufacture of pharmaceuticals for the treatment or prevention of cancer in individuals in need. A use of the pharmaceutical composition of claim 32 for the manufacture of pharmaceuticals for treating or preventing cancer in individuals in need. Use of a composition according to claim 33 for the manufacture of pharmaceuticals for the treatment or prevention of cancer in individuals in need. A use of the pharmaceutical composition of claim 34 for the manufacture of pharmaceuticals for the treatment or prevention of cancer in individuals in need. The use of claim 35, wherein the individual is a human or non-human mammal. The use of claim 36, wherein the individual is a human or non-human mammal. The use of claim 37, wherein the individual is a human or non-human mammal. For use as claimed in claim 38, wherein the individual is a human or a non-human mammal. For use as claimed in claim 39, wherein the individual is a human or a non-human mammal. The use of claim 40, wherein the individual is a human or non-human mammal. The use of claim 35, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome, myeloma, solid tumors; bladder cancer, breast cancer, lung cancer or any combination of the foregoing. The use as claimed in claim 36, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome, myeloma, solid tumor; bladder cancer, breast cancer, lung cancer or any combination of the foregoing. The use of claim 47, wherein the leukemia is lymphocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, prolymphocytic T-cell leukemia, granulocytic lymphocytic leukemia or adult T-cell leukemia; or the lung cancer is non-small cell lung cancer (NSCLC). The use of claim 48, wherein the leukemia is lymphocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, prolymphocytic T-cell leukemia, granulocytic lymphocytic leukemia or adult T-cell leukemia; or the lung cancer is non-small cell lung cancer (NSCLC). The use as claimed in claim 47, wherein the solid tumor is surrounded by a tumor stroma comprising bone marrow cells, T cells, or a combination of bone marrow cells and T cells; and/or wherein the solid tumor is infiltrated by bone marrow cells, T cells, or a combination of bone marrow cells and T cells. The use of claim 48, wherein the solid tumor is surrounded by a tumor stroma comprising bone marrow cells, T cells, or a combination of bone marrow cells and T cells; and/or wherein the solid tumor is infiltrated by bone marrow cells, T cells, or a combination of bone marrow cells and T cells. A use of an antibody or antibody fragment as claimed in any one of claims 1 to 29 for the manufacture of a pharmaceutical for inhibiting tumor growth in an individual in need thereof. Such as the use of claim 53, wherein: (i) the pharmaceutical is administered parenterally or non-parenterally, (ii) the pharmaceutical is administered intravenously, subcutaneously or orally; (iii) The dosage of the administered composition, antibody or antibody fragment is 0.1-15 mg/kg per administration; (iv) The pharmaceutical drug is administered once a week, once every two weeks, or every three weeks Once, once a month, once every 2 months, or once every 3 months; or any combination of the foregoing. An isolated nucleic acid comprising a nucleotide sequence encoding an antibody or antibody fragment as described in any one of claims 1 to 29. An expression vector comprising the nucleic acid of claim 55 operably linked to a promoter. A host cell transformed with the expression vector of claim 56. A method of producing an antibody or antibody fragment according to any one of claims 1 to 29, said method comprising culturing a host cell according to claim 57 under conditions used to produce said antibody or antibody fragment. The method of claim 58, further comprising isolating the antibody or antibody fragment. A product comprising a composition as claimed in claim 30 and a container, and further comprising a package insert or label indicating that the composition can be used to treat cancer. A product comprising a composition as claimed in claim 31 and a container, and further comprising a drug leaflet or label indicating that the composition can be used to treat cancer. For use as claimed in claim 35, it includes the treatment of a second cancer. The use of claim 62, wherein the second cancer treatment is surgery, chemotherapy, radiotherapy, biological therapy, targeted therapy, or immunomodulatory therapy or a combination thereof. A use of an antibody or an antibody fragment thereof, which is used to manufacture a pharmaceutical product for enhancing an immune response in an individual in need thereof, wherein the antibody binds to V-domain Ig inhibitor of T cell activation (VISTA), and the antibody fragment comprises an antigen binding region that binds to VISTA, wherein the antibody or antibody fragment thereof binds to VISTA, thereby enhancing an immune response to cancer; and further wherein the antibody or antibody fragment comprises: a heavy chain variable (VH) domain comprising a VH CDR1 having an amino acid sequence of SEQ ID NO: 7; a VH CDR2 having an amino acid sequence of SEQ ID NO: 8 and a VH CDR3 having an amino acid sequence of SEQ ID NO: 9; and further comprising a light chain variable (VL) domain comprising a VL CDR1 having an amino acid sequence of SEQ ID NO: 10; a VL CDR2 having an amino acid sequence of SEQ ID NO: 11; and a VL CDR3 having an amino acid sequence of SEQ ID NO: 12. CDR3. The use as claimed in claim 64, wherein the immune response is an anti-tumor immune response. Use of an antibody or antibody fragment thereof for the manufacture of pharmaceuticals that elicit a biological response in an individual in need thereof, wherein the antibody binds V-domain Ig inhibitory factor of T cell activation (VISTA), and the antibody fragment comprising an antigen-binding region that binds to VISTA, wherein the antibody or antibody fragment thereof binds to VISTA, thereby eliciting a biological response; and further wherein the antibody or antibody fragment comprises: a heavy chain variable (VH) domain comprising SEQ. VH CDR1 having the amino acid sequence of ID NO: 7; VH CDR2 having the amino acid sequence of SEQ ID NO: 8 and VH CDR3 having the amino acid sequence of SEQ ID NO: 9; and further comprising a light chain variable (VL) domain comprising an amine group having SEQ ID NO: 10 VL CDR1 with the acid sequence; VL CDR2 with the amino acid sequence of SEQ ID NO: 11; and VL CDR3 with the amino acid sequence of SEQ ID NO: 12; and wherein the biological reaction is selected from the following. Group: a. Activating monocytes; b. Inducing T-cell proliferation and cytokine secretion; c. Increasing monocyte survival; d. Inducing antibody-dependent cell-mediated cytotoxicity in cells expressing VISTA ( ADCC); and e. Inducing antibody-dependent cellular phagocytosis (ADCP) in cells expressing VISTA.

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