WO2023193794A1 - 抗vista抗体在联合用药中的应用 - Google Patents

抗vista抗体在联合用药中的应用 Download PDF

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Publication number
WO2023193794A1
WO2023193794A1 PCT/CN2023/086921 CN2023086921W WO2023193794A1 WO 2023193794 A1 WO2023193794 A1 WO 2023193794A1 CN 2023086921 W CN2023086921 W CN 2023086921W WO 2023193794 A1 WO2023193794 A1 WO 2023193794A1
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seq
antibody
antigen
cancer
amino acid
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PCT/CN2023/086921
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English (en)
French (fr)
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黄文龙
刘启源
张慧
黄贤明
陈奕藩
梁秋莲
汪志炜
陈俊有
李胜峰
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百奥泰生物制药股份有限公司
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Publication of WO2023193794A1 publication Critical patent/WO2023193794A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to combinations containing anti-VISTA antibodies and their combined use.
  • V domain immunoglobulin suppressor of T cell activation also known as PD-1H, B7-H5, DD1 ⁇ , c10orf54, Gi24 or Dies1
  • VISTA V domain immunoglobulin suppressor of T cell activation
  • PD-1H also known as PD-1H, B7-H5, DD1 ⁇ , c10orf54, Gi24 or Dies1
  • the human VISTA gene is mainly expressed in hematopoietic cell lines and tissues rich in infiltrating leukocytes.
  • PBMC peripheral blood mononuclear cells
  • CD14 + monocytes, neutrophils, myeloid CD11c + DCs, CD4 + and CD8 + T cells all express VISTA.
  • VISTA is highly conserved among different species (>80%).
  • VISTA can be used as a ligand to bind to co-inhibitory receptors (p-selectin glycoprotein ligand-1, PSGL-1) under acidic conditions, or as a receptor-binding ligand (including V-type immunoglobulin domain protein 3, VSIG-3). Studies have shown that VISTA is expressed on the surface of naive T cells and is critical for maintaining T cell quiescence and peripheral immune tolerance.
  • VISTA is involved in multiple diseases: in bladder cancer and melanoma models, VISTA monoclonal antibodies or VISTA gene knockout can significantly improve CD4 + and CD8 + T cell-mediated anti-tumor immunity; in EAE (experimental allergic reaction) In the encephalomyelitis (encephalomyelitis) model, VISTA monoclonal antibodies or VISTA gene knockout will aggravate the progression of the disease; in the ovarian cancer model, VISTA monoclonal antibodies significantly prolong the survival of mice expressing high levels of VISTA. It can be seen that VISTA is a potential target for cancer treatment. Therefore, therapeutic agents targeting VISTA would be helpful in cancer treatment.
  • the purpose of the present invention is to provide a new anti-VISTA antibody or antigen-binding fragment for use in combination with other therapeutic agents for the treatment of diseases.
  • the VISTA antibody or antigen-binding fragment of the present invention can specifically bind to VISTA, block the immunosuppressive signaling pathway downstream of VISTA, and help the immune system clear tumor cells.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment in the manufacture of a medicament for use in combination with one or more other therapeutic agents.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents in the manufacture of a medicament for the treatment of disease.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment in combination with one or more other therapeutic agents in the treatment of disease.
  • the present invention provides a method for treating a disease, such as a method for preventing, treating or improving T cell dysfunction, comprising administering to a patient in need of treatment an effective dose of an anti-VISTA antibody or antigen-binding fragment and a one or more other therapeutic agents.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents are administered simultaneously.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents can be administered in the same or different manner, or formulated as an anti-VISTA antibody or administered in combination with an antigen-binding fragment and one or more other therapeutic agents.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents are administered separately.
  • an anti-VISTA antibody or antigen-binding fragment is administered prior to one or more other therapeutic agents.
  • the anti-VISTA antibody or antigen-binding fragment is administered after one or more other therapeutic agents.
  • the invention provides a composition comprising an anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents.
  • the invention provides a method of preventing, treating or ameliorating a T cell dysfunction disorder, the method comprising administering to a patient an effective dose of an anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents. combination.
  • the additional therapeutic agent is an angiogenesis inhibitor, anti-cancer agent, targeted drug, or therapeutic antibody.
  • the anti-VISTA antibody or antigen-binding fragment specifically binds VISTA and includes one or more CDRs, such as 1, 2, 3, 4, 5, or 6 CDRs:
  • HCDR1 which contains or consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 which contains or consists of the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:2;
  • HCDR3 which contains or consists of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:3;
  • LCDR1 which contains or consists of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:4;
  • LCDR2 which contains or consists of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:5;
  • LCDR3 which contains or consists of the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:6.
  • the anti-VISTA antibody or antigen-binding fragment comprises:
  • HCDR1 which contains or consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO:2 or compared to SEQ ID NO:2 Amino acid sequences having or consisting of single-site substitutions, deletions or insertions;
  • HCDR3 which comprises or consists of an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:3.
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:1
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:2
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO:3. Amino acid sequence or consisting of.
  • the anti-VISTA antibody or antigen-binding fragment comprises:
  • LCDR1 which contains or consists of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence with a single site substitution, deletion or insertion compared to SEQ ID NO:4;
  • LCDR2 which comprises or consists of an amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:5;
  • LCDR3 which contains or consists of the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:6.
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:4
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:5
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO:6 Amino acid sequence or consisting of.
  • the anti-VISTA antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:2, HCDR3 as set forth in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 and LCDR3 as shown in SEQ ID NO:6.
  • the substitutions are conservative amino acid substitutions.
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 7 or 8, or identical to SEQ ID NO: 7 or A sequence having at least 80% identity compared to the sequence shown in SEQ ID NO: 8, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 7 or 8, or consisting of it.
  • the anti-VISTA antibody or antigen-binding fragment comprises a light chain variable region comprising a sequence set forth in SEQ ID NO: 9 that is consistent with the sequence set forth in SEQ ID NO: 9 A sequence having at least 80% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 9, or consisting of it.
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 7,
  • the light chain variable region contains the sequence shown in SEQ ID NO:9.
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region.
  • Variable region wherein the heavy chain variable region includes the sequence shown in SEQ ID NO:8, and the light chain variable region includes the sequence shown in SEQ ID NO:9.
  • the anti-VISTA antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
  • the light chain constant region is a kappa or lambda chain constant region.
  • the antibody or antigen-binding fragment thereof is one of the isotypes of IgG, IgM, IgA, IgE or IgD; preferably, the isotype is IgGl, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the Fc is a variant Fc region.
  • a variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region.
  • amino acid modifications to the Fc region alter the effector function activity relative to the activity of the parent Fc region.
  • a variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding .
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-mediated cytotoxicity
  • phagocytosis opsonization
  • Fc region amino acid modifications can alter the affinity of a variant Fc region for Fc ⁇ R (Fc ⁇ receptor) relative to the parent Fc region.
  • the Fc region is derived from IgGl or IgG4.
  • the Fc region mutation is the N297A mutation relative to the IgG1 subtype.
  • the anti-VISTA antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFv, Fab or F(ab) 2 .
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 10, or a sequence set forth in SEQ ID NO: 10 A sequence that has at least 80% identity compared to the sequence, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 10, or consists of; and/or
  • the anti-VISTA antibody or antigen-binding fragment includes a light chain constant region, the light chain constant region includes an amino acid sequence as shown in SEQ ID NO: 11, or has an amino acid sequence that is at least 80% higher than the sequence set forth in SEQ ID NO: 11. % identity sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or consisting of it.
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 10,
  • the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 11.
  • the anti-VISTA antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 12 or 13 that is identical to the sequence set forth in SEQ ID NO: 12 or 13 or consists of an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence having at least 80% identity, or having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 12 or 13; and/or
  • the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 14, a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 14, or An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 14, or consisting of it.
  • the heavy chain of the anti-VISTA antibody comprises the amino acid sequence set forth in SEQ ID NO: 12 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 14.
  • the heavy chain of the anti-VISTA antibody comprises the amino acid sequence set forth in SEQ ID NO:13 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the anti-VISTA antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody or antigen-binding fragment (e.g., a bispecific antibody or antigen-binding fragment ).
  • the anti-VISTA antibody has two sequence-identical heavy chains and two sequence-identical light chains, with the Fc regions paired to form disulfide bonds.
  • Anti-VISTA antibodies or antigen-binding fragments of the present invention are disclosed in WO2022/206677.
  • the additional therapeutic agent is selected from the group consisting of antibodies or antigen-binding fragments or antibody drug conjugates directed against: PD-L1, PD-1, CD3, such as anti-CD3 antibodies or anti-PD-L1 antibodies. or antigen-binding fragments.
  • the anti-PD-L1 antibody is Atezolizumab, Durvalumab, Avelumab, Envafolimab .
  • the anti-PD-L1 antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 24-26, respectively, and amino acid sequences as set forth in SEQ ID NOs: 27-29, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence set forth in positions 20-137 of SEQ ID NO: 19, or A sequence that is at least 80% identical to positions 20-137 of SEQ ID NO: 19, or has one or more conservative amino acid substitutions compared to positions 20-137 of SEQ ID NO: 19; and,
  • the anti-PD-L1 antibody or antigen-binding fragment includes a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in positions 23-129 of SEQ ID NO:20, or is the same as SEQ ID NO:20 A sequence with at least 80% identity compared to positions 23-129 of SEQ ID NO: 20, or an amino acid sequence with one or more conservative amino acid substitutions compared to positions 23-129 of SEQ ID NO:20.
  • the heavy chain amino acid sequence of the anti-PD-L1 antibody is as shown in positions 20-467 of SEQ ID NO:19; the light chain amino acid sequence of the anti-PD-L1 antibody is as shown in SEQ ID NO: Bits 23-236 of 20 are shown.
  • the anti-VISTA antibody or antigen-binding fragment and the other therapeutic agent are administered separately. In some embodiments, the anti-VISTA antibody or antigen-binding fragment and the other therapeutic agent are administered simultaneously.
  • the uses, methods, and compositions of the invention can be used to prevent, treat, or ameliorate T cell dysfunction disorders.
  • the T cell dysfunction condition includes tumor, cancer, or infection.
  • the anti-VISTA antibody is administered at a dose of 1-3000 mg per treatment cycle; alternatively, the anti-VISTA antibody is administered at a dose of 0.01-100 mg/kg of patient body weight per treatment cycle, Or administered at a dose of 0.01 mg/kg-40 mg/kg of patient body weight, or administered at a dose of 0.01 mg/kg-30 mg/kg of patient body weight, or administered at a dose of 0.01 mg/kg-20 mg/kg of patient body weight.
  • the anti-VISTA antibody is administered at a dose of 0.01-100 mg/kg patient body weight, or at a dose of 0.1 mg/kg-40 mg/kg patient body weight, or at 0.1 mg/kg- Or,
  • the anti-PD-L1 antibody is administered at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight, or 0.1 mg/kg to 20 mg/kg of the patient's body weight in each cycle.
  • a therapeutically effective amount of an anti-VISTA antibody or antigen-binding fragment and an anti-PD-L1 antibody or antigen-binding fragment are administered to the patient separately or simultaneously.
  • the administration cycles of the anti-VISTA antibody or antigen-binding fragment and the anti-PD-L1 antibody or antigen-binding fragment can be the same or different.
  • the T cell dysfunction disorders include, but are not limited to, infections caused by bacteria, viruses, fungi, or protozoa, as well as cancer and tumors.
  • the cancers and tumors include, but are not limited to, breast cancer, gastrointestinal/gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, genitourinary cancer, germ cell cancer, gynecological cancer, head and neck cancer, Hematology/Blood Cancer, Musculoskeletal Cancer, Neurological Cancer, Respiratory Cancer/Thorax Cancer, Bladder Cancer, Colon Cancer, Rectal Cancer, Colorectal Cancer, Lung Cancer, Endometrial Cancer, Kidney Cancer, Pancreatic Cancer, Liver Cancer, Stomach Cancer, Testicular Cancer cancer, esophageal cancer, prostate cancer, brain cancer, cervical cancer, ovarian cancer and thyroid cancer.
  • cancers and tumors include, but are not limited to, leukemia, melanoma, and lymphoma.
  • leukemias include, but are not limited to, lymphocytic leukemia or myeloid leukemia (such as, for example, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML) ), chronic myeloid leukemia (CML)), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, or adult T-cell leukemia.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • hairy cell leukemia T-cell prolymphocytic leukemia
  • large granular lymphocytic leukemia or adult T-cell leukemia.
  • lymphomas include, but are not limited to, histiocytic lymphoma, follicular lymphoma, and Hodgkin lymphoma.
  • infections include, but are not limited to, chronic infectious diseases such as HIV, HBV, HCV, HSV, and the like.
  • the uses, methods and compositions of the present invention help the immune system to eliminate tumor cells and can be used to prevent, treat or improve T cell dysfunction conditions, such as cancer or tumors.
  • Figure 1 is the SDS-PAGE pattern of the anti-VISTA antibody in Example 1 of the present invention; wherein, lane M represents the molecular marker marker, lane 1 represents the antibody P48-6-K, lane 2 represents the antibody P48-6-T, and lane 3 Represents antibody VSTB112.
  • Figure 2 shows that anti-VISTA antibodies block the binding of VSIG-3 to VISTA.
  • Figure 3 shows that anti-VISTA antibodies promote T cell proliferation; in the figure, ** represents p ⁇ 0.01, and **** represents p ⁇ 0.0001.
  • Figure 4A shows the effect of antibodies on tumor volume
  • Figure 4B shows the effect of antibodies on tumor weight
  • * represents p ⁇ 0.05
  • ** represents p ⁇ 0.01
  • *** represents p ⁇ 0.001
  • ns represents no significant sexual differences.
  • Figure 5 shows the effect of antibodies on mouse body weight.
  • an entity refers to one or more such entities, e.g. "an antibody” should be understood to mean one or more antibodies, therefore, the term “a” (or “an” ), “one or more” and “at least one” may be used interchangeably herein.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any single chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein,” “amino acid chain” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” may Used instead of or interchangeably with any of the above terms.
  • polypeptide is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence and may be produced by any means including chemical synthesis.
  • Amino acid refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code.”
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine Acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to materially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine.
  • Amide-containing side chains asparagine and glutamine
  • Aromatic side chains phenylalanine, tyrosine and tryptophan
  • Basic side chains lysine, Arginine and histidine
  • Acidic side chains aspartic acid and glutamic acid.
  • the number of amino acids of "conservative amino acid substitutions of VL and VH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values, inclusive, or any value therein.
  • the number of amino acids in the "heavy chain constant region, light chain constant region, heavy chain or light chain conservative amino acid substitution" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29 , about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive of the endpoints) or any value therein.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., such as “isolated” DNA, RNA, polypeptides, and antibodies, refers to other components in the natural environment of cells, such as DNA or RNA. one or more separated molecules.
  • isolated as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not exist in their native state and do not exist in their native state.
  • isolated is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides, antibodies, etc. are generally prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or any of these values. The range between any two values (inclusive) or any value within them.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may be combined to produce polynucleotides that do not normally exist or Peptides.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • At least 80% identical means approximately 80% identical, approximately 81% identical, approximately 82% identical, approximately 83% identical Sex, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, About 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (inclusive of the endpoints) or any value therein.
  • a polynucleotide or polynucleotide sequence has a certain percentage (eg, 90%, 95%, 98% or 99%) of "identity or sequence identity” to another sequence It refers to the percentage of bases (or amino acids) in the two sequences being compared that are identical when the sequences are compared.
  • the alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007), Current Protocols in Molecular Biology. It is preferred to use the default parameters for comparison.
  • Biologically equivalent polynucleotides are polynucleotides that share the percentage identity specified above and encode a polypeptide with the same or similar biological activity.
  • encoding when applied to a polynucleotide, refers to a polynucleotide that is said to "encode” a polypeptide that, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation can produce the polypeptide and/or fragments thereof.
  • the antibodies and antigen-binding fragments disclosed in the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, fully human antibodies, humanized antibodies, primatized antibodies, chimeric antibodies, single-chain antibodies, Epitope binding fragments (eg Fab, Fab' and F(ab') 2 ), single chain Fvs (scFv).
  • polyclonal antibodies monoclonal antibodies, multispecific antibodies, fully human antibodies, humanized antibodies, primatized antibodies, chimeric antibodies, single-chain antibodies, Epitope binding fragments (eg Fab, Fab' and F(ab') 2 ), single chain Fvs (scFv).
  • Antibody and antigen-binding fragment refer to polypeptides or polypeptide complexes that specifically recognize and bind to antigens.
  • Antibodies can be complete antibodies, any antigen-binding fragments thereof, or single chains thereof.
  • the term "antibody” thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs) of heavy or light chains or their ligand-binding portions, heavy chain variable regions (VH), light chain variable regions (VL), and heavy chain constant regions.
  • CDRs complementarity determining regions
  • the CDR region includes the CDR region of the light chain (LCDR1-3) and the CDR region of the heavy chain (HCDR1-3).
  • Antibodies and antigen-binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes of one or more (eg, two) antigens.
  • Antibodies or antigen-binding fragments that specifically recognize and bind multiple (eg, two) antigens may be referred to as multispecific (eg, bispecific) antibodies or antigen-binding fragments.
  • antibody fragment refers to a part of an antibody.
  • the composition of the antibody fragment of the present invention can be similar to F(ab') 2 , F(ab) 2 , Fab', Fab in monospecific antibody fragments. , Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody.
  • antibody fragment includes aptamers, mirrors Like isoforms and diabodies.
  • antigen-binding fragment also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
  • Single chain variable fragment refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and rich in serine or threonine to increase solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Although the protein has had the constant region removed and linkers introduced, it retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in US Patent 5,892,019.
  • antibodies includes a wide variety of biochemically distinguishable polypeptides. Those skilled in the art will understand that classes of heavy chains include gamma, mu, alpha, delta or epsilon (gamma, mu, alpha, delta, epsilon), of which there are also subclasses (eg ⁇ 1- ⁇ 4). The nature of this chain determines the "class" of the antibody: IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and the functional specificities conferred are known. All immunoglobulin species are within the scope of the invention. In some embodiments, the immunoglobulin molecule is of the IgG class.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be combined with a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequence extends from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the immunoglobulin lambda light chain is V ⁇ .
  • Both light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used according to function.
  • the light chain variable region (VL) and heavy chain variable region (VH) partially determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antibody's antigen-binding site, or amino terminus.
  • the N-terminal part is the variable region and the C-terminal part is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains respectively.
  • each antigen-binding domain is short, short, “complementarity-determining regions” or “CDRs” that form the antigen-binding domain, assuming the antibody assumes its three-dimensional configuration in an aqueous environment.
  • the remaining other amino acids in the antigen-binding domain known as the "framework” region, show less inter-molecular variability.
  • Most of the framework region adopts a ⁇ -sheet conformation, and the CDRs form a ring structure connected to it, or in some cases form part of the ⁇ -sheet structure.
  • the framework region forms a scaffold that positions the CDR in the correct orientation through non-covalent interactions between chains.
  • the antigen-binding domain with CDRs in specific positions forms a surface complementary to the epitope on the antigen that promotes non-covalent binding of the antibody to its epitope.
  • those of ordinary skill in the art can use known methods to Amino acids containing CDRs and framework regions were identified (see Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196 :901-917(1987)).
  • CDR complementarity determining region
  • CDRs defined according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbering comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDR contains based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself.
  • “Kabat number” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983).
  • Antibodies can also use the EU or Chothia numbering system.
  • the antibodies disclosed herein can be derived from any animal, including birds and mammals.
  • the antibody is a human, mouse, donkey, rabbit, goat, camel, llama, horse or chicken antibody.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • Heavy chain constant region includes at least one of a CH1 domain, a hinge (eg, upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the heavy chain constant region of an antibody can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG 1 molecule and a hinge region derived from an IgG 3 molecule.
  • the heavy chain constant region may comprise a hinge region derived in part from an IgG 1 molecule and in part from an IgG 3 molecule.
  • a portion of the heavy chain may comprise a chimeric hinge region derived in part from an IgG 1 molecule and in part from an IgG 4 molecule.
  • Light chain constant region includes a portion of the amino acid sequence derived from an antibody light chain.
  • the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.
  • a "light chain-heavy chain pair” refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.
  • a "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • a "CH1 domain” includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain.
  • Two CH2 junctions in the intact natural IgG molecule Each N297 in the domain is connected to a branched carbohydrate chain.
  • the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • the "hinge region” includes the portion of the heavy chain region that connects the CH1 domain and the CH2 domain.
  • the hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.
  • the hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).
  • the antibody or antigen-binding fragment comprises an amino acid sequence having one or more modifying groups.
  • antibodies or antigen-binding fragments may contain flexible linker sequences, or may be modified to add functional groups (eg, PEG, drugs, toxins, or tags).
  • Antibodies and antigen-binding fragments include modified derivatives, that is, modified by covalent linkage of any type of molecule to the antibody or antigen-binding fragment, where the covalent linkage does not prevent the antibody or antigen-binding fragment from binding to the epitope.
  • antibodies or antigen-binding fragments can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to Cell ligands or other proteins, etc. Any of numerous chemical modifications can be performed by existing technologies, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • Antibodies or antigen-binding fragments can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • Antibodies or antigen-binding fragments can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody or antigen-binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of a cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by disulfide bonds.
  • a “chimeric antibody” refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial, or modified) are derived from a second species.
  • the variable regions are from a non-human source (eg, mouse or primate) and the constant regions are from a human source.
  • Specific binding or “specific for” generally refers to the formation of a relatively stable complex between an antibody or an antigen-binding fragment and a specific antigen through complementary binding of its antigen-binding domain to an epitope.
  • Specificity can be expressed in terms of the relative affinity of an antibody or antigen-binding fragment for binding to a particular antigen or epitope. For example, if antibody “A” has a greater relative affinity for the same antigen than antibody “B”, antibody “A” can be considered to have higher specificity for that antigen than antibody "B”.
  • Specific binding can be described by the equilibrium dissociation constant (K D ), with a smaller K D meaning tighter binding.
  • Antibodies that "specifically bind" antigen a include those with an equilibrium dissociation constant K D for antigen a of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM, or less than or equal to about 0.5 nM. Antibody.
  • Treatment means therapeutic treatment and prophylactic or preventative measures designed to prevent, slow down, ameliorate or halt adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or undetectable
  • the results include alleviation of symptoms, reduction in disease severity, stabilization of disease status (i.e. no worsening), delay or slowdown of disease progression, improvement, alleviation, reduction or disappearance of disease status (whether partial or complete), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include patients who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of a condition or disorder for which they can or are expected to benefit from administration of an antibody or pharmaceutical composition disclosed herein for detection. , patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal for whom diagnosis, prognosis or treatment is required, including humans, dogs, cats, rabbits, rats, horses, cattle, etc.
  • the present invention provides antibodies or antigen-binding fragments thereof with high affinity for VISTA protein.
  • the antibodies or antigen-binding fragments of the present invention exhibit effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints, activate lymphocytes to release cytokines, and are used to treat various types of cancer, tumors, or infection-related diseases.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:12 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the heavy chain of the antigen-binding fragment comprises the amino acid sequence SEQ ID NO:12 except Fc, and the light chain of the antigen-binding fragment comprises the amino acid sequence SEQ ID NO:14. .
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:13 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the heavy chain of the antigen-binding fragment comprises the amino acid sequence SEQ ID NO:13 except Fc, and the light chain of the antigen-binding fragment comprises the amino acid sequence SEQ ID NO:14. .
  • an antibody of the invention contains two heavy chains (or heavy chain fragments) with identical sequences and two light chains (or light chain fragments) with identical sequences.
  • the sequences of the antibodies or antigen-binding fragments disclosed in the present invention can be replaced, and the amino acid sequence after the substitution is different from the naturally occurring amino acid sequence of the antibody.
  • the replaced amino acid sequence can be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence, for example, it can be about 80%, about 85%, or about 90% identical to the starting sequence. , about 95%, about 98%, about 99%, or a range between any two of these values (inclusive of the endpoints) or any value therein.
  • antibodies are produced that do not elicit a deleterious immune response in the animal to be treated (eg, a human).
  • the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified using art-recognized techniques to reduce their immunogenicity.
  • antibodies can be humanized, primatized, deimmunized immunization or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • CDRs complementarity-determining regions
  • framework substitutions can be identified by methods well known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues that are important for antigen binding and by sequence comparison to identify aberrant framework residues at specific positions. (See U.S. Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference).
  • Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596; Padlan, et al., Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); Roguska, et al., Proc.Natl Sci. USA 91:969-973 (1994)), and Chain Rearrangement (U.S. Patent 5,565,332), the entire contents of which are incorporated herein by reference.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering the antibody to modify a T cell epitope (see, eg, WO/9852976A1 and WO/0034317A2).
  • the heavy chain variable region sequence and the light chain variable region sequence from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated, showing the epitopes relative to complementarity determining regions (CDRs) and the location of other key residues within the sequence.
  • CDRs complementarity determining regions
  • a series of alternative heavy chain variable domain sequences and light chain variable domain sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides.
  • Genes containing the complete heavy and light chains with modified variable regions and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies.
  • the antibodies are then compared in appropriate biochemical and biological experiments to identify the optimal antibody.
  • the binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be tested by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFv The preparation of scFv can be found in the technology for producing single-chain units (U.S. Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 55: 5879-5883 (1988) and Ward et al., Nature 334:544-554 (1989) and Nie et al., Antibody Therapeutics 3(1):18-62 (2020)).
  • Single-chain units are formed by bridging the heavy and light chain segments of the Fv region with amino acids, resulting in a single-chain fusion peptide. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041(1988)).
  • scFv single chain Fv
  • antibodies include, for example, U.S. Patents 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993) and Skerra et al., Science 240:1038-1040 (1988).
  • chimeric, humanized, or fully human antibodies may be used.
  • Chimeric antibodies are a class of molecules in which different parts of the antibody are derived from different animal species, such as an antibody that has the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 -202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated by reference. Incorporated herein.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. Reference may also be made to U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, each The entire contents of the patent are incorporated herein by reference.
  • DNA encoding the desired monoclonal antibody can be isolated and analyzed using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding murine antibody heavy and light chains). Sequencing. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. in cells.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. in cells.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare sequences for the constant and variable regions of antibodies, as described in U.S. Patent 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR technology using Ig-specific primers. Suitable probes for this purpose are also described in US Patent 5,658,570.
  • one or more CDRs of the antibody of the invention can be inserted into the framework region, for example, into the human framework region to construct a humanized non-fully human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human framework regions).
  • Some polynucleotides may encode a combination of framework regions and CDRs to produce an antibody that specifically binds to at least one epitope of the target antigen.
  • One or more amino acid substitutions may be made within the framework regions, Amino acid substitutions can be selected that improve the binding of the antibody to its antigen.
  • this method can be used to substitute or delete cysteine residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby generating antibody molecules lacking one or more interchain disulfide bonds.
  • Other modifications to polynucleotides within the skill of the art are also encompassed by the present invention.
  • Antibodies can be produced using conventional recombinant DNA techniques.
  • Vectors and cell lines for producing antibodies can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the full text of which is included Supplementary content is incorporated by reference into the full text.
  • DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein according to conventional methods, placed into an expression vector, and then transfected into host cells, and the transfected host cells are cultured in culture medium to produce Monoclonal antibodies.
  • an expression antibody vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus. Promoters from other cells such as muscle can also be used. Kinesin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.
  • the inserted gene fragment needs to contain a selection marker.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection. Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired protein.
  • mutations can be introduced into the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutations.
  • Variants including derivatives encoding substitutions of less than 50 amino acids, substitutions of less than 40 amino acids, substitutions of less than 30 amino acids, substitutions of less than 30 amino acids, relative to the original heavy chain variable region and light chain variable region Substitutions of 25 amino acids, substitutions of less than 20 amino acids, substitutions of less than 15 amino acids, substitutions of less than 10 amino acids, substitutions of less than 5 amino acids, substitutions of less than 4 amino acids, substitutions of less than 3 Substitution of amino acids or substitution of less than 2 amino acids.
  • mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • the present invention also provides methods and uses of treatment.
  • methods are provided for treating or ameliorating various types of cancer, tumors, or infection-related diseases, comprising administering to a patient in need thereof an effective dose of an anti-VISTA antibody or antigen-binding fragment and a one or more other therapeutic agents.
  • the use of an anti-VISTA antibody or antigen-binding fragment in combination with one or more other therapeutic agents for treating or ameliorating cancer, tumors, or infections and other related diseases is provided.
  • the use of the anti-VISTA antibody or antigen-binding fragment together with one or more other therapeutic agents in the preparation of a medicament for treating or ameliorating cancer, tumors, or infection-related diseases is provided.
  • the other therapeutic agents are as described herein.
  • the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or derivative used, the patient's age and weight, general health, sex, and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. Judgment of these factors is left to a medical practitioner, including those with ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the desired effect.
  • the dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • the anti-VISTA antibody is administered to the patient at a dose of 0.01 mg/kg to 100 mg/kg of patient body weight per administration. In some embodiments, administration is every 1 week, 2 weeks, 3 weeks, or monthly. In some embodiments, the dosage of the antibody is 0.1-20 mg/kg per administration.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents are administered simultaneously.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents can be administered in the same or different manner, or formulated as an anti-VISTA antibody or administered in combination with an antigen-binding fragment and one or more other therapeutic agents.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents are administered separately, either by the same or different means.
  • Methods of administration of the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents independently include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection.
  • the pharmaceutical composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelium or skin mucosa (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.
  • compositions containing the antibodies or antigen-binding fragments of the invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, externally (e.g., via powder, ointment) , drops or transdermal patches), orally, or by oral or nasal spray.
  • parenteral refers to intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and Intra-articular injection and infusion modes of administration.
  • the mode of administration may be systemic or local.
  • Pulmonary administration may also be performed, for example by use of an inhaler or nebulizer, and with aerosolized formulations.
  • the anti-VISTA antibody or antigen-binding fragment and one or more other therapeutic agents may be independently administered topically to the area in need of treatment; by, but not limited to, topical application during surgery, for example, in combination with a post-operative wound dressing. , by injection, by catheter, by means of suppositories or by means of implants, which are porous, non-porous or gel-like materials, including membranes (eg silicone rubber membranes) or fibers.
  • proteins of the invention including antibodies
  • care must be taken to use materials that do not absorb the protein.
  • compositions of the present invention comprise a nucleic acid or polynucleotide encoding an antibody that can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to promote expression of the protein it encodes, The partial vector is then administered to render it intracellular, for example by using a retroviral vector (see U.S.
  • Patent 4,980,286) or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, Dupont) , or coated with lipids or cell surface receptors or transfection reagents, or administered via linkage to homeobox peptides known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci .USA 88:1864-1868) and so on.
  • the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
  • the anti-VISTA antibody is administered to the patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight, or 0.1 mg/kg to 20 mg/kg of the patient's body weight.
  • the initial dose may be followed by a second or more doses of the antibody or antigen-binding fragment at approximately the same or less than the initial dose, wherein such subsequent doses may be separated by at least 1 to 3 days; or at least one week.
  • the dose and frequency of administration of the antibodies of the invention can be reduced by enhancing the uptake and tissue penetration (eg into the brain) of the antibody through modifications such as lipidation.
  • Anti-VISTA antibodies are used in a method for testing in vitro for the treatment of disease, comprising administering an anti-VISTA antibody or derivative of the invention, then testing the desired therapeutic or preventive activity in vivo in an acceptable animal model, and finally administering to human body.
  • Suitable animal models including transgenic animals, are known to those of ordinary skill in the art.
  • in vitro assays used to demonstrate the therapeutic use of anti-VISTA antibodies, antigen-binding fragments of the invention include the effects of the antibodies on cell lines or patient tissue samples. The effects of antibodies on cell lines and/or tissue samples can be tested using techniques known to those skilled in the art, such as those disclosed elsewhere herein.
  • in vitro assays that may be used to determine whether to administer a specific antibody include in vitro cell culture experiments in which patient tissue samples are grown in culture and exposed to or otherwise administered a compound, and the effect of such compound is observed. Effects of tissue samples.
  • Various known delivery systems can be used to administer the antibodies or derivatives of the invention or polynucleotides encoding the same, e.g. encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated Endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
  • PD-1 Programmed death receptor-1
  • PD-1 is a type I transmembrane glycoprotein with a molecular weight of approximately 55kDa.
  • PD-1 is an immunosuppressive receptor expressed on activated T cells, B cells, and myeloid cells, and is a member of the CD28 immunoglobulin superfamily.
  • PD-L1 is also a type I transmembrane glycoprotein that is widely distributed: expressed on the surface of antigen-presenting cells such as B cells, T cells, dendritic cells, macrophages, and tumors. in the organization.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment described herein for the preparation of a medicament for use in combination with an anti-PD-L1 antibody or antigen-binding fragment.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment and an anti-PD-L1 antibody or antigen-binding fragment described herein in the preparation of a medicament for treating a disease.
  • the invention provides the use of an anti-VISTA antibody or antigen-binding fragment described herein in combination with an anti-PD-L1 antibody or antigen-binding fragment in the treatment of a disease.
  • the invention provides a method of treating a disease, such as a method of preventing, treating or ameliorating a T cell dysfunction disorder, comprising administering to a patient in need of treatment an effective dose of an anti-VISTA antibody or antigen-binding fragment described herein and an anti-PD -L1 antibody or antigen-binding fragment.
  • the anti-VISTA antibody or antigen-binding fragment and the anti-PD-L1 antibody are administered simultaneously.
  • the anti-VISTA antibody or antigen-binding fragment and the anti-PD-L1 antibody or antigen-binding fragment are administered simultaneously, the anti-VISTA antibody or antigen-binding fragment and the anti-PD-L1 antibody or antigen-binding fragment can be administered in the same or different ways, or made into anti-VISTA antibodies or antigen-binding fragments.
  • a combination of a VISTA antibody or antigen-binding fragment and an anti-PD-L1 antibody or antigen-binding fragment is administered.
  • the anti-VISTA antibody or antigen-binding fragment and the anti-PD-L1 antibody or antigen-binding fragment are administered separately. In some embodiments, the anti-VISTA antibody or antigen-binding fragment is administered before the anti-PD-L1 antibody or antigen-binding fragment. In some embodiments, the anti-VISTA antibody or antigen-binding fragment is administered after the anti-PD-L1 antibody or antigen-binding fragment.
  • the invention provides a composition comprising an anti-VISTA antibody or antigen-binding fragment described herein and an anti-PD-L1 antibody or antigen-binding fragment.
  • the invention provides a method of preventing, treating or ameliorating a T cell dysfunction disorder, the method comprising administering to a patient an effective dose of an anti-VISTA antibody or antigen-binding fragment described herein and an anti-PD-L1 antibody. or compositions of antigen-binding fragments.
  • an anti-VISTA antibody or antigen-binding fragment used in combination with A PD-L1 antibody is an anti-PD-L1 antibody or antigen-binding fragment described herein.
  • uses, methods, and compositions of the present invention in combination with anti-VISTA antibodies or antigen-binding fragments and anti-PD-L1 antibodies or antigen-binding fragments can also be combined with other therapeutic or preventive regimens, including administration of one or more An antibody or antigen-binding fragment of the invention, an anti-PD-L1 antibody or antigen-binding fragment, and one or more other therapeutic agents or methods are used together or in combination.
  • other treatment options include, but are not limited to, radiation therapy, chemotherapy, hormonal therapy, biologic therapy, and the like.
  • the therapeutic agents may be administered simultaneously or separately. When administered separately, these therapeutic agents can be administered in different orders.
  • therapeutic antibodies that can be administered with methods, uses or compositions in combination with the antibodies or antigen-binding fragments of the invention and anti-PD-L1 antibodies or antigen-binding fragments include, but are not limited to, anti-CD3 antibodies, anti-PD -1 antibody, anti-PD-L2 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, anti-OX40 antibody and anti-GITR antibody, etc., such as atezolizumab, avelumab, Druvalumab, Envafolimab, nivolumab, pembrolizumab, tremelimumab, ipilimumab, trastuzumab.
  • angiogenesis inhibitors that can be administered with methods, uses, or compositions in combination with the antibodies or antigen-binding fragments of the invention and anti-PD-L1 antibodies or antigen-binding fragments include, but are not limited to, angiostatin (fibrin proteolytic zymogen fragment), anti-angiogenic antithrombin III and ribozymes.
  • anti-cancer agents that can be administered with methods, uses or compositions in combination with the antibodies or antigen-binding fragments of the invention and anti-PD-L1 antibodies or antigen-binding fragments include, but are not limited to: 5-fluorouracil, acetate Visine, aldesleukin, hexamethonium, aminoglutethimide, amsacridine, anastrozole, antromycin, asparaginase, azacitidine, azatepa, azomycin, Bama Sistat, bicalutamide, bleomycin sulfate, buquina sodium, bromipirimin, busulfan, carboplatin, carmustine, carrubicin hydrochloride, carzolexin, sildifen Ge, chlorambucil, siromycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytar
  • compositions comprise an effective dose of an anti-VISTA antibody or antigen-binding fragment, one or more other therapeutic agents, and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprise 0.1%-90% anti-VISTA antibody or antigen-binding fragment.
  • the term “pharmaceutically acceptable” refers to substances approved by regulatory agencies of the government or listed in recognized pharmacopeias for use in animals, particularly humans.
  • pharmaceutically acceptable carrier generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary, etc.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient can be administered to a patient.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc.
  • the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antimicrobial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity-adjusting agents such as sodium chloride or dextrose are also contemplated.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, and the like.
  • the compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a dosage form suitable for the patient.
  • the formulation should be suitable for the mode of administration.
  • the parent preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into humans.
  • Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer.
  • the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied singly or mixed together in unit dosage form, such as as a dry lyophilized powder or anhydrous concentrate in sealed containers (such as ampoules or sachets) indicating the quantity of active agent.
  • the composition may be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline.
  • ampoules of sterile water or saline for injection may be used, allowing the active ingredients to be mixed prior to administration.
  • the compounds of the invention may be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include salts with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2- Salts formed with cations of ethylaminoethanol, histidine, procaine, etc.
  • EC 50 is the concentration for 50% of maximal effect (EC 50 ), which refers to the concentration that can cause 50% of the maximum effect.
  • the "parental Fc region” can be a naturally occurring Fc region, and the gene encoding the Fc region can be from humans, mice, rabbits, camels, and monkeys, preferably humans and mice; for example, the parent Fc region is SEQ ID NO: 10 , SEQ ID NO:12 or the Fc region in SEQ ID NO:13.
  • the antibodies prepared in this example include anti-VISTA antibodies and anti-PD-L1 antibodies.
  • the amino acid sequences and nucleic acid sequences related to the antibodies are shown in Tables 1 to 6.
  • the amino acid sequences of the heavy chain and light chain of the antibody are sequence optimized according to the codon preference characteristics of the host cell to obtain the DNA sequences of the heavy chain and light chain.
  • a signal peptide can be added to the N-terminus of the heavy and light chains.
  • the optimized and synthesized nucleic acid sequence clones are cloned into vectors (the vector can be pCDNA3.1, V79020 from Invitrogen), and then a large amount of plasmids are extracted respectively.
  • the heavy chain and light chain are transiently expressed and transformed at a plasmid molar ratio of 1:1. Stained HEK293F cells.
  • the heavy chain nucleic acid sequence (positions 58-1407 of SEQ ID NO: 18) and the light chain nucleic acid sequence (positions 61-702 of SEQ ID NO: 18) were cloned into the expression vector through enzyme digestion; wherein, the amino acid sequence of the heavy chain signal peptide is SEQ ID NO: 15, The amino acid sequence of the light chain signal peptide is SEQ ID NO:16.
  • the heavy chain nucleic acid sequence (heavy chain variable region nucleotide sequence such as positions 58-411 of SEQ ID NO: 21 shown) and the light chain nucleic acid sequence (the nucleotide sequence of the light chain variable region is shown in positions 67-387 of SEQ ID NO: 22) was cloned into the expression vector through enzyme digestion; a signal peptide sequence was added, in which the heavy The amino acid sequence of the chain signal peptide is SEQ ID NO:15, and the amino acid sequence of the light chain signal peptide is amino acids 1-22 of SEQ ID NO:20.
  • the positive reference antibody VSTB112 is an anti-VISTA antibody, and its preparation method is similar to the above (see patent application CN107922497A for the sequence). After cell expression, the culture medium was purified by immobilized metal affinity chromatography (IMAC) using a Protein A column (GE Healthcare). The purity of the purified antibody protein was >95%.
  • IMAC immobilized metal affinity chromatography
  • the purified antibodies were tested by gel electrophoresis. As shown in Figure 1, the antibodies P48-6-K and P48-6-T of the present application are single substances, and their molecular weights are consistent with the theoretical values. The purified antibody was sequenced, and the sequencing results were identical to the expected sequence. Purified antibodies are used for affinity detection and biological activity identification.
  • the Biacore T200 surface plasmon resonance instrument was used to measure the affinity constant of the antibody.
  • the main test process was as follows: Protein A chip was used for detection, and the 100nM antibody (P48-6-K, P48-6-T, VSTB112) dilution was diluted at 10 ⁇ l/min.
  • the flow rate passes through the experimental flow path (Fc2, Fc4), and the capture volume is about 560RU after 20 seconds of capture; then the flow rate is adjusted to 30 ⁇ l/min, and different concentrations (0nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, 100nM) are added in sequence.
  • VISTA-His (Acro, B75-H52H0) dilution (the diluted solvent is water) passes through the surfaces of the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time, with a binding time of 120s and a dissociation time of 300s.
  • the antibody P48-6-T of the present invention has better affinity with VISTA-His.
  • VISTA-Fc human, cynomolgus monkey, mouse species, Sino biological Inc
  • the starting concentration of anti-VISTA antibody is 2 ⁇ g/mL, 2-fold gradient dilution (the dilution solvent is PBS Buffer), place in the refrigerator at 4°C overnight
  • PBST PBS+0.05% Tween, 1 liter of PBS contains NaCl 8.0g, Na 2 HPO 4 0.9g, KH 2 PO 4 0.156g, KCl 0.125g, pH 7.2-7.4)
  • BSA bovine serum albumin
  • antibodies VSTB112, P48-6-K, and P48-6-T bind to human and cynomolgus monkey VISTA, but do not bind to mouse VISTA.
  • NA means not bound.
  • the preparation method of CHO-VISTA cells is: the full-length amino acid sequence of human VISTA (from UniProtKB-Q9H7M9): "MGVPTALEEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRH HHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYK QRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI" (SEQ ID NO:23); synthesize the nucleic acid sequence corresponding to SEQ ID NO:23, add HindIII and EcoRI restriction sites at both ends of the sequence
  • the electroporation conditions were: voltage 300V, time 17 milliseconds, 4mm electroporation cup. After 48 hours, 50 ⁇ M MSX (methionine iminosulfone) was added to select positive cells. FACS (flow cytometry) was used (Technology) detection, screen the highly expressing cell lines and collect the cells. After washing once with PBS, add 3 ⁇ g/ml antibody VSTB112, incubate at 4°C for 1 hour, wash twice with PBS, and then add 100 ⁇ l of goat anti-human antibody diluted at 1:500. IgG-Fc PE fluorescent secondary antibody (Cat. No. 12-4998-82, eBioscience), incubated at 4°C for 1 hour, washed twice with PBS, and analyzed using C6 flow cytometer; the final cells were named CHO-VISTA cells .
  • the initial concentration of anti-VISTA antibody is 5 ⁇ g/mL, 2-fold gradient dilution, 100 ⁇ L antibody dilution and 100 ⁇ L 5 ⁇ 10 5 CHO-VISTA cells per well; mix evenly and incubate at 4°C for 30 minutes; after washing the cells twice with PBS, add After using goat anti-human IgG-Fc PE fluorescent secondary antibody (1:1000 PBS dilution), incubate at 4°C for 30 minutes, wash the cells twice with PBS, then resuspend them in 200 ⁇ L PBS (Gibco Company), and measure the second channel with flow cytometer CytoFLEX. The fluorescence intensity.
  • VSIG-3 Recombinant Human VSIG3 Fc Chimera Protein, R&D, Cat. No. 9229-VS-050
  • PBST Recombinant Human VSIG3 Fc Chimera Protein
  • BSA room temperature
  • PBST 10 ⁇ g/mL biological Mix peptide-labeled VISTA
  • the antibody P48-6-K of the present invention can block the binding of VSIG-3 to VISTA in a concentration-dependent manner.
  • PBMC cells peripheral blood mononuclear cells
  • RPMI-1640 culture medium to dilute the anti-VISTA antibody (starting concentration is 6 ⁇ g/mL, 3-fold gradient dilution), add it to a U-shaped 96-well plate, 100 ⁇ L per well, 2 parallel wells; add 100 ⁇ L PBMC cell solution, and mix evenly ; Place in a 37°C, 5% carbon dioxide incubator for about 18 hours.
  • IP-10 Interferon-induced protein 10
  • the detection steps are: coat the plate with IP-10 capture antibody overnight at 4°C, wash 3 times with PBST, block with 1% BSA for 2 hours at room temperature, and wash 4 times with PBST; U-shaped 96-well plate (containing PBMC cells and antibodies) is centrifuged at 2000 rpm for 5 min, and 100 ⁇ L is taken.
  • the activity of -10 is basically close, and the secreted chemokine IP-10 can chemoattract and activate monocytes and T cells.
  • the PBMC cell isolation method is the same as in Example 6, and CD3 + T cell isolation kit (Mojosort TM Human CD3T cell Isolation Kit, Biolegend) is used to isolate CD3 positive T cells (i.e., CD3 + T cells) from PBMC.
  • CD3 + T cell isolation kit Mojosort TM Human CD3T cell Isolation Kit, Biolegend
  • CD3 + T cell isolation kit Mojosort TM Human CD3T cell Isolation Kit, Biolegend
  • CellTiter-GloTM CellTiter-GloTM, Promega
  • the antibody P48-6-K and the anti-CD3 antibody when co-coated, it can promote T cell proliferation and thereby enhance immunity.
  • Experimental mice female BALBc-hVISTA mice (VISTA humanized mice, from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).
  • Experimental cells Collect CT26.WT (colorectal cancer) cells (Jicui Yaokang) in the logarithmic growth phase, remove the culture medium, wash and inoculate; Inoculation volume: 5 ⁇ 10 5 cells/100 ⁇ L/animal; Inoculation location: mice On the right side of the back, above the thigh.
  • Group administration 3 days after the tumor cells were inoculated, the mice were randomly divided into 5 groups according to their weight, with 10 mice in each group; the day of inoculation was defined as day D0, and administration was started according to the experimental plan design on the day of grouping; the dosage and administration
  • the drug method is shown in Table 10.
  • the dosage volume is calculated as 10 ⁇ L/g.
  • tumor volume (mm 3 ) 0.5 ⁇ tumor long diameter ⁇ tumor short diameter 2
  • tumor volume inhibition rate TGITv (1-average tumor volume of the administration group/average tumor volume of the control group) ⁇ 100%
  • Tumor weight inhibition rate TGITw (1 - average tumor weight of the administration group/average tumor weight of the control group) ⁇ 100%.

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Abstract

本发明提供了含抗VISTA抗体的组合物及其在联合用药中的应用，包括联合使用抗VISTA抗体和一种或多种其它治疗剂。本发明的组合物及其应用可有助于免疫系统清除肿瘤细胞，也可以用于肿瘤或癌症的诊断和预后。

Description

抗VISTA抗体在联合用药中的应用 技术领域
本发明属于生物医药领域，尤其涉及含抗VISTA抗体的组合及其联合用药。
背景技术
T细胞活化V结构域Ig抑制蛋白(V domain immunoglobulin suppressor of T cell activation，VISTA)，又称PD-1H、B7-H5、DD1α、c10orf54、Gi24或Dies1，是一种I型跨膜蛋白，属于免疫球蛋白超家族。人类VISTA基因主要在造血细胞系和富含浸润白细胞的组织中表达。在人外周血单核细胞(PBMC)中，CD14+单核细胞、中性粒细胞、髓系CD11c+DCs、CD4+和CD8+T细胞等均表达VISTA。VISTA在不同物种间高度保守(>80％)。
在免疫反应调节过程中，VISTA既可以作为配体在酸性条件下结合共抑制受体(p选择素糖蛋白配体-1，PSGL-1)，也可以作为受体结合配体(含V型免疫球蛋白域蛋白3，VSIG-3)。研究表明，VISTA在初始T细胞表面表达，对于维持T细胞静默与外周免疫耐受至关重要。
VISTA参与多种疾病：在膀胱癌和黑色素瘤模型中，VISTA单克隆抗体或VISTA基因敲除都能显著改善CD4+和CD8+T细胞介导的抗肿瘤免疫；在EAE(实验性变态反应性脑脊髓炎)模型中，VISTA单克隆抗体或VISTA基因敲除都会加重疾病的进展；在卵巢癌模型中，VISTA单克隆抗体显著延长高水平表达VISTA的小鼠的生存期。可见，VISTA是癌症治疗的潜在靶标。因此，针对VISTA的治疗剂将有助于癌症治疗。
发明内容
本发明的目的是提供一种新的抗VISTA抗体或抗原结合片段与其它治疗剂联合使用用于治疗疾病。本发明的VISTA抗体或抗原结合片段可以特异性结合VISTA，阻断VISTA下游的免疫抑制信号通路，有助于免疫系统清除肿瘤细胞。
在一个方面，本发明提供抗VISTA抗体或抗原结合片段在制备和一种或多种其它治疗剂联合使用的药物中的用途。
另一方面，本发明提供抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂在制备用于治疗疾病的药物中的用途。
另一方面，本发明提供抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂联合使用在治疗疾病中的用途。
另一方面，本发明提供治疗疾病的方法，如预防、治疗或改善T细胞功能障碍病症的方法，包括给需要治疗的患者施用有效剂量的抗VISTA抗体或抗原结合片段和一 种或多种其它治疗剂。
在一些以上方面的实施方案中，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂同时施用。抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂同时施用时，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂可以通过同样或不同方式施用，或制成抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂的组合物施用。在一些实施方案中，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂分开施用。在一些实施方案中，抗VISTA抗体或抗原结合片段在一种或多种其它治疗剂之前施用。在一些实施方案中，抗VISTA抗体或抗原结合片段在一种或多种其它治疗剂之后施用。
另一方面，本发明提供一种组合物，所述组合物包含抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂。
另一方面，本发明提供一种预防、治疗或改善T细胞功能障碍病症的方法，所述方法包括向患者施用有效剂量的包含抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂的组合物。
在一些实施方案中，所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体。
在一些实施方式中，所述抗VISTA抗体或抗原结合片段特异性结合VISTA，并且包含以下中的一个或多个CDR，如1、2、3、4、5或6个CDR：
(a)HCDR1，其包含如SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(b)HCDR2，其包含如SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(c)HCDR3，其包含如SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(d)LCDR1，其包含如SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(e)LCDR2，其包含如SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(f)LCDR3，其包含如SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含：
(a)HCDR1，其包含如SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(b)HCDR2，其包含如SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2相比 具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；和
(c)HCDR3，其包含如SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。
在一些实施方案中，HCDR1包含SEQ ID NO:1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:3所示的氨基酸序列或由其组成。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含：
(d)LCDR1，其包含如SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；
(e)LCDR2，其包含如SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；和
(f)LCDR3，其包含如SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。
在一些实施方案中，LCDR1包含SEQ ID NO:4所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:5所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO:6所示的氨基酸序列或由其组成。
在一种实施方案中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所述的LCDR2和如SEQ ID NO:6所示的LCDR3。
在上述实施方案中，所述取代为保守氨基酸取代。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NO:7或8所示的序列，或与SEQ ID NO:7或8所示序列相比具有至少80％同一性的序列，或与SEQ ID NO:7或8所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含轻链可变区，所述轻链可变区包含SEQ ID NO:9所示的序列，与SEQ ID NO:9所示序列相比具有至少80％同一性的序列，或与SEQ ID NO:9所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:7所示的序列，所述轻链可变区包含SEQ ID NO:9所示的序列。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链可变区和轻链可 变区，其中，所述重链可变区包含SEQ ID NO:8所示的序列，所述轻链可变区包含SEQ ID NO:9所示的序列。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段还包含重链恒定区、轻链恒定区、Fc区或其组合。
在一些实施方案中，所述轻链恒定区是κ或λ链恒定区。在一些优选实施方案中，所述抗体或其抗原结合片段是IgG、IgM、IgA、IgE或IgD其中一种同种型；优选地，所述同种型是IgG1、IgG2、IgG3或IgG4。在一些优选实施方案中，所述抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
在一些实施方案中，Fc是变体Fc区。在一些实施方案中，相对于亲本Fc区，变体Fc区具有一个或多个氨基酸修饰，如取代、缺失或插入。在一些实施方案中，相对于亲本Fc区活性，Fc区的氨基酸修饰改变了效应功能活性。在一些实施方案中，变体Fc区可以具有改变的(即，增加的或降低的)抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在一些实施方案中，相对于亲本Fc区，Fc区氨基酸修饰可以改变变体Fc区对FcγR(Fcγ受体)的亲和力。在一些实施方案中，所述Fc区来源于IgG1或IgG4。在一些实施方案中，Fc区突变是相对于IgG1亚型的N297A突变。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段为分离的抗体或抗原结合片段。在一些实施方案中，所述抗体或抗原结合片段为scFv、Fab或F(ab)2。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链恒定区，所述重链恒定区包含氨基酸序列如SEQ ID NO:10所示的序列，或与SEQ ID NO:10所述序列相比具有至少80％同一性的序列，或与SEQ ID NO:10所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或
所述抗VISTA抗体或抗原结合片段包含轻链恒定区，所述轻链恒定区包含氨基酸序列如SEQ ID NO:11所示的序列，或与SEQ ID NO:11所述序列具有相比至少80％同一性的序列，或与SEQ ID NO:11所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链恒定区和轻链恒定区，其中，所述重链恒定区包含氨基酸序列如SEQ ID NO:10所示的序列，所述轻链恒定区包含氨基酸序列如SEQ ID NO:11所示的序列。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段包含重链，所述重链包含氨基酸序列如SEQ ID NO:12或13所示的序列，与SEQ ID NO:12或13所示序列相比具有至少80％同一性的序列，或与SEQ ID NO:12或13所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或
所述抗体或抗原结合片段包含轻链，所述轻链包含氨基酸序列如SEQ ID NO:14所示的序列，与SEQ ID NO:14所示序列相比具有至少80％同一性的序列，或与SEQ ID NO:14所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。
在一些实施方案中，所述抗VISTA抗体的重链包含氨基酸序列如SEQ ID NO:12所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。
在一些实施方案中，所述抗VISTA抗体的重链包含氨基酸序列如SEQ ID NO:13所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段为单克隆抗体(包括全长单克隆抗体)、多克隆抗体或多特异性抗体或抗原结合片段(例如双特异性抗体或抗原结合片段)。
在一些实施方案中，所述抗VISTA抗体具有两条序列相同的重链和两条序列相同的轻链，Fc区配对形成二硫键。
本发明所述的抗VISTA抗体或抗原结合片段在WO2022/206677中公开。
在一些实施方案中，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3，如抗CD3抗体或抗PD-L1抗体或抗原结合片段。
在一些实施方案中，所述抗PD-L1抗体为阿替利珠单抗(Atezolizumab)、度伐利尤单抗(Durvalumab)、阿维单抗(Avelumab)、恩沃利单抗(Envafolimab)。
在一些实施方案中，所述抗PD-L1抗体或抗原结合片段包含氨基酸序列分别如SEQ ID NO:24～26所示的HCDR1、HCDR2和HCDR3以及氨基酸序列分别如SEQ ID NO:27～29所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述抗PD-L1抗体或抗原结合片段包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO:19的第20-137位所示，或与SEQ ID NO:19的第20-137位相比具有至少80％同一性的序列，或与SEQ ID NO:19的第20-137位相比具有一个或多个保守氨基酸取代的氨基酸序列；以及，
所述抗PD-L1抗体或抗原结合片段包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO:20的第23-129位所示，或与SEQ ID NO:20的第23-129位相比具有至少80％同一性的序列，或与SEQ ID NO:20的第23-129位相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗PD-L1抗体的重链氨基酸序列如SEQ ID NO:19的第20-467位所示；所述抗PD-L1抗体的轻链氨基酸序列如SEQ ID NO:20的第23-236位所示。
在一些实施方案中，所述抗VISTA抗体或抗原结合片段和其他治疗剂分别给药。 在一些实施方案中，所述抗VISTA抗体或抗原结合片段和其他治疗剂同时给药。
在一些实施方案中，本发明的用途、方法及组合物可以用于预防、治疗或改善T细胞功能障碍病症。在一些实施方案中，所述T细胞功能障碍病症包括肿瘤、癌症或感染。
在一些实施方案中，每个治疗周期内所述抗VISTA抗体以1-3000mg的剂量给药；或者，每个治疗周期内所述抗VISTA抗体以0.01-100mg/kg患者体重的剂量给药，或以0.01mg/kg-40mg/kg患者体重的剂量给药，或以0.01mg/kg-30mg/kg患者体重的剂量给药，或以0.01mg/kg-20mg/kg患者体重的剂量给药；或者，每个治疗周期内所述抗VISTA抗体以0.01-100mg/kg患者体重的剂量给药，或以0.1mg/kg-40mg/kg患者体重的剂量给药，或以0.1mg/kg-30mg/kg患者体重的剂量给药，或以0.1mg/kg-20mg/kg患者体重的剂量给药；每个治疗周期内所述抗PD-L1抗体以1-1500mg的剂量给药；或者，每个周期内所述抗PD-L1抗体以0.01mg/kg至100mg/kg患者体重的剂量给药，或0.1mg/kg至20mg/kg患者体重的剂量给药。
在一些实施方案中，采用治疗有效量的抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段分别或者同时施加在患者上。抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段的给药周期可以相同或者不同。
在一些实施方案中，所述T细胞功能障碍病症包括但不限于由细菌、病毒、真菌或原生动物导致的感染，以及癌症和肿瘤。
在一些实施方案中，所述癌症和肿瘤包括但不限于乳腺癌、消化道癌/胃肠癌、内分泌癌、神经内分泌癌、眼睛癌、泌尿生殖癌、生殖细胞癌、妇科癌、头颈癌、血液学/血液癌、肌肉骨骼癌、神经癌、呼吸道癌/胸腔癌、膀胱癌、结肠癌、直肠癌、结直肠癌、肺癌、子宫内膜癌、肾癌、胰腺癌、肝癌、胃癌、睾丸癌、食道癌、前列腺癌、脑癌、宫颈癌、卵巢癌和甲状腺癌。在一些实施方案中，癌症和肿瘤包括但不限于白血病、黑色素瘤和淋巴瘤。在一些实施方案中，白血病包括但不限于淋巴细胞白血病或髓细胞性白血病(如例如急性淋巴母细胞性白血病(ALL)、慢性淋巴细胞白血病(CLL)、急性骨髓(髓细胞性)白血病(AML)、慢性髓细胞性白血病(CML))、毛细胞白血病、T细胞幼淋巴细胞白血病、大颗粒淋巴细胞白血病或成人T细胞白血病。在一些实施方案中，淋巴瘤包括但不限于组织细胞性淋巴瘤、滤泡性淋巴瘤和霍奇金淋巴瘤。在一些实施方案中，感染包括但不限于慢性感染性疾病，如HIV、HBV、HCV和HSV等。
本发明的用途、方法及组合物有助于免疫系统清除肿瘤细胞，可以用于预防、治疗或改善T细胞功能障碍病症，如癌症或肿瘤。
附图说明
图1为本发明实施例1中抗VISTA抗体的SDS-PAGE图谱；其中，泳道M表示分子标记物marker，泳道1表示抗体P48-6-K，泳道2表示抗体P48-6-T，泳道3表示抗体VSTB112。
图2示出抗VISTA抗体阻断VSIG-3与VISTA的结合。
图3示出抗VISTA抗体促进T细胞增殖；图中，**表示p<0.01，****表示p<0.0001。
图4A示出抗体对肿瘤体积的影响；图4B示出抗体对肿瘤重量的影响；图中，*表示p<0.05，**表示p<0.01，***表示p<0.001，ns表示无显著性差异。
图5示出抗体对小鼠体重的影响。
具体实施方式
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体组成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp， W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
“VL、VH的保守氨基酸取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。“重链恒定区、轻链恒定区、重链或轻链的保守氨基酸取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一 性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性或序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对和同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+Swi ssProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
本发明公开的抗体、抗原结合片段包括但不限于多克隆抗体、单克隆抗体、多特异性抗体、全人源抗体、人源化抗体、灵长类化抗体、嵌合抗体、单链抗体、表位结合片段(例如Fab、Fab'和F(ab')2)、单链Fvs(scFv)。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(LCDR1-3)和重链的CDR区(HCDR1-3)。抗体及抗原结合片段可以特异性识别和结合一个或多个(如两个)抗原的多肽或多肽复合物。特异性识别和结合多个(如两个)抗原的抗体或抗原结合片段可以被称为多特异性(如双特异性)抗体或抗原结合片段。
术语“抗体片段”或“抗原结合片段”指抗体的一部分，本发明抗体片段的组成形式可类似于单特异性抗体片段中的F(ab’)2、F(ab)2、Fab'、Fab、Fv、scFv等。不管其结构如何，抗体片段与被完整抗体识别的同一抗原结合。术语“抗体片段”包括适体、镜 像异构体和双价抗体。术语“抗原结合片段”还包括通过与特定抗原结合形成复合物起抗体作用的任何合成或基因工程蛋白质。
“单链可变片段”或“scFv”是指免疫球蛋白的重链(VH)和轻链(VL)的可变区的融合蛋白。在一些方面，这些区域与10个至约25个氨基酸的短接头肽连接。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。scFv分子通常是本领域中已知的，例如在美国专利5,892,019中有相关描述。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgD或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为Vλ。
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链可变区(VL)和重链可变区(VH)部分决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法 鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.,et al.,U.S.Department of Health and Human Services,Sequences of Proteins of Immunological Interest,(1983)和Chothia and Lesk,J.Mol.Biol.,196:901-917(1987))。
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.,U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196:901-917(1987)有相关描述，其通过引用全部并入本文。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“Sequence of Proteinsof Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。
本发明公开的抗体可以来源于任何动物，包括鸟类和哺乳动物。较佳地，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。
“重链恒定区”包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包括源自IgG1分子的CH1结构域和源自IgG3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG1分子和部分源自IgG3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG1分子和部分源自IgG4分子的嵌合铰链区。
“轻链恒定区”包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，“CH1结构域”包括免疫球蛋白重链的第一个(大部分氨基末端)恒定区。完整的天然IgG分子中两个CH2结 构域中N297各连接一个分支碳水化合物链。CH3结构域从CH2结构域开始延伸到IgG分子的C-末端，大约包含108个残基。“铰链区”包括连接CH1结构域和CH2结构域的部分重链区域。所述铰链区包含约25个残基并且是有韧性的，从而使得两个N端抗原结合区能够独立移动。铰链区可以被细分为三个不同的结构域：上、中和下铰链结构域(Rouxetal.,J.Immunol 161:4083(1998))。
在一些实施方案中，抗体或抗原结合片段包含氨基酸序列具有一个或多个修饰基团。例如，抗体或抗原结合片段可以包含有韧性的接头序列，或者可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。抗体、抗原结合片段包括被修饰的衍生物，即通过任何类型的分子与抗体或抗原结合片段的共价连接进行修饰，其中共价连接不会阻止抗体或抗原结合片段与表位结合。包括但不限制以下实例，抗体或抗原结合片段可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。抗体或抗原结合片段可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG缀合。抗体或抗原结合片段可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体或抗原结合片段的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接。
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。
“特异性结合”或“对……具有特异性”通常是指抗体或抗原结合片段与特定抗原通过其抗原结合结构域与表位互补性结合形成相对稳定的复合物。“特异性”可以用抗体或抗原结合片段与特定抗原或表位结合的相对亲和力表达。例如，如果抗体“A”比抗体“B”与同一抗原的相对亲和力大，可以认为抗体“A”比抗体“B”对该抗原具有更高的特异性。特异性结合可以用平衡解离常数(KD)来描述，较小的KD意味着较紧密的结合。确定两个分子是否特异性结合的方法是本领域内众所周知的，并包括例如平衡透析、表面等离子共振、生物膜层光学干涉测量法等。“特异性结合”抗原a的抗体包括与抗原a平衡解离常数KD小于或等于约100nM、小于或等于约10nM、小于或等于约5nM、小于或等于约1nM或小于或等于约0.5nM的抗体。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。
“患者”指需要诊断、预后或治疗的任何哺乳动物，包括人类、狗、猫、兔子、鼠、马、牛等。
抗VISTA抗体
本发明提供了对VISTA蛋白具有高亲和力的抗体或抗原其结合片段。本发明的抗体或抗原结合片段表现出有效的结合活性、生物学活性，并可用于治疗和诊断的用途。比如，这些抗体或抗原结合片段可以有效阻断抑制性的免疫检查点，激活淋巴细胞释放细胞因子，用于治疗各种类型的癌症、肿瘤或感染等相关疾病。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:12所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。在一些实施方案中，所述抗原结合片段的重链包含氨基酸序列SEQ ID NO:12中除Fc以外的序列，所述抗原结合片段的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:13所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。在一些实施方案中，所述抗原结合片段的重链包含氨基酸序列SEQ ID NO:13中除Fc以外的序列，所述抗原结合片段的轻链包含氨基酸序列如SEQ ID NO:14所示的序列。
在一些实施方案中，本发明抗体含有两条序列相同的重链(或重链片段)和两条序列相同的轻链(或轻链片段)。
本领域普通技术人员还应当理解，本发明所公开抗体或抗原结合片段序列是可以被替换的，替换后其氨基酸序列不同于该抗体的天然存在的氨基酸序列。例如，替换后的氨基酸序列可以是与起始序列相似的，比如与起始序列具有一定比例的同一性，比如它可以与起始序列的同一性是约80％、约85％、约90％、约95％、约98％、约99％，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
抗体的制备方法
在某些实施方案中，制备的抗体不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一些实施方案中，本发明公开的抗体、抗原结合片段、或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免 疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基。(参考美国专利5,585,089；Riechmann et al.,Nature 332:323(1988)；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400；WO 91/09967；美国专利5,225,539,5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596；Padlan,et al.,Molecular Immunology 28(4/5):489-498(1991)；Studnicka et al.,Protein Engineering 7(6):805-814(1994)；Roguska,et al.,Proc.Natl.Sci.USA 91:969-973(1994))，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO/9852976A1和WO/0034317A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。
本发明公开的抗体或抗原结合片段的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。
scFv的制备可参见生产单链单元的技术(美国专利4,694,778；Bird,Science 242:423-442(1988)、Huston et al.,Proc.Natl.Acad.Sci.USA 55:5879-5883(1988)和Ward et al.,Nature 334:544-554(1989)和Nie et al.,Antibody Therapeutics 3(1):18-62(2020))。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.,Science 242: 1038-1041(1988))。
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498，以及Huston et al.,Methods in Enzymology 203:46-88(1991)、Shu et al.,Proc.Natl.Sci.USA 90:1995-1999(1993)和Skerra et al.,Science 240:1038-1040(1988)中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见Morrison,Science 229:1202(1985)；Oi et al.,BioTechniques 4:214(1986)；Gillies et al.,J.Immunol.Methods 125:191-202(1989)；Neuberger et al.,Nature 372:604-608(1984)；Takeda et al.,Nature 314:452-454(1985)；和美国专利5,807,715、4,816,567和4,816,397，其全部内容通过引用并入本文。
此外，在Newman,Biotechnology 10:1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。
抗体可以通过本领域已知的多种方法制备，包括使用来自免疫球蛋白序列的抗体文库进行的噬菌体展示方法。也可参考美国专利4,444,887和4,716,111，以及PCT公布文本WO 98/46645、WO 98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735和WO 91/10741，每个专利的全部内容通过引用并入本文。
在另一实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以分离编码所需单克隆抗体的DNA并对其进行测序。分离的和亚克隆的杂交瘤细胞可以作为此类DNA的来源。一旦分离出来，DNA可以被置于表达载体中，然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不产生其他免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。
此外，使用常规重组DNA技术，可将本发明的抗体的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.,J.Mol.Biol.278:457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内可以进行一个或多个氨基酸取代， 可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。
抗体可以通过使用常规重组DNA技术制备。使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的载体及细胞系等。这些技术在各种实验室手册和主要出版物中均有描述，例如Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells，D.L.Hacker,F.M.Wurm,in Reference Module in Life Sciences,2017，其全部内容包括补充内容通过引用并入全文。
在一些实施方案中，可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生单克隆抗体。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo、pRetro-Off、pRetro-On、PLXSN、或者pLNCX、pcDNA3.1(+/-)、pcDNA/Zeo(+/-)、pcDNA3.1/Hygro(+/-)、PSVL、PMSG、pRSVcat、pSV2dhfr、pBC12MI和pCS2等。常使用的哺乳动物细胞包括HEK293细胞、Cos1细胞、Cos7细胞、CV1细胞、鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区和轻链可变区来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
治疗方法
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善各种类型的癌症、肿瘤或感染等相关疾病的方法，所述方法包括向有需要的患者施用有效剂量的抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂。在一些实施方案中，提供了抗VISTA抗体或抗原结合片段与一种或多种其它治疗剂联合使用在用于治疗或改善癌症、肿瘤或感染等相关疾病中的应用。在一些实施方案中，提供了所述抗VISTA抗体或抗原结合片段在制备用于与一种或多种其它治疗剂联合使用治疗或改善癌症、肿瘤或感染等相关疾病的药物中的应用。在一些实施方案中，提供了所述抗VISTA抗体或抗原结合片段与一种或多种其它治疗剂在制备用于治疗或改善癌症、肿瘤或感染等相关疾病的药物中的应用。在一些实施方案中，所述其它治疗剂如本文所述。
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。在一些实施方案中，所述抗VISTA抗体施用于患者的剂量为每次0.01mg/kg至100mg/kg患者体重。在一些实施方案中，每1星期、2星期、3星期、或每月给药一次。在一些实施方案中，所述抗体的剂量为每次投予0.1-20mg/kg。
在一些实施方案中，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂同时施用。抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂同时施用时，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂可以通过同样或不同方式施用，或制成抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂的组合物施用。在一些实施方案中，抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂分开施用,可以通过同样或不同方式施用。
抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂的施用方法独立的包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外和口服注射。药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，含有本发明抗体或抗原结合片段的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和 关节内注射和输注的施用方式。
施用方式可以是全身施用或局部施用。此外，可能需要通过任何合适的途径将本发明的抗体引入中枢神经系统，包括脑室内和鞘内注射；脑室内注射可以通过脑室内导管连接到如贮液囊(可以是Ommaya贮液囊)来辅助注射。也可以通过肺部给药，例如通过使用吸入器或喷雾器，以及使用雾化的制剂。
抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂独立的可以局部施用于需要治疗的区域；可以通过但不限于以下方式：手术期间局部施用，例如与手术后伤口敷料联合的局部应用，通过注射，通过导管，借助栓剂或借助植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜)或纤维。在一些实施方式中，当施用本发明的蛋白质(包括抗体)时，必须注意使用不吸收蛋白质的材料。
在一些实施方案中，本发明组合物包含编码抗体的核酸或多聚核苷酸，可以通过将其构建为合适的核酸表达载体的一部分来体内施用所述核酸以促进其编码的蛋白质的表达，然后通过下述方式施用上述部分载体使其变为胞内部分，例如通过使用逆转录病毒载体(参见美国专利4,980,286)，或通过直接注射，或通过使用微粒轰击(例如基因枪；Biolistic，Dupont)，或用脂质或细胞表面受体或转染试剂包被，或者通过与已知进入细胞核的同源异型盒类肽连接施用(参见例如Joliot et al.,1991,Proc.Natl.Acad.Sci.USA 88:1864-1868)等等。可选地，核酸可以通过同源重组在引入细胞内并整合至宿主细胞DNA中用于表达。
在一些实施方案中，抗VISTA抗体施用于患者的剂量为0.01mg/kg至100mg/kg患者体重，或0.1mg/kg至20mg/kg患者的体重。在初始剂量之后可随后给予第二剂或多剂该抗体或抗原结合片段，其剂量与初始剂量大致相同或较少，其中该随后的剂量可相隔至少1天至3天；或至少一星期。可以通过例如脂质化等修饰来增强抗体的摄取和组织穿透能力(例如进入脑内)，从而减少本发明抗体的施用的剂量和频率。
抗VISTA抗体用于进行体外测试用于治疗疾病的方法，包括施用本发明所述抗VISTA抗体或衍生物，然后在可接受的动物模型中体内测试期望的治疗性或预防性活性，最后施用于人体。合适的动物模型(包括转基因动物)是本领域普通技术人员所公知的。例如，用于证明本发明所述抗VISTA抗体、抗原结合片段的治疗用途的体外测定包括抗体对细胞系或患者组织样品的影响。抗体对细胞系和/或组织样品的作用可以利用本领域技术人员已知的技术进行检测，例如本发明其他部分公开的技术。根据本发明的内容，可用于确定是否施用特异性抗体的体外测定实验包括体外细胞培养实验，其中患者组织样品在培养物中培养，并暴露于或以其他方式施用化合物，并观察这种化合物对组织样品的影响。
各种已知输送系统可用于施用本发明抗体或衍生物或编码其的多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细胞、受体介导的内吞作用(参见例如Wu and Wu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
抗VISTA抗体和抗PD-L1抗体联合疗法
程序性死亡受体-1(PD-1)为一种I型跨膜糖蛋白，分子量约为55kDa。PD-1是表达在活化的T细胞、B细胞和髓样细胞上表达的免疫抑制性受体，属于CD28免疫球蛋白超家族成员。作为PD-1的配体，PD-L1也是一种I型跨膜糖蛋白，其分布广泛：表达在B细胞、T细胞、树突状细胞、巨噬细胞等抗原呈递细胞的表面，以及肿瘤组织中。
在一些实施方案中，本发明提供本文所述抗VISTA抗体或抗原结合片段在制备和抗PD-L1抗体或抗原结合片段联合使用的药物中的用途。
在一些实施方案中，本发明提供本文所述抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段在制备治疗疾病的药物中的用途。
在一些实施方案中，本发明提供本文所述抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段联合使用在治疗疾病中的用途。
在一些实施方案中，本发明提供治疗疾病的方法，如预防、治疗或改善T细胞功能障碍病症方法，包括给需要治疗的患者使用有效剂量的本文所述抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段。
在一些实施方案中，抗VISTA抗体或抗原结合片段和抗PD-L1抗体同时施用。抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段同时施用时，抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段可以通过同样或不同方式施用，或制成抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段的组合物施用。在一些实施方案中，抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段分开施用。在一些实施方案中，抗VISTA抗体或抗原结合片段在抗PD-L1抗体或抗原结合片段之前施用。在一些实施方案中，抗VISTA抗体或抗原结合片段在抗PD-L1抗体或抗原结合片段之后施用。
在一些实施方案中，本发明提供一种组合物，所述组合物包含本文所述抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段。
在一些实施方案中，本发明提供一种预防、治疗或改善T细胞功能障碍病症方法，所述方法包括向患者施用有效剂量的包含本文所述抗VISTA抗体或抗原结合片段和抗PD-L1抗体或抗原结合片段的组合物。
在一些实施方案中，与本文所述抗VISTA抗体或抗原结合片段联合使用的抗 PD-L1抗体为本文所述抗PD-L1抗体或抗原结合片段。
在一些实施方案中，本发明抗VISTA抗体或抗原结合片段与抗PD-L1抗体或抗原结合片段的联合使用的用途、方法及组合物还可以结合其它治疗或预防方案，包括施用一种或多种本发明抗体或抗原结合片段，抗PD-L1抗体或抗原结合片段以及一种或多种其它治疗剂或方法一起使用或组合使用。在一些实施方案中，其他治疗方案包括但不限于放射疗法、化学疗法、激素疗法、生物制品疗法等。对于组合治疗，这几种治疗剂可同时或分开施用。当分开施用时，这几种治疗剂可以通过不同顺序施用。
在一些实施方案中，可以与本发明的抗体或抗原结合片段及抗PD-L1抗体或抗原结合片段联合的方法、用途或组合物一起施用的治疗性抗体包括但不限于抗CD3抗体、抗PD-1抗体、抗PD-L2抗体、抗TIM-3抗体、抗LAG-3抗体、抗OX40抗体和抗GITR抗体等，如阿替利珠单抗(atezolizumab)、阿维单抗(avelumab)、度伐利尤单抗(druvalumab)、恩沃利单抗(Envafolimab)、纳武单抗、派姆单抗、替西木单抗(tremelimumab)、伊匹木单抗、曲妥珠单抗。
在一些实施方案中，可以与本发明抗体或抗原结合片段及抗PD-L1抗体或抗原结合片段联合的方法、用途或组合物一起施用的血管生成抑制剂包括但不限于血管抑制素(血纤蛋白溶解酶原片段)、抗血管生成抗凝血酶III和核酶。
在一些实施方案中，可以与本发明抗体或抗原结合片段及抗PD-L1抗体或抗原结合片段联合的方法、用途或组合物一起施用的抗癌剂包括但不限于：5-氟尿嘧啶、阿西维辛、阿地白介素、六甲蜜胺、氨鲁米特、安吖啶、阿那曲唑、安曲霉素、天冬酰胺酶、阿扎胞苷、阿扎替派、阿佐霉素、巴马司他、比卡鲁胺、硫酸博来霉素、布喹那钠、溴匹立明、白消安、卡铂、卡莫司汀、盐酸卡柔比星、卡折来新、西地芬戈、苯丁酸氮芥、西罗霉素、顺铂、克拉屈滨、甲横酸克立那托(crisnatol mesylate)、环磷酰胺、阿糖胞苷、达卡巴嗪、更生霉素、盐酸柔红霉素、地西他滨、右奥马铂、地扎胍宁、甲磺酸地扎胍宁(dezaguanine mesylate)、地吖醌、多西他赛、多柔比星、盐酸阿霉素、屈洛昔芬、柠檬酸屈洛昔芬、丙酸屈他雄酮、达佐霉素、依达曲沙、盐酸依氟鸟氨酸、恩洛铂、恩普氨酯、依匹哌啶、盐酸表柔比星、厄布洛唑、盐酸伊索比星、雌莫司汀、雌莫司汀磷酸钠、依他硝唑、依托泊苷、磷酸依托泊苷、法扎拉滨、芬维A胺、氟尿苷、磷酸氟达拉滨、氟尿嘧啶、氟西他滨、磷喹酮、福司曲星钠、吉西他滨、盐酸吉西他滨、羟基脲、盐酸伊达比星、异环磷酰胺、伊莫福新、白介素II(包括重组白介素II)、干扰素α-2a、干扰素α-2b、干扰素α-m、干扰素α-n3、干扰素β-Ia、干扰素γ-I b、异丙铂、盐酸伊立替康、醋酸兰瑞肽、来曲唑、亮丙瑞林乙酸盐、盐酸利阿唑、洛美曲索钠、洛莫司汀、盐酸洛索蒽醌、马索罗酚、盐酸氮芥、醋酸甲地孕酮、乙酸甲烯雌醇、美法仑、美诺立尔、巯基嘌呤、甲氨蝶呤、甲氨蝶呤钠、氯苯氨 啶、美妥替哌、丝裂霉素、米托司培、米托坦、盐酸米托蒽醌、麦考酚酸、诺考达唑、奥马铂、紫杉醇、培门冬酶、紫菜霉素(porfromycin)、泼尼莫司汀、盐酸丙卡巴肼、嘌呤霉素、罗谷亚胺、盐酸沙芬戈、司莫司汀、辛曲秦、司泊索非钠、司帕霉素、螺莫司汀、螺铂、链黑霉素、链脲菌素、磺氯苯脲、太利苏霉素、替加氟、盐酸替洛蒽醌、替莫泊芬、替尼泊苷、替罗昔隆、睾内酯、硫咪嘌呤、硫鸟嘌呤、噻替派、噻唑呋林、替拉扎明、拓扑替康、三甲曲沙、葡萄糖醛酸三甲曲沙、曲普瑞林、乌拉莫司汀、乌瑞替派、伐普肽、维替泊芬(verteporfn)、硫酸长春碱、硫酸长春新碱、长春地辛、硫酸长春地辛、硫酸长春匹定、硫酸长春甘酯、硫酸长春罗辛、酒石酸长春瑞滨、硫酸长春罗定、硫酸长春利定、伏氯唑、折尼铂、净司他丁和盐酸佐柔比星等。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗VISTA抗体或抗原结合片段、一种或多种其它治疗剂以及药学上可接受的载体。在一些实施方案中，药物组合物包含0.1％-90％的抗VISTA抗体或抗原结合片段。
在一些实施方案中，术语“药学上可接受的”是指由政府的监管机构批准的或公认药典中列出的用于动物，特别是用于人类的物质。此外，“药学上可接受的载体”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“载体”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右旋葡萄糖也是可以预见的。这些组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合片段，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。亲本制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
在一些实施方案中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物组合物。用于静脉内给药的组合物通常是在无菌等渗水性缓冲液中的溶液。组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有无菌药用级水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括衍生自如盐酸、磷酸、乙酸、草酸、酒石酸等的与阴离子形成的盐，以及衍生自如钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的与阳离子形成的盐。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“EC50”即半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
本发明中“亲本Fc区”可以为天然存在的Fc区，编码Fc区的基因可来自人、鼠、兔、骆驼、猴子，优选为人和小鼠；例如，亲本Fc区为SEQ ID NO:10、SEQ ID NO:12或SEQ ID NO:13中Fc区。
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改或调整仍属于本发明的保护范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1抗体制备方法
本实施例制备的抗体包括抗VISTA抗体和抗PD-L1抗体，抗体相关的氨基酸序列和核酸序列见表1-表6。
将抗体的重链和轻链的氨基酸序列依照宿主细胞密码子偏好性特点进行序列优化，得到重链和轻链的DNA序列。为了便于在宿主细胞中表达，可在重链和轻链的N端添加信号肽。
将优化并合成的核酸序列克隆分别克隆至载体(载体可选pCDNA3.1，来源于Invitrogen的V79020)中，然后分别抽提大量质粒，重链和轻链按质粒摩尔比1:1瞬时表达转染HEK293F细胞。以抗体P48-6-K为例，将重链核酸序列(SEQ ID NO:17 的第58-1407位)和轻链核酸序列(SEQ ID NO:18的第61-702位)通过酶切克隆至表达载体中；其中，重链信号肽的氨基酸序列为SEQ ID NO:15，轻链信号肽的氨基酸序列为SEQ ID NO:16。以抗PD-1抗体L1-R2-4-71(序列来自专利申请CN114057877A)为例，将重链核酸序列(重链可变区核苷酸序列如SEQ ID NO:21的第58-411位所示)和轻链核酸序列(轻链可变区核苷酸序列如SEQ ID NO:22的第67-387位所示)通过酶切克隆至表达载体中；添加信号肽序列，其中，重链信号肽的氨基酸序列为SEQ ID NO:15，轻链信号肽的氨基酸酸序列为SEQ ID NO:20的第1-22位氨基酸。阳性参照抗体VSTB112为抗VISTA抗体，其制备方法与上述类似(序列见专利申请CN107922497A)。细胞表达后，培养液由采用Protein A柱(GE Healthcare)的固定化金属亲和层析(IMAC)进行纯化，纯化后的抗体蛋白的纯度＞95％。
对纯化后抗体进行凝胶电泳检测，如图1所示，本申请抗体P48-6-K和P48-6-T为单一物质，分子量与理论值是一致的。对纯化后抗体进行测序，测序结果与预计的序列相同。纯化后抗体用于亲和力检测和生物活性鉴定等。
表1抗体的组成
表2抗VISTA抗体的CDR区
表3抗VISTA抗体的可变区和恒定区

表4抗VISTA抗体的重链和轻链(重链的Fc区用单下划线标出)

表5抗VISTA抗体相关核酸序列(信号肽的核酸序列用双下划线标出)


表6抗PD-L1抗体L1-R2-4-71的氨基酸和核苷酸序列表(双下滑线为信号肽氨基酸序列，单下划线为恒定区序列)

实施例2抗体亲和力的测定
采用Biacore T200表面等离子体共振仪测定抗体的亲和力常数，主要试验过程如下：用Protein A芯片进行检测，100nM抗体(P48-6-K、P48-6-T、VSTB112)稀释液以10μl/min的流速通过实验流路(Fc2、Fc4)，捕获20s使捕获量约为560RU；之后流速调为30μl/min，依次进不同浓度(0nM、1.23nM、3.7nM、11.1nM、33.3nM、100nM)的VISTA-His(Acro，B75-H52H0)稀释液(稀释的溶剂为水)，同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面，结合时间120s，解离时间300s，最后进Glycine 1.5对芯片进行再生并进入下一个循环；用数据分析软件Evaluation Software3.1对试验结果进行分析，将样品实验流路采集所得传感信号进行参比流路、样品空白双扣减，并选用动力学“1:1”模型进行拟合，得出动力学参数(Kon(Ka)：结合速率常数；Koff(Kd)：解离速率常数；KD：结合解离平衡常数)。
1)如表7所示，抗体VSTB112与本发明抗体P48-6-K同VISTA-His的亲和力相当。
表7抗体P48-6-K与VISTA-His结合的亲和力常数
2)如表8所示，与抗体VSTB112相比，本发明抗体P48-6-T与VISTA-His具有更好的亲和力。
表8抗体P48-6-T与VISTA-His结合的亲和力常数
实施例3抗体与不同种属VISTA结合情况的检测
100μL、1μg/ml的VISTA-Fc(人、食蟹猴、小鼠种属，Sino biological Inc)包板，抗VISTA抗体的起始浓度为2μg/mL，2倍梯度稀释(稀释的溶剂为PBS缓冲液)，4℃冰箱放置过夜；PBST(PBS+0.05％吐温，1升PBS含NaCl 8.0g，Na2HPO4 0.9g，KH2PO4 0.156g，KCl 0.125g，pH 7.2-7.4)洗涤3次，5％BSA(牛血清白蛋白)室温封闭2h，PBST洗涤4次；2μg/mL抗体室温孵育1.5h，PBST洗涤4次；洗涤后再用羊抗人kappa轻链-过氧物酶抗体(SIGMA，货号为A7164)孵育0.5h；PBST洗涤6次，加入100μL四甲基联苯胺(TMB)，37℃孵育5min，加入50μL 0.1M硫酸终止；酶标仪读取450nm吸光值。
如表9所示，抗体VSTB112、P48-6-K、P48-6-T与人、食蟹猴的VISTA结合，且不与小鼠VISTA结合。
表9抗体与VISTA-FC结合的EC50(μg/mL)
注：NA表示不结合。
实施例4抗体与表达VISTA细胞的结合力的检测
CHO-VISTA细胞的制备方法为：人VISTA全长氨基酸序列(来自UniProtKB-Q9H7M9)：“MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYK QRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI”(SEQ ID NO:23)；合成SEQ ID NO:23对应的核酸序列，并在序列两端添加HindIII和EcoRI酶切位点，然后构建到pcDNA3.1表达载体(Invitrogen，V79020)上，接着通过电转的方法转染到CHO细胞中，电转条件为：电压300V，时间17毫秒，4mm电转杯，48h后加入50μM的MSX(蛋氨酸亚氨基代砜)筛选阳性细胞。利用FACS(流式细胞术)检测，筛选高表达的细胞株并收集细胞，用PBS洗一遍后，加入3μg/ml抗体VSTB112，4℃孵育1h后，用PBS洗2遍，再加入100μl 1:500稀释的羊抗人IgG-Fc PE荧光二抗(货号为12-4998-82，eBioscience)，4℃孵育1h后，用PBS洗2遍，采用C6流式细胞仪分析；将最终得到的细胞命名为CHO-VISTA细胞。
抗VISTA抗体起始浓度为5μg/mL，2倍梯度稀释，每孔100μL抗体稀释液和100μL 5×105个CHO-VISTA细胞；混合均匀，4℃孵育30min；PBS洗两次细胞后，加入羊抗人IgG-Fc PE荧光二抗(1:1000PBS稀释)后，4℃孵育30min，PBS洗两次细胞；再用200μL PBS(Gibco公司)重悬，用流式细胞仪CytoFLEX测量第二通道荧光强度。
结果显示，抗体VSTB112、P48-6-K和P48-6-T与VISTA-CHO细胞的结合的EC50值分别为1.52μg/mL、1.01μg/mL和1.22μg/mL。
实施例5抗体阻断VISTA与配体VSIG-3的结合
10μg/mL VSIG-3(Recombinant Human VSIG3 Fc Chimera Protein，R&D，货号9229-VS-050)包板4℃过夜；PBST洗涤3次，5％BSA室温封闭2h，PBST洗涤4次；10μg/mL生物素标记的VISTA(Acro，货号B75-H82F3)与抗体P48-6-K稀释液混合(以15μg/mL为起始浓度，2倍梯度稀释)，加入板上，室温孵育1.5h，PBST洗涤4次；洗涤后再用链霉亲和素标记的辣根过氧化物酶(Streptavidin-HRP)孵育0.5h；PBST洗涤6次，加入100μL四甲基联苯胺(TMB)，37℃孵育5min，加入50μL 0.1M硫酸终止；酶标仪读取450nm吸光值。
如图2所示，本发明抗体P48-6-K可以阻断VSIG-3与VISTA结合，且有浓度依赖关系。
实施例6细胞因子的检测
采集健康成年人外周血，用生理盐水(四川科伦药业)1:1稀释，缓慢加入等体积人外周血淋巴细胞分离液(达科为生物技术股份有限公司，达优)中，800g离心30min，吸取中间白色层，PBS洗两次；重悬在含10％FBS(胎牛血清，Gibco公司)的RPMI-1640培养基(Gibco公司)中并对细胞进行计数，调整PBMC细胞(外周血单核细胞)浓度为106个/mL。
采用RPMI-1640培养基稀释抗VISTA抗体(起始浓度为6μg/mL，3倍梯度稀释)，加入U型96孔板中，每孔100μL，2个平行孔；加入100μL PBMC细胞溶液，混合均匀；放入37℃、5％二氧化碳培养箱中培养约18h。
使用人CXCL10/IP-10ELISA试剂盒(R&D Systems，货号DY266)检测IP-10(干扰素诱导蛋白10)。检测步骤为：IP-10捕获抗体包板4℃过夜，PBST洗涤3次，1％BSA室温封闭2h，PBST洗涤4次；U型96孔板(包含PBMC细胞和抗体)2000rpm离心5min，取100μL上清加入ELISA板中，配制适宜浓度梯度的标准品，室温孵育1.5h，PBST洗涤4次，加入100μL生物素标记的IP-10检测抗体溶液室温孵育1.5h，PBST洗涤4次；加入100μL链霉亲和素标记的辣根过氧化物酶溶液，室温孵育0.5h，PBST洗涤6次；加入100μL四甲基联苯胺(TMB)，37℃孵育5min，再加入50μL 0.1M硫酸终止；酶标仪读取450nm吸光值。
结果显示，抗体VSTB112、抗体P48-6-T促进PBMC细胞分泌趋化因子IP-10对应的EC50值分别为：0.0052μg/ml和0.0054μg/ml，两者促进PBMC细胞分泌趋化因子IP-10的活性基本接近，而分泌的趋化因子IP-10可趋化活化单核细胞和T细胞。
实施例7 T细胞增殖实验
PBMC细胞分离方法同实施例6，用CD3+T细胞分离试剂盒(Mojosort TM Human CD3T cell Isolation Kit,Biolegend)从PBMC中分离CD3呈现阳性的T细胞(即CD3+T细胞)。实验前一日用1μg/ml抗CD3抗体(Recombinant Anti-CD3mAb，novoprotein，GMP-A018)分别和10μg/ml抗体VSTB112、P48-6-K、同型对照IgG1(HG1K，义翘神州)共同铺板，4℃过夜，PBS洗两次后按10万细胞/孔加入CD3+T细胞，体积100μL，3天后用CellTiter(CellTiter-GloTM，Promega)检测细胞增殖情况。
如图3所示，抗体P48-6-K和抗CD3抗体共包板情况下，能促进T细胞增殖，从而增强免疫能力。
实施例8抗肿瘤作用
实验小鼠：雌性BALBc-hVISTA小鼠(VISTA人源化的小鼠，来源于江苏集萃药康生物科技有限公司)。实验细胞：收集对数生长期的CT26.WT(结直肠癌)细胞(集萃药康)，去除培养液、洗涤后接种；接种量：5×105细胞/100μL/只；接种位置：小鼠右侧背部大腿上方。分组给药：当肿瘤细胞接种3天后，根据小鼠体重随机分成5组，每组10只；接种当天定义为D0天，并于分组当天，根据实验方案设计开始给药；给药剂量和给药方式如表10所示，给药体积：按照10μL/g计算。
细胞接种后，每周常规监测肿瘤对动物正常行为的影响。具体内容：实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。试验过程中观察到的临床症状均记录在原始数据中。开始给药后，第一周称量体重2次， 测量瘤体积2次；随后每周称量体重三次，测量瘤体积三次。瘤体积计算方式为：肿瘤体积(mm3)＝0.5×肿瘤长径×肿瘤短径2，肿瘤体积抑制率TGITv＝(1-给药组平均肿瘤体积/对照组平均肿瘤体积)×100％，肿瘤重量抑制率TGITw＝(1-给药组平均肿瘤重量/对照组平均肿瘤重量)×100％。
表10试验设计表
肿瘤体积抑制的结果如图4A、4B和表11A、11B所示，与G1组相比，在接种后第30天，抗体P48-6-T能显著抑制肿瘤生长，尤其是本发明抗体P48-6-T与抗体L1-R2-4-71的联合给药后能进一步抑制肿瘤的生长。如图5所示，各组小鼠的体重之间无显著性差异。
表11A第30天的肿瘤体积抑制率
表11B第30天的肿瘤重抑制率

Claims (13)

  1. 一种组合物，包含抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂，其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3中一个或多个；所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体；例如，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3。
  2. 抗VISTA抗体或抗原结合片段在制备和一种或多种其它治疗剂联合使用的药物中的用途，其包括向有需要的患者施用抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂；其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3中一个或多个；所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体；例如，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3。
  3. 抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂联合使用的用途，其包括向有需要的患者施用抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂；其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3中一个或多个；所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体；例如，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3。
  4. 抗VISTA抗体或抗原结合片段和一种或多种其它治疗剂在制备用于治疗疾病的药物中的用途，其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3中一个或多个；所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体；例如，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3。
  5. 一种预防、治疗或改善疾病的方法，其包括向有需要的患者施用抗VISTA抗 体或抗原结合片段和一种或多种其它治疗剂；其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3中一个或多个；所述其它治疗剂为血管生成抑制剂、抗癌剂、靶向药物或治疗性抗体；例如，所述其它治疗剂选自针对以下靶点的抗体或抗原结合片段或抗体药物偶联物：PD-L1、PD-1、CD3。
  6. 根据权利要求1-5任一项所述的组合物、用途或方法，其中，所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3；和/或
    所述抗VISTA抗体或抗原结合片段包含如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3；
    例如，所述抗体或抗原结合片段包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2、如SEQ ID NO:3所示的HCDR3、如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3。
  7. 根据权利要求1-6任一项所述的组合物、用途或方法，其中，
    (1)所述抗VISTA抗体或抗原结合片段包含重链可变区，其中，所述重链可变区包含SEQ ID NO:7或8所示的序列，或与SEQ ID NO:7或8所示序列具有相比至少80％同一性的序列，或与SEQ ID NO:7或8所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或
    所述抗VISTA抗体或抗原结合片段包含轻链可变区，其中，所述轻链可变区包含SEQ ID NO:9所示的序列，或与SEQ ID NO:9所示序列相比具有至少80％同一性的序列，或与SEQ ID NO:9所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；或
    (2)所述抗VISTA抗体或抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示；或
    所述抗VISTA抗体或抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO:8所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示；
    所述抗VISTA抗体或抗原结合片段可选的还包含重链恒定区，所述重链恒定区包含氨基酸序列如SEQ ID NO:10所示的序列，或与SEQ ID NO:10所述序列相比具有至少80％同一性的序列，或与SEQ ID NO:10所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或轻链恒定区，所述轻链恒定区包含氨基酸序列如SEQ ID NO:11所示的序列，或与SEQ ID NO:11所述序列相比具有至少80％同一性的序列，或与SEQ ID NO:11所示序列相比具有一个或多个保守氨基酸取代的氨基酸序 列，或由其组成；或
    (3)所述抗VISTA抗体重链的氨基酸序列如SEQ ID NO:12所示，所述抗VISTA抗体轻链的氨基酸序列如SEQ ID NO:14所示；或
    所述抗VISTA抗体重链的氨基酸序列如SEQ ID NO:13所示，所述抗VISTA抗体轻链的氨基酸序列如SEQ ID NO:14所示。
  8. 根据权利要求1-7任一项所述的组合物、用途或方法，其中，所述其它治疗剂为抗CD3抗体或抗PD-L1抗体或抗原结合片段；例如，所述抗PD-L1抗体为阿替利珠单抗、阿维单抗、度伐利尤单抗或恩沃利单抗；
    或者，所述抗PD-L1抗体或抗原结合片段包含氨基酸序列分别如SEQ ID NO:24～26所示的HCDR1、HCDR2和HCDR3以及氨基酸序列分别如SEQ ID NO:27～29所示的LCDR1、LCDR2和LCDR3。
  9. 根据权利要求8所述的组合物、用途或方法，其中，所述抗PD-L1抗体或抗原结合片段包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO:19的第20-137位所示，或与SEQ ID NO:19的第20-137位相比具有至少80％同一性的序列，或与SEQ ID NO:19的第20-137位相比具有一个或多个保守氨基酸取代的氨基酸序列；以及，
    所述抗PD-L1抗体或抗原结合片段包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO:20的第23-129位所示，或与SEQ ID NO:20的第23-129位相比具有至少80％同一性的序列，或与SEQ ID NO:20的第23-129位相比具有一个或多个保守氨基酸取代的氨基酸序列。
  10. 根据权利要求8所述的组合物、用途或方法，其中，所述抗PD-L1抗体的重链氨基酸序列如SEQ ID NO:19的第20-467位所示；所述抗PD-L1抗体的轻链氨基酸序列如SEQ ID NO:20的第23-236位所示。
  11. 如权利要求1-9任一项所述的组合物、用途或方法，所述抗VISTA抗体或抗原结合片段和其它治疗剂分别或同时给药。
  12. 权利要求1-11任一项所述的组合物的应用，所述应用为：
    制备用于预防、治疗或改善T细胞功能障碍病症的药物中的应用；或
    权利要求1-11任一项所述的用途，其中，所述用途为预防、治疗或改善T细胞功能障碍病症；或
    权利要求1-11任一项所述的方法，其中，所述疾病为T细胞功能障碍病症。
  13. 根据权利要求12所述的应用、用途或方法，其中，所述T细胞功能障碍病症包括肿瘤、癌症或感染；
    例如，所述癌症或肿瘤选自乳腺癌、消化道癌/胃肠癌、内分泌癌、神经内分泌癌、眼 睛癌、泌尿生殖癌、生殖细胞癌、妇科癌、头颈癌、血液学/血液癌、肌肉骨骼癌、神经癌、呼吸道癌/胸腔癌、膀胱癌、结肠癌、直肠癌、结直肠癌、肺癌、子宫内膜癌、肾癌、胰腺癌、肝癌、胃癌、睾丸癌、食道癌、前列腺癌、脑癌、宫颈癌、卵巢癌和甲状腺癌、白血病、淋巴瘤、骨髓发育不良症候群或骨髓瘤。
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