TW202017946A - 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 T-cell activated V-domain Ig inhibitory factor (VISTA), and methods for their preparation and use.

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

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

The antibody or antibody fragment binds to VISTA to modulate or promote the immune response. Antibody fragments can include, for example, Fab, F(ab') ₂ or scFv antibody fragments. The antibody or antibody fragment may comprise an antibody constant region. Antibodies or antibody fragments can bind to hematopoietic cells (such as bone marrow cells) and/or lymphocytes, monocytes or neutrophils, T cells, natural killer (NK) cells, natural killer T (NKT) cells, tumors VISTA on 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 complementarity determining regions (CDRs) and/or one or more light chain CDRs, including one or more CDRs (eg all three) of any anti-VISTA antibody described herein Heavy chain CDR, all 3 light chain CDRs or all 6 CDRs), these antibodies include the names VSTB112 (S2), VSTB116 (S5), VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 ( S47) antibody. In some specific examples, the antibody or fragment thereof is selected from the group consisting of VSTB112, VSTB95, VSTB116, VSTB50, VSTB53, and VSTB60. In a 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 specific examples, the antibody or antibody fragment may further comprise at least one heavy chain and at least one light chain of any anti-VISTA antibody described herein. In some specific examples, the antibody or antibody fragment comprises at least one heavy chain comprising a heavy chain variable region sequence and/or at least one light chain comprising a light chain variable region sequence. In some specific examples, the antibody comprises a human framework region. In some specific examples, the antibody is a whole antibody. In some specific examples, the fragment is an anti-VISTA binding member. In some specific examples, the heavy chain CDR of an 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 specific examples, the amino acid sequences of the heavy chain and light chain variable regions are represented by SEQ ID NOs: 7 and 8, respectively.

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

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

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

In some specific examples, the antibody or antibody fragment is a monoclonal antibody. In some specific examples, the antibody is a chimeric, humanized, or human antibody. In some specific examples, the antibody or antibody fragment comprises a human constant region. In some specific examples, the antibody or antibody fragment is specific to the epitope of VISTA, for example within the amino acid sequence SEQ ID NO:9. In some specific examples, the antibody or antibody fragment binds to the antibody with an affinity of at least 1×10 ^-7 liters/mole (eg, at least 1×10 ^-8 liters/mole, such as at least 1×10 ^-9 liters/mole) The epitope of VISTA.

In some specific examples, modulating the immune response includes increasing CD45+ white blood cells, CD4+ T cells, and/or CD8+ T cells. In some specific examples, modulating the immune response includes increasing (eg, T cells) the production of cytokines (eg, IFNγ, IL 10, TNFα, IL-17), increasing the T cell response, and/or modulating Foxp3 performance.

The present invention also relates to a composition comprising the antibody or antibody fragment (eg, anti-VISTA antibody) described herein and a pharmaceutically acceptable carrier, diluent, or excipient. For example, the composition may include a VISTA antagonist, the VISTA antagonist includes an antibody or antibody fragment thereof, the antibody or antibody fragment includes an antigen binding region bound to VISTA; and a vaccine (such as a viral vector vaccine, bacterial vaccine, 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 present invention also relates to a method of treating or preventing cancer, which comprises administering an effective amount of at least one antibody, antibody fragment, or composition described herein to an individual in need (eg, a mammal, such as a human or non-human animal).

In some specific examples, the antibody or antibody fragment binds to VISTA, thereby modulating or promoting an immunogenic response to cancer. In some specific examples, the cancer is leukemia, lymphoma, myelodysplastic syndrome, and/or myeloma. In some specific examples, the cancer may be any type or type of leukemia, including lymphocytic leukemia or myeloid leukemia, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute bone marrow (marrow) Sex) leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large-particle lymphocytic leukemia, or adult T-cell leukemia. In some specific examples, the lymphoma is histiocytic lymphoma, and in some specific examples, the cancer is multiple myeloma. In some specific examples, the cancer is a solid tumor, such as melanoma or bladder cancer. In some embodiments, the cancer is lung cancer (eg, non-small cell lung cancer (NSCLC)). Some treatments further include the administration of vaccines (such as viral vector vaccines, bacterial vaccines, cell-based vaccines, DNA vaccines, RNA vaccines, peptide vaccines, or protein vaccines). The invention also relates to a method of inhibiting tumor growth in an individual in need, which method comprises administering an effective antibody or antibody fragment or composition described herein.

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

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

The invention also includes methods for preparing antibodies, fragments, and compositions, such as methods for preparing the antibodies or fragments described herein, which include culturing the host cells under the conditions under which the antibodies are prepared. The method may further comprise isolating the antibody. The invention also relates to nucleic acids comprising nucleotide sequences encoding antibodies and fragments (eg isolated nucleic acids), expression vectors comprising such nucleic acids (eg they are operably linked to promoters) and transformations using such expression vectors Host cell.

The present invention also relates to kits and products that include a composition and container containing an anti-VISTA antibody, and further include a package insert or label indicating that the composition can be used to treat cancer.

The present invention also provides an isolated antibody or antibody fragment thereof, which comprises an antigen binding region that binds to T cell activated V domain Ig inhibitory factor (VISTA), wherein the antibody includes an antibody VH domain, and the antibody VH domain includes a SEQ ID VH CDR1 of the amino acid sequence of NO:25, VH CDR2 having the amino acid sequence of SEQ ID NO:26 and VH CDR3 having 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 having the amino acid sequence of SEQ ID NO: 28, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 30 . In some specific examples, the antibody or antibody fragment comprises one or more humanized or human framework regions. In a specific embodiment, the antibody or antibody fragment comprises the antibody VH domain comprising SEQ ID NO: 37 and/or the antibody VL domain comprising SEQ ID NO: 44. In some specific examples, the antibody comprises a heavy chain constant region (eg, human heavy chain constant region) and/or a light chain constant region (eg, human light chain constant region, such as the light chain constant region present in SEQ ID NO: 56 ). The heavy chain constant region is preferably an IgG1 heavy chain constant region (for example, the IgG1 heavy chain constant region present in SEQ ID NO: 61). In a specific embodiment, the IgG1 heavy chain constant region is modified to increase the antibody's protease resistance. An example of an IgG1 heavy chain constant region modified to increase protease resistance is the IgG1 heavy chain constant region present in SEQ ID NO:60. In some specific examples, 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 comprising SEQ ID NO: 56 Light chain. In specific embodiments, antibodies or antibody fragments are 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 antibody fragment thereof, the antibody or antibody fragment thereof comprising an antibody VH domain, the antibody VH domain comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 25, having SEQ ID VH CDR2 of the amino acid sequence of NO: 26 and VH CDR3 having the amino acid sequence of SEQ ID NO: 27, and the antibody or antibody fragment thereof further comprises an antibody VL domain, which comprises the SEQ ID NO: 28 VL CDR1 of the amino acid sequence, VL CDR2 having the amino acid sequence of SEQ ID NO: 29 and VL CDR3 having the amino acid sequence of SEQ ID NO: 30; and a pharmaceutically acceptable carrier, Diluent or excipient.

In another specific example, the present invention relates to a method of treating cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of an antibody or antibody fragment that binds to T cell activated V domain Ig inhibitory factor (VISTA) , Wherein the antibody comprises an antibody VH domain, the antibody VH domain comprises 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 SEQ ID NO: 27 VH CDR3 of the amino acid sequence of the amino acid sequence, and the antibody further comprises an antibody VL domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 28, and an amino acid sequence having 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 a specific embodiment, 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 comprises 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 antibody fragment thereof, which comprises an antigen-binding region that binds to the T-cell activated V-domain Ig inhibitory factor (VISTA), wherein the antibody binds to a conformational epitope in VISTA (such as human VISTA). In some specific examples, the conformational epitopes include residues 103-111 (NLTLLDSGL (SEQ ID NO: 62)) and 136-146 (VQTGKDAPSNC (SEQ ID NO: 63) of human VISTA (SEQ ID NO: 46)) ) Or exist in these residues. In another specific example, the conformational epitope comprises residues 24-36 (LLGPVDKGHDVTF (SEQ ID NO: 64)) of human VISTA (SEQ ID NO: 46), 54-65 (RRPIRNLTFQDL (SEQ ID NO: 65 )) and 100-102 (TMR) may exist in these residues. In another specific example, the conformational epitope comprises an amino acid residue in the FG loop of human VISTA (SEQ ID NO: 46).

In addition, the present invention relates to a method of improving the immune response of an individual in need thereof, which comprises administering to the individual a therapeutically effective amount of an antibody or antibody fragment thereof that binds to T cell activated V domain Ig inhibitory factor (VISTA), which includes binding In the antigen binding region of VISTA, thereby promoting the immune response to cancer. In a specific embodiment, the immune response is an anti-tumor immune response.

In another specific example, the present invention provides a method of initiating 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 to T cell activated V domain Ig inhibitory factor (VISTA) , Which contains an antigen-binding region that binds to VISTA, thereby promoting an immune response to cancer. Examples of biological reactions include activation of mononuclear spheres; induction of T cell proliferation and cytokine secretion; antibody-dependent cell-mediated cytotoxicity (ADCC) of cells expressing VISTA; and antibody-dependent cell phagocytosis of cells expressing VISTA ( ADCP).

The description of illustrative specific examples of the present invention is as follows.

The present invention relates to an antibody to a novel immunoglobulin family ligand (Genbank: JN602184) named V-domain immunoglobulin inhibitory factor (VISTA) activated by T cells (Wang et al., 2010, 2011). VISTA has homology with PD-L1, but the display is limited to the unique performance pattern of the hematopoietic compartment. In particular, VISTA is constitutively and highly expressed on CD11b ^high bone marrow cells, and is expressed on CD4 ⁺ and CD8 ⁺ T cells at a lower level. Human homologues share approximately 85% homology with murine VISTA and have similar expression patterns (Lines et al., Cancer Research 74:1924, 2014). VISTA expressed on antigen presenting cells (APC) inhibits CD4 ⁺ and CD8 ⁺ T cell proliferation and cytokine production independently of PD-1 via homologous receptors. In a passive EAE (experimental autoimmune encephalomyelitis) disease model, VISTA-specific monoclonal antibodies increase T cell-dependent immune responses and aggravate the disease. The excessive expression of VISTA on tumor cells weakens the protective anti-tumor immunity of the tumor-bearing host. The human VISTA study confirmed its inhibitory function on human T cells (Lines et al., Cancer Research 74:1924, 2014). Research from Flies et al. also identified VISTA (called PD-1H) as an effective immunosuppressive molecule (Flies et al., 2011). VISTA is described in further detail in US Published Application US 20130177557 A1 and US Patent Nos. 7,919,585 and 8,236,304, the entire contents of which are incorporated herein by reference.

VISTA is a novel checkpoint negative regulator that suppresses immune responses. As described in Example 12 herein, treatment with a VISTA-specific monoclonal antibody in a murine tumor model has been shown to reverse the inhibitory characteristics of the tumor immune microenvironment and improve protective anti-tumor immunity, thus exhibiting VISTA monoclonal antibodies as cancer immunity The potential of novel therapeutic agents for therapy.

Antibodies and fragments of the invention

The term "antibody" is intended to include multiple antibodies, monoclonal antibodies (mAb), chimeric antibodies, humanized antibodies, human antibodies, and anti-Id antibodies and by any known technique (such as ( But not limited to) fragments, regions or derivatives provided by enzymatic cleavage, peptide synthesis or recombinant technology. The anti-VISTA antibody of the present invention can bind to the part of VISTA that regulates, regulates or enhances the immune response. In some specific examples, the antibodies competitively inhibit one or more of the anti-VISTA antibodies described herein. Methods for determining whether two or more antibodies compete for binding to the same target are known in the art. For example, competitive binding analysis can be used to determine whether one antibody blocks the binding of another antibody to the target. Typically, competitive binding analysis involves the use of a purified target antigen (eg PD-1) bound to a solid substrate or cell, unlabeled test binding molecules, and labeled reference binding molecules. Competitive inhibition is measured by determining the amount of marker bound to a solid surface or cell in the presence of the test binding molecule. Usually test binding molecules are present in excess. Typically, when a competitive binding molecule is present in excess, it will inhibit the specific binding of the reference binding molecule to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75 % Or more than 75%. In some specific examples, competitive inhibition is determined using competitive inhibition ELISA analysis.

Multiple antibodies are a heterogeneous group of antibody molecules derived from the serum of animals immunized with an antigen. Monoclonal antibodies contain a substantially homogeneous group of antibodies of specific antigens, which group contains 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); US Patent No. 4,376,110; edited by Ausubel et al., 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); Edited by Colligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), where the owner’s content is in full text The way cited is incorporated herein. Such antibodies may have any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD, and any subclasses thereof. The fusion tumor producing the monoclonal antibody of the present invention can be cultured in a test tube, on the spot, or in vivo.

The invention also encompasses digested fragments, designated parts and variants thereof, including antibody mimetics or antibody parts that include the structure and/or function of mimic antibodies or designated fragments or parts thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to mammalian VISTA protein. For example, the present invention covers antibody fragments or parts thereof that can bind to VISTA, including (but not limited to) Fab (eg, by papain digestion), Fab' (eg, by pepsin digestion and partial reduction), and F( ab') ₂ (for example by pepsin digestion), facb (for example by plasmin digestion), pFc' (for example by pepsin or plasmin digestion), Fd (for example by pepsin digestion, Partial reduction and re-agglutination), Fv or scFv (for example by molecular biology techniques) fragments. The antibody fragments of the present invention also include the antibody fragments described in Aaron L. Nelson, mAbs 2:1, 77-83 (January/February 2010), the contents of which are incorporated by reference in their entirety.

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 a variety of truncated forms using antibody genes that have one or more stop codons introduced upstream of the natural stop site. The various parts of the antibody can be joined together chemically by conventional techniques, or they can be made into contiguous proteins using genetic engineering techniques.

In a specific example, the amino acid sequence of the immunoglobulin chain or a portion thereof (eg, variable region, CDR) is about 70-100% identical to the amino acid sequence of the corresponding variable sequence chain described herein ( 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). Preferably, a suitable computer algorithm known in the art is used to determine 70-100% amino acid consistency (eg 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value).

This article provides examples of heavy and light chain variable region sequences.

The antibody or variant of the present invention specifies that the variant may comprise any number of contiguous amino acid residues from the antibody of the present invention, wherein the number is selected from the group consisting of an integer consisting of 10-100% of the number of contiguous residues in the anti-TNF antibody . As appropriate, the length of the subsequence adjacent to the amino acid 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 thereof. In addition, the number of such subsequences may be any integer selected from the group consisting of 1 to 20, such as at least 2, 3, 4, or 5.

Those skilled in the art should understand that the present invention includes at least one biologically active antibody of the present invention. The specific activity of biologically active antibodies is at least 20%, 30% or 40% of natural (non-synthetic), endogenous or related and known antibodies, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95%-100%. Methods for analyzing and quantifying the measured values of enzyme activity and substrate specificity are well known to those skilled in the art.

Substantially similar means that the compound has at least 85% (eg, at least 95%) identity with natural (non-synthetic), endogenous, or related and known antibodies, and has at least 85% (eg, 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 (eg CDR, framework, CL, CH domain (eg CH1, CH2, CH3), hinge (VL, VH)) It is essentially non-immunogenic in humans, where only a small amount of sequence changes or changes. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees and their analogs), rodents (mice, rats and their analogs), and other mammals refer to such species, subgenus, genus, and subtype Section-specific antibodies. In addition, chimeric antibodies can include any combination of the above. Such alterations or changes are optional 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 pointed out that human antibodies can be prepared from non-human animals or prokaryotic or eukaryotic cells that can functionally express rearranged human immunoglobulin (eg heavy and/or light chain) genes. In addition, when the human antibody is a single chain antibody, it may include a linker peptide that is not present in the natural human antibody. For example, the Fv may include a connecting peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. It is believed that such connecting peptides have human origin.

Bispecific, heterospecific, heterobinding or similar antibodies can also be used, which are single, preferably human or humanized antibodies with binding specificities for at least two different antigens. 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. The recombinant preparation of bispecific antibodies can be based on the co-presentation of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). See also WO 93/08829; US Pat. Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083, 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 92/00373, EP 03089; Traunecker and others , EMBO J. 10:3655 (1991); Suresh et al., Methods in Enzymology 121:210 (1986), each of which is incorporated herein by reference in its entirety.

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

Human antibodies or fragments thereof specific for human VISTA protein can be cultured against appropriate immunogenic antigens such as VISTA protein or parts thereof (including synthetic molecules such as synthetic peptides).

Other specific or universal mammalian antibodies can be similarly cultured. The preparation of immunogenic antigens and the preparation of monoclonal antibodies can be carried out using any suitable technique.

For example, fusion tumors are suitable for immortal cell lines (eg, myeloma cell lines, 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 analogs thereof, or hybrid myeloma, its fusion products or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art, see eg www .atcc.org) prepared with antibody-producing cells. Antibody-producing cells may include isolated or colonized spleen, peripheral blood, lymph, tonsils, or other immune cells (such as B cells) or those that exhibit constant or variable heavy chain or light chain or framework or complementarity determining region (CDR) sequences Any other cell. Such antibody-producing cells may be recombinant or endogenous cells, and may also be prokaryotic or eukaryotic cells (eg, mammals such as rodents, horses, sheep, goats, sheep, primates). See, for example, Ausubel above and Colligan, Immunology above, Chapter 2, which is incorporated herein by reference in its entirety.

Antibody-producing cells can also be obtained from the peripheral blood or better spleen or lymph nodes of humans or other suitable animals that have been immunized with the relevant antigen. Any other suitable host cell can also be used to express heterogeneous or endogenous nucleic acids encoding the antibodies of the invention, designated fragments or variants thereof. Fusion cells (fusion tumors) 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 that produce antibodies with the desired specificity can be selected by suitable analysis (eg, 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 necessary specificity can be used, including (but not limited to) from peptide or protein libraries (such as (but not limited to) bacteriophage, Ribosomes, oligonucleotides, RNA, cDNA or its analogs, presentation libraries; 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, for example, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93 /00605; PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835; WO90/14443; WO90/14424; WO90/14430; PCT/U594/1234; WO92/18619; WO96/07754; EP 614 989 ; WO95/16027; WO88/06630; WO90/3809; US 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-US 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 by reference in its entirety Here) select recombinant antibodies or transgenic animals that depend on the lineage that enables the production of human antibodies (eg SCID mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998), and related patents and applications are each incorporated herein by reference in their entirety) methods of immunization. Such techniques include (but are not limited to) ribosome presentation (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci . USA, 95: 14130-14135 (November 1998)); Single cell antibody preparation technology (US 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 droplets 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, Volume 5, In Vitro Immunization in Hybridoma Technology, edited by Borrebaeck, Elsevier Science Publishers BV, Amsterdam, Netherlands (1988)).

Methods of engineering or humanizing non-human or human antibodies can also be used and are well known in the art. In general, humanized or engineered antibodies have one or more amino acid residues from non-human sources (such as, but not limited to, mice, rats, rabbits, non-human primates, or other mammals) . 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. Reveal known human Ig sequences, such as 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 known in the art, such introduced sequences can be used to reduce immunogenicity or reduce, increase or change binding, affinity, affinity, specificity, half-life, or any other suitable characteristic. In general, part or all of the non-human or human CDR sequences are maintained, and part or all of the non-human sequences of the framework and/or constant regions are replaced with human or other amino acids. The three-dimensional immunoglobulin model known to those skilled in the art can also be used to humanize antibodies as appropriate, while maintaining high affinity for antigens and other favorable biological properties. A computer program that illustrates and presents the possible three-dimensional configuration of the selected candidate immunoglobulin sequence can be used. Testing these presentations makes it possible to analyze the possible role of residues in the function of the candidate immunoglobulin sequence, that is, to analyze the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected and combined from the common and imported sequences so that the desired antibody characteristics (such as increased affinity for the target antigen) are achieved. In general, CDR residues are directly and most substantially involved in affecting antigen binding. Humanization or engineering of the antibodies of the present invention can be performed using any known method, such as (but not limited to) the methods described in the following documents, such as Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)); Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 ( 1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); U.S. Patent Nos. 5,723,323, 5,976862, No. 5,824514, No. 5,817483, No. 5,814476, No. 5,763,192, No. 5,723,323, No. 5,766,886, No. 5,714,352, No. 6,204,023, No. 6,180,370, No. 5,693,762, No. 5,530,101, Nos. 5,585,089, 5,225,539; 4,816,567, each of which is incorporated herein by reference in its entirety, including references cited therein.

As described herein and/or known in the art, anti-VISTA antibodies can also be used by transgenic animals (eg, mice, rats, rabbits, hamsters, non-human primates, etc.) capable of producing human antibody lineages, as appropriate. Its analogues) are generated by immunization. Cells producing human anti-VISTA antibodies can be isolated from such animals and immortalized using suitable methods, such as the methods described herein.

Transgenic animals that can produce a lineage of human antibodies that bind to human antigens can be prepared by known methods (eg, 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. U.S. Patent No. 5,545,807, Bruggemann et al. WO 90/04036, Bruggemann 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). In general, these mice contain at least one transgene containing DNA from at least one human immunoglobulin locus that is functionally rearrangeable or can be functionally rearranged. Endogenous immunoglobulin loci in such mice can be disrupted or deleted to remove the animal's ability to produce antibodies encoded by endogenous genes.

Screening for antibodies or fragments that specifically bind to similar proteins can be conveniently achieved using peptide presentation libraries. This method involves screening a large number of peptides for individual members with the desired function or structure. Antibody screening of peptide presentation libraries is well known in the art. The length of the peptide sequence presented may be 3 to 5000 or more amino acids, usually 5 to 100 amino acids long, and usually 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 includes the presentation of peptide sequences on the surface of phages or cells. Each phage or cell contains a nucleotide sequence that encodes 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 the appearance of in-tube chemical synthesis and recombinant methods. See PCT Patent Publication Nos. 92/05258, 92/14843 and 96/19256. See also U.S. Patent No. 5,658,754; and 5,643,768. Peptide presentation libraries, vectors and screening kits can be purchased from suppliers such as Invitrogen (Carlsbad, Calif.) and Cambridge antibody Technologies (Cambridgeshire, UK). See for example 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 issued to Dyax No. 5,223,409, No. 5,403,484, No. 5,571,698, No. 5,837,500, No. 5,427,908, No. 5,580,717 granted to Cambridge antibody Technologies; No. 5,885,793; No. 5,750,373, No. 5,618,920, No. 5,595,898 awarded to Genentech No. 5,576,195, 5,698,435, 5,693,493 and 5,698,417.

The antibodies of the present invention can also be prepared using at least one nucleic acid encoding an anti-VISTA antibody to obtain transgenic animals (such as goats, cows, sheep, and the like), which produce such antibodies in milk. Such animals can be provided using known methods. See, for example (but not limited to) US 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 cited 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. The above references are each incorporated by reference in their entirety.

The antibody of the present invention can bind human VISTA with a wide range of affinity (K _D ). In a preferred embodiment, at least one human monoclonal antibody of the present invention can bind human VISTA with high affinity as appropriate. For example, human monoclonal antibodies may be equal to or less than about 10 ^-7 M of K _D (such as (but not limited to) 0.1-9.9 (or any range or value ^{where) × 10 -7, 10 -8,} 10 - ⁹ , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 or any range or value) combined with human VISTA. In some specific examples, the antibody or antibody fragment can bind human VISTA with an affinity of 1×10 ^-7 liters/mole (for example, at least 1×10 ^-8 liters/mole, for example, at least 1×10 ^-9 liters/mole) .

The affinity or affinity of an antibody for an antigen can be experimentally determined using any suitable method. (See, for example, Berzofsky et al., "Antibody-Antigen Interactions," Fundamental Immunology, Paul, WE, Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992) ; And the method described in this article). If measured under different conditions (such as salt concentration, pH), the measured affinity of the specific antibody-antigen interaction may vary. Therefore, measuring the affinity and other antigen-binding parameters _{(K D, K a, K} d) is preferably standardized solutions of antibody and antigen, and a standardized buffer for.

Nucleic acid molecule

Use the information provided herein, such as or at least 70-100% of the nucleotide sequences encoding contiguous amino acids encoding at least one of the specified fragments, variants or common sequences thereof, or containing at least one of these sequences The deposited vector can use the method described herein or known in the art to obtain the nucleic acid molecule of the present invention encoding at least one anti-VISTA antibody, the anti-VISTA antibody comprising the heavy chain variable CDRs of SEQ ID NOs: 1, 2 and 3 All of the regions and/or all of the light chain variable CDR regions of SEQ ID NO: 4, 5 and 6.

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

The isolated nucleic acid molecule of the present invention may include a nucleic acid molecule comprising an open reading frame (ORF), such as (but not limited to) at least one designated portion of at least one CDR, such as CDR1, CDR2 of at least one heavy or light chain And/or CDR3; a nucleic acid molecule that contains the coding sequence of an anti-VISTA antibody or fragment (eg, a fragment that includes a variable region); and a nucleotide sequence that is different from the above but still encodes at least one such as the degeneracy of the genetic code Nucleic acid molecules of anti-VISTA antibodies described herein and/or known in the art. It is conventional for those skilled in the art to generate such degenerate nucleic acid variants encoding specific anti-VISTA antibodies of the 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 present 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 of an entire antibody or part thereof; an antibody, fragment or part Coding sequences and other sequences, such as the coding sequence of at least one signal leader sequence or fusion peptide in the presence or absence of the above other coding sequences, such as at least one intron, and other non-coding sequences, including (but not limited to) non-coding sequences Encoding 5'and 3'sequences, such as transcribed untranslated sequences that play a role in transcription, mRNA processing (including splicing), and polyadenylation signals (eg, ribosomal binding and stability of mRNA); encoding other amines Other coding sequences of base acids, such as coding sequences that provide other functions. Thus, the sequence encoding the antibody can be fused with a labeling sequence (such as a sequence encoding a peptide that facilitates purification of the fused antibody containing the antibody fragment or part).

The human genes encoding the constant (C) regions of the antibodies, fragments and regions of the invention can be derived from human fetal liver libraries by known methods. The human C region gene can be derived from any human cell, including human cells that express and produce human immunoglobulins. Human C _H region can be derived from any human H chain of known classes or isotypes, including γ, μ, α, δ or ε type, and secondly, such as G1, G2, G3 and G4. Since the H chain isotype is responsible for the various effector functions of an antibody, it will select the desired effector functions (such as complement fixation) or antibody dependent cellular cytotoxicity (ADCC) in the active region of the C _H boot.

Composition

The pharmaceutical composition disclosed herein is prepared according to standard procedures and is administered in a dose selected to treat (eg, reduce, prevent, or remove the treated condition or slow or interrupt its progression) (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, NY, the contents of which are incorporated herein by reference for a general description of methods for administering various agents for human treatment). Compositions including the disclosed antibodies and agents can be delivered using controlled or sustained release delivery systems (eg, capsules, biodegradable matrices). Examples of delayed-release delivery systems for drug delivery suitable for administration of the disclosed compounds are described in, for example, US Patent Nos. 5,990,092; 5,039,660; 4,452,775; 3,854,480, the entire teachings of which are incorporated by reference Into this article.

For preparing pharmaceutical compositions from anti-VISTA antibodies and/or fragments of the invention, pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, lozenges, pills, capsules, cachets, suppositories, and dispersible granules. For example, the compounds of the present invention can be in powder form for reconstitution on delivery. The solid carrier can be one or more substances, which can also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, lozenge disintegrant, or encapsulating material. When in powder form, the carrier is a finely powdered solid, which forms a mixture with the finely powdered active ingredient.

Powders and lozenges preferably contain from about 1 to about 70% active ingredient. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter and Its analogs. Lozenges, powders, cachets, buccal tablets, fast-melt strips, capsules and pills can be used as solid dosage forms containing active ingredients 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 can be formulated in aqueous polyethylene glycol solution.

The pharmaceutical composition may be in unit dosage form. In such forms, the composition is subdivided into unit doses containing appropriate amounts of active ingredients. The unit dosage form can be an encapsulated preparation, the package containing individual quantities of unit dose. The dosage can vary depending on the patient's requirements, the severity of the condition being treated, the compound used and the route of administration. It is within the skill of this technique to determine the appropriate dose for a particular situation.

In addition, if necessary, the pharmaceutical composition may contain other compatible agents, such as drugs, therapeutic agents, or prophylactic agents. Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimics, synthetic drugs, inorganic molecules, and organic molecules. Examples of the class of such agents (eg, anticancer agents) include (but are not limited to) cytotoxins, angiogenesis inhibitors, immunomodulators, tumor immune agents, and those used to relieve pain or counteract the toxic effects of one or more therapeutic agents Medicines (for example, bisphosphonates that reduce the hypercalcemia effect of glucocorticoids).

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

Immunomodulators and therapies suitable for the compositions and methods described herein include (but are not limited to) anti-T cell receptor antibodies, such as anti-CD3 antibodies (eg Nuvion (Protein Design Laboratories), OKT3 (Johnson & Johnson) or anti-CD20 antibody Rituxan (IDEC)), anti-CD52 antibody (eg Campas 1H (CAMPATH 1H) (Ilex)), anti-CD11a antibody (eg 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 (eg MRA (Chugai)) And anti-IL-12 antibodies (CNTO1275 (Janssen)), anti-TNFα antibodies (Remicade (Janssen)) or TNF receptor antagonists (Enbrel (Immunex)), anti-IL-6 antibodies (BE8 (Diaclone)) and siltuximab (CNTO32 (Centocor)) and antibodies that specifically bind to tumor-associated antigens (eg trastuzimab (Genentech)).

Tumor immune agents suitable for the compositions and methods described herein include (but are not limited to) ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), Pembrolizumab (anti-PD-1), anti-PD-L1 antibody and anti-LAG-3 antibody.

The composition is preferably prepared in the form of a dosage unit containing a therapeutically effective amount of antibody or fragment. Examples of dosage units are lozenges and capsules. For therapeutic purposes, in addition to the active ingredients, tablets and capsules may contain conventional carriers, such as binding agents, such as gum arabic, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, such as phosphoric acid Calcium, glycine, lactose, corn starch, 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 oil 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, glycerin, hydrogenated edible fat, lecithin, methyl cellulose, methylparaben or parahydroxyl 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, US Patent No. 7,820,169, the entire contents of which are incorporated herein.

treatment method

Those skilled in the art (such as clinicians) can consider the selected agents, pharmaceutical formulations and routes of administration, various patient factors, and other considerations to determine the appropriate dosage and route for administration of a particular antibody, fragment or composition for administration to an individual To. The dosage is preferably not to cause or produce minimal or no adverse side effects. In a standard multiple dosing regimen, pharmacological agents can be administered in a dosage time course designed to maintain a predetermined or optimal plasma concentration in the individual undergoing treatment. Antibodies, fragments and compositions can be added in any suitable dosage range or therapeutically effective amount, such as 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg /kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 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 mg/kg, 20.0 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg 60 mg/kg, 70 mg/kg, 80 mg/ kg, 90 mg/kg and 100 mg/kg. In a specific example, the dose of the composition, antibody or fragment administered is 0.1-15 mg/kg per administration.

The antibody or fragment can be administered once, at least once, twice, at least twice, three times, or at least three times per day. The antibody or fragment can 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 can be administered once a month, at least once a month, twice a month, at least twice a month, three times a month, or at least three times a month. Antibodies or antibody fragments 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 Or at least six times a year.

Anti-VISTA antibodies, fragments and compositions can be administered, for example, by parenteral or enteral methods, including (but not limited to) intravenous, subcutaneous, oral, rectal, intramuscular, intraperitoneal, transmucosal, transdermal, Intrathecal, nasal or superficial administration. One of ordinary skill will recognize that the following dosage forms may contain the compound or the corresponding pharmaceutically acceptable salt of the compound of the present invention as the active ingredient. In some specific examples, the dosage form may contain the compound or the 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, administered together with each other or with one or more other therapeutic agents). The compounds of the present invention can be administered before, after, or simultaneously with one or more other therapeutic agents. In some embodiments, the compounds of the present invention and other therapeutic agents can be co-administered simultaneously (eg, simultaneously) in separate formulations or in combination formulations. Alternatively, the agents can be administered sequentially in separate composition forms within a suitable time frame determined by a skilled clinician (for example, a time sufficient to overlap the drug effects of the therapy). The compound of the present invention and one or more other therapeutic agents can be administered in a single dose or multiple doses in an order and time course suitable for achieving the desired therapeutic effect.

The invention also provides a method for regulating or treating at least one malignant disease of cells, tissues, organs, animals or patients. In some specific examples, 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, digestive tract cancer, gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, urogenital cancer, germ cell cancer, gynecological cancer, head and neck cancer, blood/blood cancer, musculoskeletal cancer , Neuronal 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 cancer described herein. In some specific examples, the solid tumor is infiltrated with bone marrow and/or T cells. In some specific examples, the cancer is leukemia, lymphoma, myelodysplastic syndrome, and/or myeloma. In some specific examples, the cancer may be any type or type of leukemia, including lymphocytic leukemia or myeloid leukemia, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute bone marrow (marrow) Sex) leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large-particle lymphocytic leukemia, or adult T-cell leukemia. In some specific examples, the lymphoma is histiocytic lymphoma, follicular lymphoma, or Hodgkin lymphoma (Hodgkin lymphoma), and in some specific examples, the cancer is multiple myeloma. In some specific examples, 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 of cells, tissues, organs, animals or patients, 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 myelogenous leukemia (AML), chronic myelogenous 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 Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome/malignant hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic Diseases, cancer-related bone resorption, cancer-related bone pain and similar diseases. In some specific examples, the solid tumor is infiltrated with bone marrow and/or T cells. In a specific embodiment, the solid tumor is lung cancer, such as non-small cell lung cancer (NSCLC).

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

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

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

In another specific example, 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 can be administered to individuals in need to prevent (including preventing the recurrence of cancer) or treat (eg, control or ameliorate cancer or one or more symptoms) cancer. Any medicine or therapy (eg chemotherapy, radiation therapy, targeted therapy (such as imatinib, sorafenib and vemurafenib)), hormonal therapy and/or biological therapy or immunity Therapy) can be used in combination with the compounds or compositions of the invention described herein. Anticancer agents include (but are not limited to) 5-fluorouracil; acivicin; adesleukin; aldesleukin; altretamine; aminoglutethimide; amsacrine ; Anastrozole; anthramycin; asparaginase; azacitidine; azetepa; azotomycin; azotomycin; parmes Batimastat; bicalutamide; bleomycin sulfate; brequinar sodium; brequinar sodium; bropirimine; busulfan; carboplatin ( carboplatin); carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; siromycin (Cirolemycin); cisplatin; cladribine; cristnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin ); daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; deaquinone mesylate (Diaziquone); docetaxel (docetaxel); cranberries; cranberries hydrochloride; droloxifene (droloxifene); droloxifene citrate; dromostanolone propionate (dromostanolone propionate); Dazazomycin; edatrexate; eflornithine hydrochloride; enloplatin; enpromate; epipropidine; Epirubicin hydrochloride; erbulozole; essobicin hydrochloride (es orubicin hydrochloride); estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; fazarabin (Fazarabine); fenretinide; fluorouridine (fluxuridine); fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fosquidone (Fostriecin sodium); gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant Interleukin II or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-m; interferon alpha-n3; interferon beta-I a; interferon gamma-I b; isopropyl platinum ( iproplatin); irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liaprozole hydrochloride; lometrix Lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; mechlorethamine hydrochloride; megestrol acetate ; Melenestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; sodium methotrexate; metoprine; metoprine Piper (meturedepa); mitomycin (mitomycin); mitosper (mitosper); mitotane (mitotane); mitoxantrone hydrochloride (mitoxantrone hydrochloride); mycophenolic acid (mycophenolic acid); nocodazole (Nocodazole); omaplatin (ormapl atin); Paclitaxel; pegaspargase; porfromycin; prednimustine; procarbazine hydrochloride; puromycin; rogludimine (Rogletimide); safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparfosate sodium; sparsomycin; spirostatin (Spiromustine); spiroplatin; streptonigrin; streptozocin; sulofenur; sulofenur; talisomycin; tegafur; hydrochloric acid Teloxantrone hydrochloride; temoporfin; teniposide; tenoxoside; teroxirone; testolactone; thiamiprine; thioguanine (Thioguanine); thiotepa; tiazofurin; tiazofurin; tirapazamine; topotecan; trimetrexate; trimetrexate glucuronate ); triptorelin; uracil mustard; uredepa; uredepa; vapreotide; verteporfin; vinblastine sulfate Vincristine sulfate; vindesine; vindesine sulfate; vindidine sulfate; vinepidine sulfate; vinlyrosate sulfate; vinlyrosate sulfate; vinleurosine sulfate; tartaric acid Vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zenozplatin; net statin (Zinostatin); zorubicin hydrochloride. Targeted therapies include (but are not limited to) tyrosine kinase inhibitors (such as imatinib, sorafenib, and velopinib). The present invention also encompasses the combination of administration of the anti-VISTA compound of the present invention and radiotherapy, which includes the use of x-rays, gamma rays, and other sources of radiation to kill cancer cells. Cancer treatment is known in the art and described in documents such as Physician's Desk Reference (57th Edition, 2003).

The anti-VISTA antibodies described herein are also suitable, for example, for the treatment of chronic infectious diseases (such as especially HIV, HBV, HCV, and HSV).

Various characteristics 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 all-human or humanized anti-human VISTA antibodies

*VSTB140、VSTB149及VSTB174中之恆定區序列加有下劃線。^Δ VSTB149之重鏈中的賦予蛋白酶抗性之胺基酸殘基以粗體表示。Table 1B: Selected heavy and light chain sequences of all human or humanized anti-human VISTA antibodies

*The constant region sequences in VSTB140, VSTB149 and VSTB174 are underlined. ^Δ Amino acid residues conferring protease resistance in the heavy chain of VSTB149 are shown in bold.

實施例Table 2: Dissociation constants (K _D ) of selected anti-VISTA antibodies

Examples

Example 1: Analysis of VISTA performance on human hematopoietic cells

method:

Prepare and stain fresh human PBMC for VISTA performance

VISTA performance was tested on freshly isolated human PBMC (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 Company (Colmar, PA) and analyzed on the same day. Express 10 ml of whole blood containing heparin sulfate for analysis.

Sample Preparation

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

Staining scheme: Use a frozen vial containing 5×10 ⁷ PBMCs for compensation control and use as a staining control.

Use the following reagents and/or consumables:

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

Cell surface staining protocol

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

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

Subsequently, live/dead cells were stained 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 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. The plate was incubated at 4°C for 15 minutes. The wells were then washed with 150 μl FACs staining buffer and centrifuged at 1300 rpm for 3 minutes at 4°C. Discard the supernatant.

A mixture containing the following antibodies was then added to each well for surface staining: the mixture is described in Tables 3-6 below. Depending on the relevant group, each mixture is used separately from other mixtures.

Table 3: Pedigree staining

Table 4: T cell staining

Table 5: DC staining

Table 6: Bone marrow staining

After surface staining, the cells were washed twice with FACS staining buffer as previously described and centrifuged at 1300 rpm for 5 minutes at 4°C. Resuspend the sample in 50 μl of FACS staining buffer containing appropriately fluorescently labeled streptavidin. The samples were incubated at 4°C for 30 minutes. The cells were washed with 150 μl FACS staining buffer and centrifuged at 1300 rpm for 5 minutes at 4°C. Repeat this washing step, then resuspend the sample in 250 μl FACS staining buffer. The samples were analyzed on the same day on the BD LSRFortessa™ cell analyzer (BD Biosciences).

ANALYSE information

Use FlowJo Model 9 software to analyze flow cytometry data to gate specific phenotypic populations. An enumeration of geometric means is used to compare VISTA performance in different cell subpopulations. Each population was normalized to the background by subtracting the isotype control value from the mean value of anti-VISTA treated samples. Use Prism to make the curve and use the Student's T-test for statistics when comparing only two samples or use one-way ANOVA and Bonferroni post-test for statistics.

result:

VISTA performance on human bone marrow and lymphatic subgroups:

As shown in Figures 2A-2E, 3A-3G, 4, 5A-5B, and 6A-6C, the performance of VISTA on the CD14 ⁺ mononuclear ball was significantly different from that of all other groups (p<0.001). No significant differences were found between other groups. The mononuclear ball shows the highest level of VISTA in the surrounding blood, and the CD14 ⁺ CD16 ^- subpopulation has significantly higher performance than CD14 ^lo CD16 ⁺ cells. Although APC showed moderate performance, lymphatic subgroups showed low performance.

VISTA performance on human T and NK subgroups:

As shown in FIGS. 7A-7E, 8A-8G, and 9, for the NK subpopulation, CD56 ^lo cells exhibit significantly higher VISTA performance than CD56 ^Hi NK cells. Among the subpopulations of T cells, CD8 ⁺ memory cells showed the highest performance, but they were 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 performance were found; DC and basophilic spheres showed low VISTA performance, with plasma cell-like dendritic cells (pDC) generally higher but to a greater extent Not significant.

Conclusion: These results show that VISTA manifests on various immune cell subpopulations and VISTA manifests highest on mononuclear spheres, some of which appear on different T cell subpopulations and NK cells and hardly on B cells.

Example 2: VISTA performance on peripheral blood cells

method:

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

Peripheral blood mononuclear cells (PBMC) staining: Ficoll gradient was used to isolate peripheral blood mononuclear cells from whole blood. Freshly separated 1×10 ⁶ PBMCs were stained with 100 μl of antibody mixture in staining buffer. The samples were incubated at 4°C for 30 minutes and then washed once with staining buffer. Resuspend cells in 100 μl staining buffer. Data was collected using 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-bound mouse anti-human VISTA (pure line GG8) was prepared from ImmuNext (Lebanon, NH).

in conclusion:

VISTA performance on blood cells around healthy humans

Multicolor flow cytometry was used to analyze the VISTA performance of whole blood and peripheral blood mononuclear cells. As shown in Figures 13A and 13B, the highest level of VISTA performance was detected on a single-core ball, followed by a neutrophilic ball. CD4+ and CD8+ T cells show low levels of VISTA, as shown in Figures 13C and 13D.

VISTA performance on blood cells around cancer patients

As shown in Figures 14A-C, peripheral blood mononuclear cells (PBMCs) from lung cancer patients were analyzed. 14A is a representative flow cytometry bone marrow graph showing the analysis results of CD14 ⁺ mononuclear balls and CD15 ⁺ bone marrow-derived suppressor cells (MDSC). The results indicate that the phenotype CD15 ⁺ cells are MDSCs derived from neutrophils. In addition, these cells are not present in healthy blood samples. 14B is a representative histogram of VISTA performance on mononuclear spheres derived from healthy and cancer patients, indicating that a higher level of VISTA is expressed on cancer patient cells compared to healthy controls. Similarly, a higher level of VISTA was found on the MDSC of cancer patients, as shown in FIG. 14C.

15A is a representative FACS diagram showing the presence of neutrophil-derived MDSC in the blood of colon cancer patients. 15B and 15C are representative histograms showing that VISTA performance on cancer patients' mononuclear spheres is higher than that of healthy donor blood samples.

VISTA performance on blood cells around crab-eating macaques

As shown in FIGS. 16A and 16B, flow cytometry analysis of monkey whole blood revealed a VISTA performance pattern similar to human cells. Compared with CD4 ⁺ (Figure 16C) and CD8 ⁺ (Figure 16D) T cells, both mononuclear and neutrophils showed the highest level of VISTA.

Example 3: VISTA performance at the RNA level and the 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 includes RNAseq analysis results of approximately 140 hematological malignant cell lines (some cell lines are repeated in the analysis). The information is shown in Figure 17.

The RNAseq values are listed as FPKM (fragment per thousand bases of exons per 1 million located fragments) values.

Essentially, this means counting all reads that belong to the exon region outside the gene, and is normalized by the length of the gene and the total number of reads per sample (to calculate the difference between samples). The cut-off value is 1; VISTA is positive (RNA level) above 1, while VISTA is negative.

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

Example 4: Production of monoclonal antibodies against VISTA

Phage panning

Twenty-four phage panning experiments were performed to enrich phage that are active against Cyno VISTA-His. The macaque VISTA protein was used for these experiments because it exhibited better biotin binding than the human VISTA protein. To confirm the success of the phage experiment, phage pools from individual rounds of panning were added to a neutral streptavidin disc coated with biotin-labeled cyno VISTA-His and detected with anti-M13 antibodies that bind HRP. Individual colonies were picked from phage selection rounds and Fab proteins were prepared in 96-well dishes. The binding of Fab supernatant expressed by biotin-labeled cyno VISTA-His was analyzed. This operation resulted in more than 200 successful results.

Amplify the VH and VL regions from the Fab disk, submit for DNA sequencing and export as a FASTA file. When picking pure lines that will be converted to MAB and tested in the form of MAB, these pure lines are selected based on sequence diversity and having a limited risk of post-translational modification and as few hydrophobic residues as possible.

VH and VL from phage pure lines were sub-colonized in mammalian IgG1/κ expression vector and transfected into HEK293 cells. The antibody was purified on protein A agarose fast flow affinity resin. The concentration of bacteriophage MAB was determined by quantitative ELISA using Nanodrop measurement. The antibody group performed at a high level. SDS-PAGE analysis revealed the integrity of each manifested antibody variant.

In-line maturation of phage antibodies was performed by amplifying the VH domains of multiple antibody mixtures from the last round of panning to colonize phage vectors with VL diversity. This operation results in an enriched VH pool, which is sampled with additional VL diversity. The phages were subjected to 1-2 rounds of strict panning, with a view to identifying extremely high-affinity binders of VISTA ECD His protein. Operate a single Fab ELISA to determine the success of maturation. The ELISA and performance data were standardized to 100% of the reference pure lines from the original rebirth panning experiment, and the identification of mature mature lines with higher affinity for the cyno VISTA antigen than the reference pure lines. This process yielded several pure lines that showed up to 200% binding when screened at low antigen concentration (1 nM). The pure lines with the highest affinity were sequenced and made into MAB.

Fusion tumor generation

A group of BALB/cAnNCrl mice received an intraperitoneal (IP) injection of 50 μg Hu VISTA-Ig recombinant protein (Sino) emulsified in Complete Freund's Adjuvant, and then received once every two weeks Intraperitoneal injection of 50 μg Hu VISTA-Ig recombinant protein emulsified in Incomplete Freund's Adjuvant. Two weeks later, mice received an intraperitoneal injection of 50 μg of cyno VISTA-Fc recombinant protein emulsified in Incomplete Adjuvant. All mice received the last injection of PBS containing 25 μg human and 25 μg cyno VISTA at the bottom of the tail, and spleens were collected for fusion five days later.

Another group of BALB/cAnNCrl mice received an intraperitoneal injection of 50 μg of Hu VISTA-His recombinant protein emulsified in pass 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, the mice received an intraperitoneal injection of 50 μg of Cyno VISTA-His recombinant protein emulsified in Incomplete Adjuvant. After two weeks, all mice received the last injection of PBS containing 25 μg Hu VISTA-His and 25 μg Cyno VISTA-His, and spleens were collected three days later for fusion.

On the day of fusion, the mice were euthanized by CO ₂ asphyxiation, the spleen was removed and placed in 10 mL cold phosphate buffered saline. A single-cell suspension of spleen cells was prepared by grinding the spleen through a fine mesh sieve with a small pestle and rinsing with PBS at room temperature. The cells were washed once with PBS and subjected to RBC lysis. Briefly, cells were resuspended in 3 mL RBC lysis buffer (Sigma #R7757)/spleen and placed on ice for 5 minutes. The cells were washed once more with PBS at room temperature and labeled for magnetic sorting. 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) according to the manufacturer’s instructions, followed by Use MS column sorting with Midi MACS. The negative cell elution fraction (the positive cell elution fraction was discarded) was fused with FO cells. Mouse myeloma cells and viable spleen cells were fused in a 1:1 ratio. Briefly, spleen and myeloma cells were mixed together, granulated and washed once with 50 mL PBS. The agglomerated pellets were resuspended with 1 mL of polyethylene glycol (PEG) solution (2 g PEG (molecular weight 4000), 2 mL DMEM, 0.4 mL DMSO) per 10e8 spleen cells at 37°C for 30 seconds. The cell/fusion mixture was then immersed in a 37°C water bath for about 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, and then centrifuged at 150×g for 5 minutes. The cells were then resuspended in medium E-HAT (MediumE, StemCell Technologies catalog number 03805) containing HAT (Sigma catalog number H0262) and seeded in 96-well flat bottom polystyrene tissue culture plates (Corning # 3997).

The fusion tumor supernatant was screened using capture EIA against antibodies specific for cyno VISTA. In short, the goat anti-mouse IgG (Fc) antibody (Jackson #115-006-071) used in the coating buffer (Thermo 28382) was coated at 4 μg/ml (Nunc-Maxisorp #446612) for at least 60 minutes. The plate was blocked with 200 μl of 0.4% (w/v) bovine serum albumin (BSA) in PBS at room temperature for 30 minutes. The dishes were washed once, 50 μl of fusion tumor supernatant was added to each well and incubated at room temperature for at least 30 minutes. Wash the dish once, add 50 μl 0.1 μg/mL cyno VISTA-huIg per well and incubate at room temperature for 30 minutes. The dish was washed once, and a solution of 1:40,000 streptavidin HRP (Jackson 016-030-084) in 0.4% BSA/PBS was added to the dish and incubated at room temperature for 30 minutes. Wash the plate 3 times, then use 100 μl TMB Turbo substrate (Thermo Scientific 34022) for color development in each well, and incubate at room temperature for about 10 minutes. The reaction was stopped using 25 μl 4 N sulfuric acid per well and the absorbance was measured at 450 nm using an automatic disk spectrophotometer. Fifteen primary success results were selected for secondary colonization by limiting dilution and screened in the same primary screening format.

Human VISTA-Ig was used to cross-screen all cyno VISTA active fusion tumor cell lines to assess cross-reactivity. Briefly, goat anti-ms Fc (Jackson#115-006-071) used in a 0.1 M sodium carbonate-bicarbonate buffer (pH 9.4, Pierce 28382BupH™) at 4 μg/mL coating pan (Nunc- Maxisorp #446612) at 4°C overnight. Without washing, the wells were blocked with 200 μl of blocking agent (0.4% BSA (Sigma) (w/v) in PBS (Invitrogen), and overnight at 4°C. After removing the blocking solution, the undiluted fusion tumor supernatant was incubated on the coated disk for 30 minutes at room temperature. The dishes 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 blocker for 30 minutes. The plate was washed once and probed with goat anti-human Fc-HRP (Jackson #109-036-098) diluted 1:10,000 with blocking agent for 30 minutes at room temperature. The dishes were washed again, and then each well was developed with 100 μl TMB Turbo substrate (Thermo Scientific 34022), and incubated at room temperature for about 10 minutes. The reaction was stopped using 25 μl 4 N sulfuric acid per well and the absorbance was measured at 450 nm using an automatic disk spectrophotometer.

The V region antibody sequences of fusion tumors exhibiting activity between human and cynomolgus monkey VISTA were screened. Prior to the reverse transcriptase (RT) reaction, Invitrogen's SuperScript III cell direct cDNA system was used to prepare fusion tumor cells. 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 a plastic film and rotated at 700 rpm for 3 minutes. Discard PBS and add 10 μl of resuspension buffer and 1 μl of lysis enhancer to each well. The disk was sealed, incubated at 75°C for 10 minutes and stored at -80°C.

For RT reactions, each well contains 5 μl water, 1.6 μl 10×DNase buffer, 1.2 μl 50 mM EDTA, 2 μl Oligo(dT)20 (50 mM) and 1 μl 10 mM dNTP mixture. Incubate the plate at 70°C for 5 minutes, then incubate on ice for 2 minutes, then add the following reagents to each well: 6 μl 5×RT buffer, 1 μl RNaseOUT™ (40 U/μl), 1 μl SuperScript™ III RT (200 U/μl) and 1 μl 0.1 M DTT. The disk was sealed, placed on a thermocycler preheated to 50°C and incubated at 50°C for 50 minutes, and then inactivated (incubated at 85°C for 5 minutes). The reaction was frozen on ice and the single strand cDNA was stored at -80°C until further use.

For amplification of the V region, 20 μl PCR reaction was provided. 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 dNTP, 0.15 μl 100 μM forward primer mixture, 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 mixture, seal the disk and handle the amplification reaction; for VH, the formula is (i) 94°C for 1 minute, (ii) 94°C for 15 seconds, ( iii) 55°C for 30 seconds, (iv) 68°C 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 formula 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.

The forward primer was premixed and this mixture was used with the reverse primer at a ratio of 3:1. The PCR product was checked on an agarose gel. The reactants were prepared for infusion colonization by adding enhancers (In-Fusion HC colonization kit, catalog number 639650, Clontech). Five microliters of PCR reaction was transferred to the PCR plate, and then 2 μl of enhancer per well was transferred. The disk was sealed and incubated in a thermal cycler (15 minutes at 37°C and 15 minutes at 80°C). The target vector (vDR243 or vDR301) was prepared by Esp3I digestion; (digest 1.5 μg of the carrier in 3 μl Tango buffer, 2 l Esp3I and water in 30 μl reaction at 37°C for 2 hours).

For infusion colonization, the PCR product treated with 2 μl enhancer was mixed with 100 ng Esp3I digested vector and 2 μl 5×infusion enzyme (Clontech). Perform the infusion reaction in a 96-well dish format. Incubate the plate on the PCR machine at 50°C for 15 minutes and transform the Stella competent cells by heat shock at 42°C without shaking for 40 seconds and spread with the selected antibiotic Incubate on LB agar plates at 37°C overnight. The next day, the colonies were picked in 96-well deep well dishes containing LB/Carbenicillin medium and grown overnight at 37°C. Frozen stocks were made from overnight cultures mixed with an equal volume of 30 w/v% glycerol. The V region was sequenced using sequencing primer SPF0052. The sequence was analyzed, and one positive well was selected for each fusion tumor vH and vL, then arranged in a new plate and grown overnight in an enriched medium with ampicillin. The pure lines were then miniprep DNA prepared and transfected in 96-well dishes on a small scale.

Forty-eight selected mouse fusion tumor sequences of the heavy chain and light chain were adapted for human architecture using an internal software program. Choose a human architecture for each of mouse vH or vL. The DNA sequence of the V region was obtained through reverse translation. Synthetic DNA regions corresponding to HFA amino acid sequences are sequenced by integrated DNA technology (Coralville, IA). The pre-cleaved vDR149 and vDR157, human IgG1 and human kappa were screened respectively. Prepared using Qiagen Endo-free Maxi-prep kit. This antibody group was expressed using Expi293 (100 ml) culture.

Example 5: Protocol for human VISTA-IGT cell suppression analysis in a test tube

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

Example 6: Adaptation of human framework regions of anti-VISTA antibodies

Internal software programs were used to adapt the human architecture of mouse fusion tumor sequences of heavy and light chains by CDR grafting (Jones et al., Nature , 321: 522-525 (1986)). The program is based on Kabat definition (Wu, TT & Kabat, EA (1970). J Exp Med, 132, 211-50) to delineate the complementarity determining region (CDR) of the V region sequence and use Blast to compare the framework region and human germline genes. The human reproductive system with the highest sequence identity to the mouse architecture was selected as the recipient gene for human architecture adaptation (HFA). In some cases, based on the aforementioned experience of a well-performing human architecture, the closely related human germline genes are selected as replacements. The DNA sequence of the human architecture selected for each of the mouse vH or vL V regions was obtained via reverse translation. Synthetic DNA regions corresponding to HFA amino acid sequences are sequenced by integrated DNA technology (Coralville, IA). They were cloned into human IgG1 and human κ, respectively.

Example 7: Anti-VISTA antibody construct

Plastid and sequence information of molecules used in cell line production: Plastid constructs are generated for VSTB112 variable region and IgG1κ constant region (VSTB174, new number due to heterotypic change of constant region), IgG2δ constant region (VSTB140) or IgG1 anti-protease constant region (VSTB149) anti-VISTA antibody.

Lonza vector

Established pEE6.4 and pEE12.4 Chinese Hamster Ovary (CHO) expression vector system (Lonza Biologics Public Co., Ltd.) in Biologics Research (BR) and Pharmaceutical Development & Manufacturing Sciences (PDMS) to produce therapeutic mAb in mammalian expression cell lines Main performance system. Each vector contains a human cytomegalovirus (huCMV-MIE) promoter that drives heavy chain (HC) or light chain (LC) expression and contains an ampicillin resistance gene. The pEE12.4 vector also includes a gene encoding glutamic acid synthase (GS). Growth conditions that require glutamate synthetase activity exert selective pressure on cells to maintain the expression vector (GS Gene Expression System Manual Version 4.0). HC genes were selected using pEE6.4 as a single gene vector and LC genes were selected using pEE12.4 as a single gene vector. From these two Lonza single gene vectors, Lonza double gene plastids were produced.

Selected VISTA mAb Amino acid sequence of variable heavy chain region

＞ VSTB112 heavy chain (SEQ ID NO: 37)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS

＞ VSTB50 heavy chain (SEQ ID NO: 38)

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSS

＞ VSTB53 heavy chain (SEQ ID NO:39)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDYYAMDYWGQGTLVTVSS

＞ VSTB95 heavy chain (SEQ ID NO: 40)

EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSS

Selected VISTA mAb Amino acid sequence of 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)

DIQMTQSPSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQGTKVEIK

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

These experiments are used to determine the ability of antibodies produced in ELISA to bind to human or cynomolgus monkey VISTA protein, and to use FACS screening to determine whether the antibody binds to VISTA protein on the surface of K562 cells (human myeloid leukemia cell line) expressing human or cynomolgus monkey VISTA protein. The ability.

method:

Overview of the ELISA procedure: The discs were coated with 1 μg/ml SB0361 (human) or SB0361 (cyno (cynomolgus)) proteins at 4°C overnight, these proteins being the extracellular domain of VISTA from various species. The antibody was gradually diluted from the initial concentration of 1:4 to 1 μg/ml, a total of 4 concentrations were obtained, and incubated at room temperature (RT) for 2 hours. Mouse anti-human IgG1-HRP (horseradish peroxidase) was used for detection and incubated at room temperature for 1 hour. All washings were performed using PBS-Tween (0.05%).

FACS Overview: with each test antibody 5 μg / ml (human) or K562-C7 (cynomolgus) and stained cells incubated at 4 ℃ th to 2 × 10 ⁵ K562-G8 30 minutes. 5 μg/ml goat anti-human IgG1-PE (phycoerythrin) antibody was used as secondary detection antibody. Cells were manipulated on BD Fortessa and FlowJo software (Tree Star, Inc., Ashlang, OR) was used to analyze the MFI (Mean Fluorescence Intensity) of the live population.

Data analysis/results: For each antibody, ELISA and FACS analysis for each of the 4 analyses gives a subjective score (yes/no) on whether the antibodies are firmly bound. If the antibody gives a "no" result for the binding in any analysis, the analysis is repeated to confirm that it is negative. The results are shown in Table 7 below and Figures 19A-19F and 20A-20F.

Table 7.

Example 9: Screening of anti-human VISTA antibodies using mixed lymphocyte reaction (MLR) and Staphylococcus ( STAPHYLOCOCCUS ) enterotoxin B (SEB) activation analysis

The purpose of this study is to provide information that supports the identification of multifunctional α-VISTA antibodies that enhance cellular immune responses in mixed lymphocyte reaction (MLR) analysis and staphylococcal enterotoxin B activation (SEB) analysis.

Mixed lymphocyte reaction (MLR) is a standard immunoassay that depends on MHC Class I and Class II mismatches to drive allogeneic T cell responses. The peripheral blood mononuclear cells are separated from the two mismatched individuals and cultivated together, and due to these mismatches, proliferation and cytokine production occur.

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-glutamic acid (100×) and 10 ml HEPES (1 M) AB medium. The medium is stored for no more than 14 days. 1 mCi tritiated thymidine was prepared by diluting 0.2 ml thymidine stock solution (1 mCi/ml) with 9.8 ml RPMI.

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

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

Mixed lymphocyte reaction (MLR) 96-well plate setting: Determine the appropriate number of "stimulator cells" and "responder cells" required for analysis based on the number of samples to be analyzed. The stimulation population was seeded with 0.5×10 ⁵ cells per well in a 96-well U-chassis and the reaction population was seeded with 1.0×10 ⁵ cells per well. All conditions must be repeated three times. Pipette an appropriate number of "stimulating cells" into a new Erlenmeyer flask and centrifuge as described previously. The cells were resuspended in 10 ml and irradiated with 4000 rad. The cells were centrifuged as described previously, resuspended in 10% AB serum medium at a concentration of 1×10 ⁶ cells/ml and 50 μl was added to appropriate wells. Isolate the desired number of reactive cells, centrifuge as described previously, resuspend in 10% AB serum medium at a concentration of 2 × 10 ⁶ cells/ml and add 50 μl to appropriate wells. The 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. Transfer the cells to a 96-well micro scintillation dish according to the manufacturer's instructions. Count using a micro scintillation counter according to the manufacturer's instructions. IFN-γ concentration was determined by ELISA (eBioscience catalog number 88-7316-88) using the manufacturer's protocol.

Data analysis: Calculate the average count per minute (CPM) or IFN-γ concentration of untreated wells. Calculate the average CPM or IFN-γ of each test group. Convert the data set to Log ₁₀ . Using the 12 MLR multiple-score values of each compound, calculate the average value of the 12 test groups of each compound: the average score of 12 experiments = Σ[(log ₁₀ (the average CPM of three repeated experiments of the test compound))-( log ₁₀ (average CPM of three untreated repeated experiments))]/12.

Acceptance criteria: Test all test reagents and appropriate controls for endotoxin before operation analysis and the content should be <0.1 EU/mg. The average CPM count of the individual reaction cells was lower than 700 CPM, indicating that the cells were quiescent when incubated alone. The CPM of the MLR group was at least 2 times the CPM of the reaction cells incubated alone, indicating that the reaction occurred and donor mismatch. All MLR analyses included human IgG1 negative control protein. Based on the use of Stodden's t test, the results of the human IgG1 negative control are not statistically different from the untreated samples.

Screening of anti-VISTA antibodies in MLR: initial screening of all compounds. Before operating MLR with anti-VISTA antibody, confirm that the antibody binds 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 in this initial screening, but are still being tested in the analysis. For the purpose of preliminary screening, all antibodies were tested at 10 μg/ml in MLR, in which the measured parameters were proliferation and IFN-γ (Figures 21A-21D and 22A-22B).

Select the first 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 MLR: protocol adjustment. The protocol is the same as previously described, but with some adjustments: the antibody is diluted to the following concentrations: 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 μg/ml.

Determination of IC ₅₀ value: The original CPM count and IFN-γ concentration were used to determine the IC ₅₀ value of each antibody. Calculate the IC ₅₀ value by using the program "EZ-R stats." IC ₅₀ values measured using six individual responders. Individual CPM counts and IFN-γ concentrations were obtained in MLR, and the previous candidates were dose titrated.

Table 8: MLR ₅₀ CPM value for the IC and IFN-γ

** Value is log ₁₀ of antibody concentration.

Conclusion: Preliminary screening shows that multiple VISTA specific antibodies can improve the immune response of MLR cells. The antibodies were graded based on efficacy and changes and based on these results, VSTB112, VSTB116, VSTB95, VSTB50, VSTB53, and VSTB60 were selected for evaluation in dose titration experiments. In the dose titration experiment, VSTB60 induced a weaker response than the other five antibodies.

Staphylococcal enterotoxin B (SEB) activation analysis: SEB is a bacterial superantigen that induces the activation of specific Vβ+ T cells. The peripheral blood mononuclear cells are isolated and incubated with the SEB antigen in the culture, thereby inducing stable cell hormone production. This analysis was performed on the top five candidates.

The preparation of 10% AB medium, the preparation of 1 mCi tritiated thymidine, the preparation of soluble VISTA antibody and the isolation of leukocytes were all carried out as described in the MLR above.

SEB 96-well plate setting: Determine the appropriate number of reaction cells required for analysis based on the number of samples to be analyzed. The reaction population was seeded with 2.0×10 ⁵ cells per well in a 96-well U-chassis. All conditions must be repeated three times. The cells were centrifuged as described previously and resuspended in 10% AB serum medium at a concentration of 4×10 ⁶ cells/ml and 50 μl was added to the appropriate wells. Add 50 μl of 10% AB serum medium containing SEB antigen at a concentration of 40 ng/ml. In the experiment, SEB was obtained from Sigma Aldrich (catalog number S0812). The final concentration in the well is 10 ng/ml. The cells were incubated at 37°C and 5% CO ₂ for 3 days. On the third day, 30 μl of supernatant was removed to analyze IFN-γ production. 25 μl of 1 mCi/ml tritiated thymidine solution was added to each well and incubated at 37° C. and 5% CO ₂ for 8 hours. Transfer the cells to a 96-well micro scintillation dish according to the manufacturer's instructions. Count using a micro scintillation counter according to the manufacturer's instructions. IFN-γ concentration was determined by ELISA (eBioscience catalog number 88-7316-88) using the manufacturer's protocol.

Scheme: data analysis. Calculate the average count per minute (CPM) or IFN-γ concentration for each antibody at all concentrations. The acceptance criteria are implemented as described previously. The IC ₅₀ value determination was performed as described. Individual CPM counts and IFN-γ concentrations were obtained in SEB analysis, and the previous candidates were dose titrated.

Table 9: SEB values for CPM ₅₀ and IFN-γ of the IC.

**Value is log ₁₀ of antibody concentration.

Conclusion: In SEB analysis, VISTA-specific antibodies increased cytokine production and proliferation in a dose-dependent manner. The IC ₅₀ value from the SEB study is generally similar to the results of the MLR dilution study.

Example 10: Analysis of epitope binning

Method: ProteOn XPR36 system (BioRad) was used to perform epitope grouping. ProteOn GLC wafers (BioRad, catalog number 176-5011) were coated with two sets of 6 monoclonal antibodies (mAb) using the manufacturer's instructions for the amine coupling chemistry (BioRad, catalog number 176-2410).

Pre-incubate excess competitive mAb (250 nM final concentration) with human VISTA (25 nM final concentration) for 4 hours at room temperature, and operate 6 at a time on the wafer coated with the coated mAb group, where the association time 4 minutes, followed by dissociation for 5 minutes. After each operation, the wafer was regenerated with 100 mM phosphoric acid.

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

Non-competitive mAb is defined as the same binding signal or >A1 signal (only binds to human VISTA).

Competitive mAb is defined as the binding signal << A1 signal (ie, only binds to human VISTA).

Results: In the exemplary sensor map shown in FIG. 23, the VSTB85 antibody was coated on the Proteon SPR wafer and the VISTA protein pre-incubated with the designated competitor was passed on the wafer. VSTB50 is an example of a non-competitive antibody, because a positive reaction can be seen when handling the VISTA/VSTB50 complex. GG8, VSTB49 and VSTB51 complexed with VISTA do not bind to VSTB85 coated on the wafer, so they are classified as competing with VSTB85 for the same binding site on VISTA.

Table 10:

Example 11: PROTEON affinity determination

Anti-IgG Fc-coated surfaces were used to capture antibodies on ProteOn wafers. The test antibody binds to the extracellular domain (ECD) of humans and cynomolgus monkeys (VISTA) with VISTA protein concentrations ranging from 0.39 nM to 100 nM. The antigen was allowed to bind/associate with the antibody-coated wafer for 4 minutes and then monitored for dissociation for 30 minutes. The wafer was regenerated twice by treatment with 100 mM phosphoric acid for 18 seconds. All experiments were performed at 25°C and the data was fitted 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 started. Tumor growth was subsequently monitored 3 times/week. According to IACUC regulations, after any size of the tumor reaches 15 mm, the mice are sacrificed.

For each experiment, a frozen vial of MB49 cells was thawed and grown in RPMI 1640 (+ L-Glut) with 10% serum and penicillin/streptomycin antibiotic. 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 acclimatize for a day, then shave its right belly and mark the tail. They are injected three to five days later.

Tumor injection (intradermal): Inject 50 μl of MB49 cell suspension (approximately 250,000 cells) intramuscularly (i.d.) through the shaved side of the stomach.

To monitor tumor growth: use an electronic caliper gauge to first measure the tumor growth across the widest dimension (L) and secondly at the 90° angle (W) of the first measurement. The tumor volume is obtained as follows:

Volume = (L ² _* W ² )/2 .

After the tumor reaches a diameter of about 5 mm (volume of about 60 mm ³ ), it is considered to form a tumor. After the formation, start processing. During the treatment, tumor growth was measured three times a week and until the experiment was terminated.

Anti-VISTA treatment: Intraperitoneal injection of 10 mg/kg chimeric 13F3-mIgG2a monoclonal antibody. The injection schedule is three times a week for four weeks.

Euthanasia of mice: According to IACUC requirements, after the longest size of animal tumor reaches 15 mm, the animal is euthanized.

Analysis efficacy: Excel was used for data management and GraphPad Prism plot was used to analyze mouse tumor volume. Use the macro of R statistical calculation software for statistical analysis.

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 remission (PR) in 30% of animals (n=7) (Table 11 and Figure 25B). In contrast, all control mIgG2a-treated mice displayed progressive tumor growth (6/6) (Figure 25A). These data show that anti-VISTA treatment can have a great effect on tumor growth.

Table 11: Complete response (CR) and partial response (PR)

Human VISTA sequences are shown in Figures 26 and 27, modified from the aforementioned Wang et al., 2011, the entire contents of this document are incorporated herein.

Example 13: Hydrogen/deuterium (H/D) exchange study for epitope localization of anti-VISTA antibodies

To identify epitopes on human VISTA against VSTB50, 60, 95 and 112, solution hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed using the corresponding Fab. For H/D exchange, the procedure for analyzing Fab disturbances is similar to that previously described (Hamuro et al., J. Biomol. Techniques 14:171-182, 2003; Horn et al., Biochemistry 45:8488-8498, 2006) , But with some modifications. Fab was prepared by papain digestion from IgG and Pierce Fab preparation kit (Thermo Scientific, catalog number 44985) was used to capture protein A. The human VISTA protein sequence contains six N-linked glycosylation sites. To improve sequence coverage, the protein was deglycosylated with PNGaseF. The deglycosylated VISTA protein is incubated in a deuterated aqueous solution for a predetermined time so that deuterium is incorporated at the exchangeable hydrogen atom. The deuterated VISTA protein was complexed with any Fab of VSTB50, VSTB60, VSTB95, or VSTB112 in 46 μL of deuterium oxide (D ₂ O) at 4°C for 30 seconds, 2 minutes, 10 minutes, and 60 minutes. The exchange reaction is quenched by lowering the pH and the protein is 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 did not complex with the Fab molecule. The regions bound to the Fab are presumed to be relatively difficult to exchange and therefore contain higher fractions of deuterium compared to the reference VISTA protein. About 94% of proteins can be localized to specific peptides.

The solution HDX-MS disturbance positioning of VISTA with VSTB50/VSTB60 and VSTB95/VSTB112 is shown in the upper and lower panels of FIG. 28, respectively. Identify two epitopes. Anti-VISTA VSTB50 and VSTB60 recognize the same epitope; VSTB95 binds to another epitope region bounded by VSTB112 on VISTA. Anti-VISTA VSTB50 and 60 share the same epitope, which includes 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, which include 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 by VSTB95 and 112, including residues 39-52 and 118-134. However, the reduction is not as strong as the aforementioned areas (27-36 and 54-65) in different positioning. Although a peptide 100TMR102 showing strong disturbances caused by VSTB95 and 112 is located on the other surface of VISTA surface, it is far away from epitope regions 27-36 and 54-65. This perturbation may be due to other effects. These HDX-MS results provide peptide-level epitopes for anti-VISTA antibodies. There is no overlapping epitope region for these two epitopes. These results are consistent with the aforementioned competitive grouping data in that they do not compete with each other.

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

In the epitope and paratope devoted to determining the structure of VISTA and delineating the interaction between the extracellular domain (ECD) of VISTA and the Fab fragment of the first antibody VSTB112, the complex is crystallized and the structure is determined with a resolution of 1.85Å . Dedicated to determine the structure of ECD in human VISTA complexed with the Fab fragment of antibody VSTB112 to determine the structure of VISTA ECD itself and define the epitope/paratope for this interaction. This structure reveals that VISTA adopts IgV folding, and its chain topology is similar to the TCR Vα chain. In addition to the standard disulfide bonds bridging the B and F strands in the back and front sides of the beta sandwich, the structure reveals that the ECD has two additional disulfide bonds, one tethering the CC' ring to the front panel and the second at A' And G'shares. Although there is crystal contact between VISTA molecules, it is a small amount and there is no evidence of dimer of VISTA ECD based on this structure. The VSTB112 epitope was shown to contain the VISTA BC, CC' and FG loops and the residue on the front β sheet closest to their loops (C'CFG). Paratopes are mainly biased towards heavy chain interactions, where CDR L3 produces the least contact.

Define VISTA: VSTB112 interaction epitope/paratope

VSTB112 Fab buried 1024.3 Å2 surface area after combining VISTA ECD, and the buried heavy chain surface accounted for 715.3 Å2 of this total. Seven hydrogen bonds and 4 salt bridge interactions are formed between VISTA and VSTB112 light chain, and 10 hydrogen and 2 salt bridge interactions are formed between VISTA and VSTB112 heavy chain. VSTB112 recognizes the residues in the proximal end of the FG loop and the residues in the BC, FG and CC' loops of the front stocks C', C, F and G (Figures 29 and 30). The interaction with the CC' loop accounts for most of the contact with the Fab light chain having residues E125 and R127 only in the FG loop, resulting in additional light chain interaction. Residues 119 to 127 corresponding to the VISTA FG loop accounted for 38% of the total 1034.8 Å2 of the surface area buried after binding to VSTB112. Notably, this loop has high polarity and it contains the following sequence-IRHHHSEHR- (SEQ ID NO: 76). In addition, W103 in VSTB112 CDR H3 is well encapsulated against the backbone 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 epitope regions identified by crystallography and HDX is shown in FIG. 31.

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

The functional effects of anti-VISTA antibodies were evaluated in two in-tube analyses (mixed white blood cell reaction (MLR) and SEB (staphylococcal enterotoxin B)). Both analyses measure T cell proliferation and cytokine induction as their main readings, but these effects are due to different mechanisms. In MLR, peripheral blood mononuclear cells (PBMC) from two different human donors are cultivated together, the main histocompatibility complex between T cells of one donor and dendritic cells of the other donor ( MHC) mismatch causes T cell proliferation and interferon (IFNγ) production. In SEB analysis, PBMCs from a single donor are incubated with bacterial superantigens, which directly connect MHC class II proteins on the surface of antigen presenting cells (APC) to T cell receptors (TCR) on T cells , Thereby causing T cell activation, proliferation and cytokine secretion. In both analyses, VSTB112, which is the parent molecule of VSTB174, exhibited dose-dependent induction of T cell proliferation and cytokine production and was most effective among candidates (Figures 21A-21D, Table 12).

Table 12. EC50 values of MLR analysis reading results. VSTB112 (the parent molecule of VSTB174) is the most effective molecule.

Mononuclear Activation Analysis

The analysis data shown in Table 12 was generated using the parent molecule VSTB112 of VSTB174. In order to fully understand the activity of VSTB174, single-core ball activation analysis was performed. The results showed that incubation of VSTB174 with whole PBMC induced the up-regulation of activation markers (CD80 and HLA-DR) on CD14+ mononuclear spheres, indicating the role of antibodies bound to immune cell subsets known to express high levels of VISTA (Figure 32) . Another question is whether the effect on mononuclear ball activation in whole PBMC can be promoted by any antibody that binds VISTA and has IgG1 Fc. Antibodies VSTB103 and VSTB63 bind to VISTA with high affinity (KD 6.36E-10 and 8.30E-10, respectively) and bind to cells expressing VISTA protein similarly to VSTB112 and VSTB111. VSTB103 and VSTB112 are in the same epitope group, while VSTB63 is in a different epitope group; neither antibody promotes mononuclear ball activation. Taken together, these results show that one mechanism by which VSTB174 can exert its effect on T cell activation/proliferation is the activation of mononuclear cells promoted by NK cells.

Preparation medium

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

Preparation of soluble VISTA and control antibodies

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

Add 100 μl of the appropriate antibody solution to the appropriate wells of a 96-well bottom plate (Falcon, 353077). After adding various cell populations in 100 μl, the final concentration of each antibody was 1, 0.1 or 0.01 g/ml. The IgG1 control antibody CNTO 3930 (Lot No. 6405, ENDO <0.1 EU/mg) was added at a final concentration of 1 μg/ml.

Separate PBMC.

Donors are at least 18 years old, generally healthy and randomly selected from local groups.

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

Place 15 ml Ficoll 1077 (SIGMA, 10771) below, being careful not to mix with blood. Do this for every 25 ml of blood.

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

White blood cells were separated at the interface of Ficoll and serum and the cells were diluted with 40 ml Hank's balanced salt solution (HBSS).

The cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4°C.

The cells were resuspended in 50 ml HBSS and counted by transferring 500 μl to each eppendorf tube.

In addition, according to the manufacturer's instructions (catalogue number 130-096-537), a pan mononuclear ball isolation kit from Miltenyi was used to isolate CD14+ cells by negative selection in several treatment groups.

In-tube culture settings

Based on the number of samples to be analyzed, determine the appropriate number of cells for analysis. The reaction population was seeded with 2.0×10 ⁵ cells per well in a 96-well U-chassis. For the CD14 negative selection population, 0.5×10 ⁵ cells were inoculated. All conditions were repeated three times.

The cells were centrifuged as described above, re-suspended in 10% AB serum medium at a concentration of 2 × 10 ⁶ cells/ml for the entire PBMC population and at a concentration of 0.5 × 10 ⁶ cells/ml for the CD14-negative selection population and applied to appropriate wells Add 100 μl to make the total volume in each well 200 l.

Cells were incubated at 37°C and 5% CO ₂ for 1, 2 or 3 days.

Antibody staining and flow cytometry

The 96-well U-chassis was centrifuged at 453 g for 5 minutes and the supernatant was removed.

Wash the 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 HCD14) 1:250 (Biolegend catalog number 325608) ․ HLA-DR-PE Cy7 (pure line L243) 1:250 (Biolegend catalog number 307616) ․ CD80-PE (pure 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 l of PBS buffer was added and analyzed via FACS.

The samples were operated on a Miltenyi MACSQuant 10 parameter flow cytometer and FlowJo 9.7.5 was used to analyze the performance of HLA-DR and CD80 on the CD14+ population. The geometric intensity of fluorescence intensity (MFI) (a statistical parameter that defines the central trend of a set of numbers) is used as the specified statistical parameter for processing comparisons.

Statistical Analysis

All statistics are performed in Prism GraphPad model 6. At each time point, a one-way ANOVA was used to compare pairs between groups, and Tukey correction was performed on diversity. The p-value for all tests was below 0.05 and the comparison 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 IgG1 Fc, which can confer antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) activity. Two types of analysis were performed and showed that VSTB174 may lyse or phagocytose K562-VISTA cells (Figures 33-34), but not K562 myeloma cell line mother cells (data not shown). Another mechanism by which VSTB174 regulates the inhibitory effect of VISTA can be the lysis or phagocytosis of cells that exhibit high levels of VISTA, so they are removed from the local microenvironment.

Example 17: ADCP activity of anti-VISTA antibody

In vitro phagocytosis analysis was used to study the phagocytosis of cells with anti-human VISTA mAb (VSTB173 and VSTB174) to enhance macrophage-mediated ectopic expression of VISTA. These mAbs were colonized in different Fc main chains (IgG1 WT (wild type), IgG1 PR (protease resistance) and IgG2σ) and it was assumed that they might have different activities to increase phagocytosis. 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.

In ADCP analysis, K562 mother cells and K562-VISTA target cells were used to test anti-VISTA antibodies. As shown in Figures 35-36, VSTB174, VSTB149, VSTB173, and VSTB145 increase hMac phagocytosis of K562-VISTA cells. VISTA antibodies VSTB140 or VSTB132 with IgG2σFc that does not bind Fc receptors did not increase phagocytosis as expected. VISTA mAb VSTB174 and VSTB173 with IgG1 Fc showed more phagocytosis than VSTB149 and VSTB145 with IgG1PR Fc (for EC ₅₀ values, see Tables 13 and 14).

Table 13. Anti-human VISTA mAb EC ₅₀ values.

Table 14. Anti-human mAb EC ₅₀ values.

VSTB174 and VSTB173 showed weakly increased phagocytosis of K562 mother cells at the highest concentration (Figure 35-36), which can be attributed to the low VISTA performance of K562 cells. Other anti-VISTA antibodies cannot increase the phagocytosis of K562 cells.

In the K562-VISTA phagocytosis analysis, the negative control antibody was tested at two different concentrations each, but it did not induce any phagocytosis. This result indicates that anti-VISTA antibody-mediated phagocytosis is specific and attributable to the VISTA antigen expression of K562-VISTA cells.

Example 18: ADCC activity of other anti-VISTA antibodies

To test its 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 dish at six different concentrations and repeated three times to obtain a total of six data points.

VSTB174, VSTB149, and VSTB174.LF of 10, 1, 0.1, and 0.01 μg/mL each exhibited measurable ADCC activity, while only LF antibody exhibited measurable ADCC activity at 0.001 μg/mL; at 0.0001 μg/mL None of the lower antibodies exhibit ADCC. This result can be expected when these antibodies each have IgG1 or IgG1 variant Fc. The LF antibody exhibited increased ADCC efficacy, as evidenced by the lower EC ₅₀ value of the LF antibody curve (0.002293 μg/mL) compared to the conventional IgG1 antibody curve (0.02381 μg/mL). The EC ₅₀ value of the IgG1 PR antibody curve is similar to the conventional IgG1 curve (0.01846 μg/mL).

Table 15. ADCC as determined by analysis of the three antibodies tested against EC VISTA ₅₀ values (μg / mL).

At antibody concentrations of 10, 1, 0.1, and 0.01 μg/mL, human IgG1, human IgG1 PR, and human IgG1 LF antibodies all showed measurable ADCC-mediated killing, while only LF antibody was at 0.001 μg/mL antibody Show the killing effect under the concentration. Anti-VISTA antibodies showed no killing effect at the antibody concentration of 0.0001 μg/mL.

As can be seen in the EC ₅₀ value, the LF antibody exhibited about 10 times the effective ADCC killing effect compared to the conventional IgG1 antibody or IgG1 PR antibody.

Example 19: VSTB174's affinity for humans and rhesus monkey VISTA

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

Example 20: VISTA antibody exhibits efficacy in murine 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.

The VSTB174 variable region (VSTB123) grafted on mouse Fc IgG2a was used to generate anti-human VISTA antibodies to enable testing in VISTA-KI mice.

MB49 bladder cancer was evaluated in VISTA KI mice.

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

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

MB49 efficacy studies were conducted in female VISTA-KI mice, and VSTB123 was tested at several doses ranging from 1-10 mg/kg. On day 0, 250,000 MB49 tumor cells were injected intradermally. On Day 6, dosing started as shown in Figure 37 (10 mg/kg isotype control mIgG2a or specified dose VSTB123; 10 mice/group).

As shown in Figure 37, VSTB123 is more effective at higher and lower doses. The doses of 10 mg/kg and 7.5 mg/kg are equivalent, and tumor growth is faster in mice given 5 or 1 mg/kg.

Example 21: Detection of VISTA performance in human tumors with anti-VISTA antibodies Figure 1 shows the VISTA performance of AML tumor cells-and the RNA sequence performance data in FIG. 17 supports the idea that VISTA is expressed by AML cells and that anti-VISTA drugs are used for immunomodulation or antibody-mediated killing Cells are targeted and effective.

Data were obtained from lung tumor samples from surgical resection to assess the performance of VISTA in lung cancer. Dissociate and characterize cells for the performance of VISTA and various other markers. The results showed that 13/13 lung tumors (squamous or adenocarcinoma) contained CD14+VISTA+ bone marrow cells (Figure 38).

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

Immunohistochemical analysis was performed using pure line GG8 (an anti-human VISTA mouse IgG1). Use this mAb to study the staining of VISTA in non-small cell lung cancer (NSCLC) FFPE tumor sections.

FFPE tumor sections were treated with standard antigen repair methods before staining. GG8 mouse anti-human VISTA antibody was used at a dilution of 1:500. Multiple rabbit anti-mouse antibodies and anti-rabbit polymer HRP were used in sequence to detect GG8 binding. Then hematoxylin was used for contrast staining, and then tumor sections were scored.

VISTA performance in lung cancer is mainly 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. Recombinant his-tagged VSTB174 Fab was internally expressed and purified. Crystals were generated and used to collect higher resolution data of the VISTA ECD:VSTB174 Fab complex using synchrotron radiation, and a combination of homology modeling and electron density analysis was used to resolve the structural determination (Figure 29 (top)).

The structure of VISTA ECD:VSTB174 Fab complex was determined by X-ray crystallography, with a resolution of 1.85Å, to obtain the first structure of VISTA ECD and depict the epitope and paratope of VSTB174. VISTA ECD uses IgV folding, and its topology is similar to CTLA-4 ECD, but has a unique G'strand that extends before the beta sandwich. A'and G'are additionally chemically tethered via a disulfide bridge formed between residue C12 in the A'strand and C146 in the G'strand. It was found that six cysteines participate in three intramolecular disulfide bonds, and based on crystal contact, there is no evidence of dimerization of VISTA.

VSTB174 recognizes the residues at the proximal end of the FG loop and the residues in the BC, FG and CC' loops in the front strands C', C, F and G.

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

Although the present invention has been specifically shown and described with reference to its illustrative specific examples, those skilled in the art should understand that forms and forms can be made therein without departing from the scope of the invention covered by the scope of the accompanying patent application Various changes in details.

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The patent or application file contains at least one color drawing. After applying and paying the necessary fees, the Patent Office will provide a copy of the patent or patent application publication in color.

Figures 1A-1C: Curves showing VISTA performance on TF1 AML cells. The expression of VISTA protein obtained by flow cytometry was displayed in the TF-1 AML cell line.

Figures 2A-2E: Curves of staining and gating strategies for identifying human bone marrow and lymph subgroups.

Figures 3A-3G: Curves showing the performance of VISTA on human bone marrow and lymph subpopulations from healthy normal donors.

Figure 4: Curves showing the performance of VISTA on human bone marrow and lymph subgroups of multiple healthy normal donors.

Figures 5A-5B: Curves showing staining and gating strategies for identifying the performance of VISTA on human monocytes and macrophages.

6A-6C: Curves showing the performance of VISTA on human monocytes and macrophages.

Figures 7A-7E: Curves showing staining and gating strategies for identifying the performance of VISTA on human T and NK cell subsets.

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

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

Figures 10A-10D: Curves showing staining and gating strategies for identifying the performance of VISTA on human dendritic cell subsets.

11A-11C: Curves showing the performance of human dendritic cell subsets and VISTA on basophiles from healthy normal donors.

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

Figures 13A-13D: Analysis of VISTA performance on blood cells surrounding healthy humans. Characteristics of VISTA performance on healthy human peripheral blood cells obtained using multicolor flow cytometry analysis: analysis of single-core spheres (SSC ^lo, CD11b ^hi CD14 ^hi CD16 ^-ve CD33 ^+ve ) from whole blood samples from 2 different individuals HLA-DR ^+ve CD19 ^-ve ) (Figure 13A), neutrophil (SSC ^hi CD177 ⁺ CD11b ^hi CD14 ^lo CD16 ^+ve CD33 ^+ve HLA-DR ^-ve CD19 ^-ve ) (Figure 13B) VISTA performance . Ficoll gradient was used to isolate peripheral blood mononuclear cells to analyze CD4+ T cells (CD3 ^+ve CD4 ^+ve ) (Figure 13C) and CD8+ T cells (CD3 ^+ve CD8 ^+ve ) (Figure 13D).

14A-14C: Analysis of VISTA performance on peripheral blood cells from lung cancer patients and healthy control donors. Multi-color flow cytometry analysis was used to obtain the characteristics of VISTA on blood cells surrounding lung cancer patients: a representative FACS diagram from an individual was displayed (Figure 14A). Isolate the peripheral blood mononuclear cells by Ficoll and analyze the mononuclear cells (CD14+CD11b+CD33+HLADR+CD15-) (Figure 14B) and bone marrow-derived inhibitor cells (CD14-CD11b+CD33-HLADR-CD15+CD16+) (Figure 14C) VISTA performance.

Figures 15A-15C: Characteristics of VISTA performance in peripheral blood cells from patients with colon cancer obtained using multicolor flow cytometry analysis: showing a representative FACS graph from an individual (Figure 15A). Isolate the peripheral blood mononuclear cells by Ficoll and analyze the mononuclear cells (CD14+CD11b+CD33+HLADR+CD15-) (Figure 15B) and bone marrow-derived inhibitor cells (CD14-CD11b+CD33-HLADR-CD15+CD16+) (Figure 15C) VISTA performance.

Figure 16A-16D: Characteristics of VISTA performance on blood cells around cynomolgus monkeys obtained using multicolor flow cytometry analysis: analysis of mononuclear spheres (SSC ^lo CD11b ^hi CD14 ^hi HLA- DR ^hi CD16 ^-ve CD19 ^-ve ) (Figure 16A) and neutrophil CD11b ^hi CD14 ^lo HLA-DR ^-ve CD16 ^-ve CD19 ^-ve (Figure 16B) VISTA performance. Ficoll gradient was used to isolate peripheral blood mononuclear cells from three monkeys 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: Demonstrates the absolute performance of VISTA RNA in heme cell lines.

Figure 18: Stable transfection of mouse A20 cells with GFP or human VISTA. It was incubated with ova peptide and DO11.10 T cells. The CD25 performance of T cells was measured 24 hours after the start of incubation. A20-huVISTA cells inhibited the CD25 expression of T cells, but the results of this readout were significantly restored by incubation with VSTB95.

Figures 19A-19F: Curves showing the results of human VISTA ELISA.

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

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

Figures 22A-22B: SEB analysis (individual CPM counts and IFN-g concentration) from 30 μg/ml to 0.0 μg/ml of six candidate anti-VISTA antibody dilution studies.

Figure 23: Sensor diagram obtained using anti-VISTA antibody VSTB85 and VISTA protein coated on Proteon SPR wafers, where designated competitors (competitors listed in Table 16) pass on the wafer.

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

Figure 25A-25B: MB49 tumor growth in female C57BL/6 mice. The curve illustrates the tumor growth of 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 to VSTB50 and VSTB60 antibodies (top) or VSTB95 and VSTB112 antibodies (bottom) as determined by HDX.

Figure 29: VISTA epitope binding to VSTB112. (Top) Show VISTA with a sketch and mark the shares. Dyes the residue with at least one atom within 5Å of VSTB112 in the complex in blue. The blue and orange balls highlight the chain break, and the cyan and green balls mark the N and C ends of the VISTA structure, respectively. (Bottom) The sequence of the VISTA construct used in the structural determination. As calculated by PISA, the circles under the sequence are used to indicate only the residues that bring the main chain into contact with VSTB112, the triangles indicate the side chain contacts, and the squares indicate the side chain contacts that produce hydrogen bonds or salt bridge interactions. The shape is stained to indicate the CDR with the largest number of atoms in contact with a given residue, where the CDR color is as defined in Figure 59. The secondary structural elements are defined by the formula MOE, where the yellow arrow represents the β strand and the red rectangle indicates the α helix.

Figure 30: VSTB112 paratope. (Top) The VISTA antigen is displayed in an illustrative manner, and VSTB112 within 5 Angstroms (Å) of VISTA is displayed in color on the surface to indicate the CDR identity specified in the following sequence. The residues of the contact structure adjacent to the CDR are stained similar to the corresponding CDR (lower) sequence of the VSTB112 Fv region. The color background designates the CDR as defined by Kabat. As calculated by PISA, the circles under the sequence are used to indicate the residues that only bring the main chain into contact with VISTA, the triangle indicates the side chain contact, and the square indicates the side chain contact that produces hydrogen bonding or salt bridge interaction.

Figure 31: Comparison of epitope regions identified by crystallography and hydrogen deuterium exchange (HDX). Sequence of VISTA constructs used in structural determination. As calculated by PISA, the circles under the sequence are used to indicate only the residues that bring the main chain into contact with VSTB112, the triangles indicate the side chain contacts, and the squares indicate the side chain contacts that produce hydrogen bonds or salt bridge interactions.

Figure 32: Activation of CD14+ mononuclear spheres in the entire PBMC 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 picture shows the CD80 MFI; the right picture shows the HLA-DR MFI (two donors tested to show representative results).

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

Figure 34: A graph showing the ADCP activity of VSTB174 against K562-VISTA cells. The two antibodies depicted have the same Fab, but VSTB174 has IgG1 Fc and VSTB140 has Fc-silenced IgG2.

Figure 35: A graph showing the phagocytosis mediated by KVS-VISTA by VSTB174, VSTB149 or VSTB140 mAb. Each mAb was tested at 7 semi-logarithmic doses ranging from 0.0008 μg/ml to 0.56 μg/ml.

Figure 36: A graph showing the phagocytosis mediated by VSTB174, VSTB149 or VSTB140 mAb against myeloma cell line K562 cells. Each mAb was tested at 7 semi-logarithmic doses ranging from 0.0008 μg/ml to 0.56 μg/ml.

Figure 37: Evaluation of MB49 tumor efficacy in VSTB123 1, 5, 7.5 and 10 mg/kg in female VISTA-KI mice. The tumor volume was approximately 50 mm ³ when the drug was started on the 6th day after implantation. VSTB123 is VSTB112 Fab grafted on the mouse Fc backbone and binds to human VISTA in VISTA-KI mice.

Figure 38: The curve shows that CD14+ cells with high/intermediate levels of VISTA were found in 13/13 lung cancer samples as well as the patient’s distal lung tissue and surrounding blood.

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

Claims (102)

An isolated antibody or antibody fragment thereof, comprising an antigen binding region that binds to T cell activated V domain Ig inhibitory factor (VISTA), wherein the binding of the antibody or antibody fragment to VISTA regulates or promotes an immune response, and wherein the antibody contain: a) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 1, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 2, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 3, and b) VL domain, which includes: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 4, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 5, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 6, or c) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 7, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 8, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 9, and d) VL domain, which contains: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 10, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 11, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 12; or e) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 13, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 14, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 15, and f) VL domain, which includes: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 16, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 17, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 18, or g) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 31, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 32, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 33 and h) VL domain, which contains: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 34, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 35, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 36; or i) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 19, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 20, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 21 and j) VL domain, which contains: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 22, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 23, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 24; or k) VH domain, which includes: i. VH CDR1, which has the amino acid sequence of SEQ ID NO: 25, ii. VH CDR2, which has the amino acid sequence of SEQ ID NO: 26, and iii. VH CDR3, which has the amino acid sequence of SEQ ID NO: 27; and l) VL domain, which includes: i. VL CDR1, which has the amino acid sequence of SEQ ID NO: 28, ii. VL CDR2, which has the amino acid sequence of SEQ ID NO: 29, and iii. VL CDR3, which has the amino acid sequence of SEQ ID NO: 30. 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 according to claim 1, which comprises an antibody constant region. The antibody or antibody fragment of any one of claims 1-3, which further comprises at least one heavy chain and at least one light chain. The antibody or antibody fragment of claim 4, wherein at least one heavy chain comprises the heavy chain variable region sequence contained in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 60, or 61. The antibody or antibody fragment of claim 4, wherein at least one light chain comprises the light chain variable region sequence contained in SEQ ID NO: 48, 50, 52, 54, 56, or 58. The antibody or antibody fragment of claim 5, wherein at least one heavy chain includes the heavy chain variable region sequence contained in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 60, or 61, and wherein At least one light chain variable region sequence is contained in SEQ ID NO: 48, 50, 52, 54, 56, or 58. The antibody or antibody fragment of claim 7, wherein the heavy chain comprises the sequence of SEQ ID NO: 47 and the light chain comprises the sequence of SEQ ID NO: 48; or the heavy chain comprises the sequence of SEQ ID NO: 49 and the light The chain contains the sequence of SEQ ID NO: 50; or the heavy chain contains the sequence of SEQ ID NO: 51 and the light chain contains the sequence of SEQ ID NO: 52; or the heavy chain contains the sequence of SEQ ID NO: 53 and the light The chain contains the sequence of SEQ ID NO: 54; or the heavy chain contains the sequence of SEQ ID NO: 55 and the light chain contains the sequence of SEQ ID NO: 56; or the heavy chain contains the sequence of SEQ ID NO: 57 and the light The chain comprises the sequence of SEQ ID NO: 58; or the heavy chain comprises the sequence of SEQ ID NO: 59 and the light chain comprises the sequence of SEQ ID NO: 56; or the heavy chain comprises the sequence of SEQ ID NO: 60 and the light The chain comprises the sequence of SEQ ID NO: 56; or the heavy chain comprises the sequence of SEQ ID NO: 61 and the light chain comprises the sequence of SEQ ID NO: 56. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody is a monoclonal antibody. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody is a humanized antibody. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody comprises a human constant region. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody comprises a human IgG1 or IgG2 constant region. The antibody or antibody fragment of claim 12, wherein the human IgG1 Fc region contains a mutation that prevents proteolysis or glycosylation. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody molecule is specific for the epitope within the amino acid sequence SEQ ID NO: 46. The antibody or antibody fragment according to any one of claims 1-3, wherein the antibody binds to the epitope of VISTA with an affinity of at least 1×10 ^-9 liter/mole. The antibody or antibody fragment according to any one of claims 1-3, wherein the antibody binds to the epitope of VISTA with an affinity of at least 1×10 ^-8 liter/mole. The antibody or antibody fragment according to any one of claims 1-3, wherein the antibody binds to the epitope of VISTA with an affinity of at least 1×10 ^-7 liter/mole. The antibody or antibody fragment of any one of claims 1-3, wherein the modulation of the immune response comprises an increase in CD45 ⁺ leukocytes, CD4 ⁺ T cells or CD8 ⁺ T cells or a combination thereof, or a decrease in immune cells expressing VISTA . The antibody or antibody fragment of any one of claims 1-3, wherein the modulation of the immune response includes increasing cytokine production, increasing T cell response, and/or modulating Foxp3 performance. The antibody or antibody fragment according to any one of claims 1 to 3, which is used for the treatment of cancer in an individual. The antibody or antibody fragment of claim 20, wherein the individual is a mammal. The antibody or antibody fragment of claim 20, wherein the individual is a human. The antibody or antibody fragment of any one of claims 1 to 3, wherein the antibody or antibody fragment binds to VISTA, thereby modulating or enhancing the immune response against cancer. The antibody or antibody fragment of claim 23, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome or myeloma or a combination thereof. The antibody or antibody fragment of claim 24, wherein the leukemia is lymphocytic leukemia or myeloid leukemia. The antibody or antibody fragment according to claim 24, wherein the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (myeloid) (AML), chronic myelogenous leukemia (CML ), hairy cell leukemia, pre-lymphocytic T-cell leukemia, extended granular lymphocytic leukemia or adult T-cell leukemia. The antibody or antibody fragment of claim 23, wherein the cancer is acute bone marrow (myeloid) leukemia (AML). The antibody or antibody fragment of claim 23, wherein the cancer is chronic myelogenous leukemia (CML). The antibody or antibody fragment of claim 23, wherein the cancer is a solid tumor. The antibody or antibody fragment of claim 29, wherein the solid tumor is surrounded by a tumor matrix comprising bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. The antibody or antibody fragment of claim 29, wherein the solid tumor is infiltrated by bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. The antibody or antibody fragment of claim 23, which is adjusted for administration with the vaccine. The antibody or antibody fragment of any one of claims 1-3, which is used to inhibit tumor growth in an individual in need. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody or fragment is adapted for parenteral or non-parenterly administration, such as intravenous, subcutaneous, or oral . The antibody or antibody fragment of any one of claims 1-3, wherein the dose of the antibody or fragment adjusted for administration is 0.1-15 mg/kg per administration. The antibody or antibody fragment of any one of claims 1-3, wherein the antibody or fragment is adjusted to be administered once a week, once every two weeks, once every three weeks, once a month, Give once every 2 months or once every 3 months. The antibody or fragment thereof of claim 1, wherein the light chain or the heavy chain comprises a human framework region. The antibody or fragment thereof according to claim 1, wherein the antibody is a whole antibody. The antibody or antibody fragment of any one of claims 1-3, which is adjusted for administration with a second cancer treatment. The antibody or antibody fragment of claim 39, wherein the second cancer treatment is surgery, chemotherapy, radiation therapy, biological therapy, target therapy, or immunomodulatory therapy, or a combination thereof. The antibody or antibody fragment of claim 39, wherein the cancer is lung cancer. The antibody or antibody fragment of claim 41, wherein the cancer is non-small cell lung cancer (NSCLC). The antibody or antibody fragment of any one of claims 1-3, which comprises a constant heavy chain IgG1 region, which has been modified to improve the protease resistance of the antibody. The antibody or antibody fragment of claim 43, wherein the heavy chain constant IgG1 region modified to improve the protease resistance of the antibody comprises the amino acid sequence of the IgG1 heavy chain constant region present in SEQ ID No: 60. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antibody fragment is expressed in a cell lacking a fucosylation enzyme. The antibody or antibody fragment of claim 45, wherein the cell is a Chinese hamster ovary cell (CHO). The antibody or antibody fragment according to any one of claims 1 to 3, which binds to a conformational epitope which contains or exists in the following: residues of human VISTA (SEQ ID No: 46) 24-36 (LLGPVDKGHDVTF (SEQ ID No: 64)) and residues 54-65 (RRPIRNLTFQDL (SEQ ID No: 65)). The antibody or antibody fragment according to any one of claims 1 to 3, which binds to a conformational epitope which is contained or present in the FG loop of human VISTA (SEQ ID No: 46). The antibody or antibody fragment according to any one of claims 1 to 3, which is used to enhance the immune response in an individual in need. The antibody or antibody fragment of claim 49, wherein the immune response is an anti-cancer immune response. The antibody or antibody fragment according to any one of claims 1-3, which is used to elicit a biological response in an individual in need, and wherein the biological response is selected from the group consisting of: a. Activate single core ball; b. Induces T lymphocyte proliferation and cytokine secretion; c. Improve single-core ball survival; d. Inducing cell-mediated antibody-dependent cytotoxicity (ADCC) in cells expressing VISTA; and e. Inducing antibody-dependent cellular phagocytosis (ADCP) in cells expressing VISTA. The antibody or antibody fragment of claim 50, wherein the cancer is bladder cancer. The antibody or antibody fragment of claim 50, wherein the cancer is breast cancer. A composition comprising the antibody or antibody fragment according to any one of claims 1-19 and a pharmaceutically acceptable carrier, diluent or excipient. The composition of claim 54 which is used to treat cancer in an individual. The composition of claim 55, wherein the individual is a mammal. The composition of claim 55, wherein the individual is a human. The composition of any one of claims 54-57, wherein the antibody or antibody fragment binds to VISTA, thereby modulating or enhancing the immune response against cancer. The composition of claim 58, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome, or myeloma, or a combination thereof. The composition of claim 59, wherein the leukemia is lymphocytic leukemia or myeloid leukemia. The composition of claim 59, wherein the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (myeloid) (AML), chronic myelogenous leukemia (CML), Hairy cell leukemia, prolymphocytic T-cell leukemia, large-particle lymphocytic leukemia, or adult T-cell leukemia. The composition of claim 58, wherein the cancer is acute bone marrow (myeloid) leukemia (AML). The composition of claim 58, wherein the cancer is chronic myelogenous leukemia (CML). The composition of claim 58, wherein the cancer is a solid tumor. The composition of claim 64, wherein the solid tumor is surrounded by a tumor matrix comprising bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. The composition of claim 64, wherein the solid tumor is infiltrated by bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. The composition of claim 58 additionally contains a vaccine adjusted for administration. The composition of any one of claims 54-57, wherein the composition is adjusted for parenteral or enteral administration, such as intravenous, subcutaneous, or oral administration. The composition of any one of claims 55-57, wherein the composition is adjusted for administration at a dose of 0.1-15 mg/kg per administration. The composition of any one of claims 55-57, wherein the composition is adjusted to be administered once a week, once every two weeks, once every three weeks, once a month, every two Give once a month or once every 3 months. The composition of any one of claims 55-57, which is adjusted to be administered with a second cancer treatment. The composition of claim 71, wherein the second cancer treatment is surgery, chemotherapy, radiotherapy, biological therapy, target therapy, or immunomodulatory therapy or a combination thereof. The composition of claim 71, wherein the cancer is lung cancer. The composition of claim 73, wherein the cancer is non-small cell lung cancer (NSCLC). A use of the antibody or antibody fragment according to any one of claims 1-19, which is for preparing a medicine for treating cancer in an individual. Use of the composition as claimed in claim 54 for the preparation of a medicament for the treatment or prevention of cancer in an individual. The use of claim 75 or 76, wherein the individual is a mammal. The use of claim 75 or 76, wherein the individual is a human. Use according to claim 75 or 76, wherein the antibody or antibody fragment binds to VISTA, thereby modulating or enhancing the immune response against cancer. The use according to claim 79, wherein the cancer is leukemia, lymphoma, myelodysplastic syndrome or myeloma or a combination thereof. The use according to claim 80, wherein the leukemia is lymphocytic leukemia or myeloid leukemia. The use according to claim 80, wherein the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (myeloid) (AML), chronic myelogenous leukemia (CML), Mao Cellular leukemia, T-lymphocytic leukemia, large-particle lymphocytic leukemia, or adult T-lymphocytic leukemia. The use according to claim 80, wherein the cancer is acute bone marrow (myeloid) leukemia (AML). The use according to claim 80, wherein the cancer is chronic myelogenous leukemia (CML). The use according to claim 80, wherein the cancer is a solid tumor. The use according to claim 85, wherein the solid tumor is surrounded by a tumor matrix comprising bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. The use according to claim 85, wherein the solid tumor is infiltrated by bone marrow cells, T lymphocytes, or a combination of bone marrow cells and T lymphocytes. For the use of claim 78, it additionally contains a vaccine adjusted for administration. Use of the antibody or antibody fragment according to any one of claims 1 to 19 for the preparation of a medicament for inhibiting tumor growth in an individual in need. Use according to any one of claims 75, 76 and 89, wherein the composition, antibody or fragment is adapted for parenteral or enteral administration, such as intravenous, subcutaneous or oral administration. Use according to any one of claims 75, 76 and 89, wherein the composition, antibody or fragment is adjusted for administration at a dose of 0.1-15 mg/kg per administration. Use according to any one of claims 75, 76 and 89, wherein the composition, antibody or fragment is adjusted to be administered once a week, once every two weeks, once every three weeks, once a month Once, every 2 months or once every 3 months. The use of any one of claims 75, 76, or 89, which further comprises a second cancer treatment adjusted for administration. The use of claim 93, wherein the second cancer treatment is surgery, chemotherapy, radiation therapy, biological therapy, target therapy, or immunomodulatory therapy or a combination thereof. The use according to claim 93, wherein the cancer is lung cancer. The use according to claim 95, wherein the cancer is non-small cell lung cancer (NSCLC). An isolated nucleic acid comprising a nucleotide sequence encoding the antibody of claim 1. An expression vector comprising a nucleic acid such as claim 97 operably linked to a promoter. A host cell transformed with the expression vector as in claim 98. A method for preparing the antibody or fragment thereof according to claim 1, which method comprises cultivating the host cell according to claim 99 under the conditions for producing the antibody or fragment. The method of claim 100, further comprising isolating the antibody. An article of manufacture comprising the composition and container of claim 54 and also a packaging insert or label indicating that the composition can be used to treat cancer.

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