WO2022206677A1 - 抗vista抗体及其应用 - Google Patents

抗vista抗体及其应用 Download PDF

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WO2022206677A1
WO2022206677A1 PCT/CN2022/083390 CN2022083390W WO2022206677A1 WO 2022206677 A1 WO2022206677 A1 WO 2022206677A1 CN 2022083390 W CN2022083390 W CN 2022083390W WO 2022206677 A1 WO2022206677 A1 WO 2022206677A1
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antibody
seq
amino acid
antigen
binding fragment
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PCT/CN2022/083390
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French (fr)
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黄文龙
刘启源
张慧
黄贤明
陈振埕
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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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/12—Antivirals
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to an anti-VISTA antibody and application thereof.
  • 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
  • Human VISTA genes are predominantly 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 (>80%) across species.
  • VISTA can either act 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 ligands) Immunoglobulin Domain Protein 3, VSIG-3). Studies have shown that VISTA is expressed on the surface of naive T cells and is essential for maintaining T cell silencing and peripheral immune tolerance.
  • both VISTA monoclonal antibody or VISTA gene knockout significantly improved CD4 + and CD8 + T cell-mediated antitumor immunity; in EAE (experimental allergic encephalomyelitis) model , VISTA monoclonal antibody or VISTA gene knockout aggravated disease progression; in ovarian cancer models, VISTA monoclonal antibody significantly prolonged the survival of mice expressing high levels of VISTA.
  • VISTA is a potential target for cancer therapy.
  • the present invention provides anti-VISTA antibodies or antigen-binding fragments, which can specifically bind to VISTA, block the immunosuppressive signal pathway downstream of VISTA, and help the immune system to clear tumor cells.
  • HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:2;
  • HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:3;
  • LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:4;
  • LCDR2 comprising or consisting of the amino acid sequence shown 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 comprising or consisting 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.
  • HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:2;
  • HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:3.
  • HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1
  • HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2
  • HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 amino acid sequence or consist of it.
  • LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:4;
  • LCDR2 comprising or consisting of the amino acid sequence 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 comprising or consisting 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 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4
  • LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5
  • LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6 amino acid sequence or consist of it.
  • HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:2;
  • HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:3;
  • LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO:4;
  • LCDR2 comprising or consisting of the amino acid sequence 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 comprising or consisting 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 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 shown in NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.
  • the substitutions are conservative amino acid substitutions.
  • Some embodiments provide an antibody or an antigen-binding fragment that specifically binds VISTA, the heavy chain variable region of the antibody or antigen-binding fragment comprising the sequence shown in SEQ ID NO: 7 or 8, A sequence having at least 80% identity compared to the sequence shown in SEQ ID NO: 7 or 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 an amino acid sequence consisting of its composition.
  • Some embodiments provide an antibody or an antigen-binding fragment that specifically binds VISTA, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:9, which is the same as SEQ ID NO: 9.
  • SEQ ID NO:9 A sequence having at least 80% identity compared to the sequence shown in ID NO: 9, 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.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:7
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:9 the sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:8, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:9 the sequence shown.
  • the 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.
  • the isotype is IgGl, IgG2, IgG3, or IgG4.
  • the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the Fc is a variant Fc region.
  • the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parent Fc region.
  • the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region.
  • variant Fc regions may have altered (ie, increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding .
  • the Fc region amino acid modifications can alter the affinity of the variant Fc region for Fc ⁇ Rs (Fc ⁇ receptors) relative to the parent Fc region.
  • the Fc region is derived from IgGl or IgG4.
  • the Fc region mutation is N297A.
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a scFv, Fab, or F(ab) 2 . In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 10, or is at least 80% identical to the sequence set forth in SEQ ID NO: 10 Sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 10, or consisting of; and/or
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 80% identity compared to the sequence set forth in SEQ ID NO: 11, or a sequence with SEQ ID NO: 11
  • the sequence shown in ID NO: 11 is compared to or consists of an amino acid sequence having one or more conservative amino acid substitutions.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as set forth in SEQ ID NO: 10
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as SEQ ID NO: 10 The sequence shown in NO:11.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 12 or 13, a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 12 or 13, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 12 or 13, or consisting of; and/or
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 14, a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO: 14, or a sequence shown in SEQ ID NO: 14 compared to, or consisting of, amino acid sequences having one or more conservative amino acid substitutions.
  • 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 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 antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).
  • the antibody has two heavy chains that are identical in sequence and two light chains that are identical in sequence, and the Fc regions are paired to form a disulfide bond.
  • the antigen-binding fragment is a Fab, Fv or scFv.
  • the present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment.
  • the nucleic acid is an isolated nucleic acid.
  • the present invention also provides a vector comprising the nucleic acid.
  • the vector is an isolated vector.
  • the present invention also provides a host cell comprising the nucleic acid or vector.
  • the host cell is an isolated host cell.
  • the host cells are CHO cells, HEK cells (eg, HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells, or murine L cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the present invention also provides methods and uses for preventing or treating tumors, cancers or infections.
  • a method for treating or ameliorating a T cell dysfunctional disorder is provided, the method comprising administering to a patient an effective dose of the antibody or antigen-binding fragment.
  • use of the antibody or antigen-binding fragment for the treatment or amelioration of a T cell dysfunctional disorder is provided.
  • use of the antibody or antigen-binding fragment in the manufacture of a medicament for the treatment or amelioration of T cell dysfunction is provided.
  • the T cell dysfunctional disorders include, but are not limited to, infections caused by bacteria, viruses, fungi, or protozoa, as well as cancers 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, nerve cancer, respiratory cancer/thoracic cancer, bladder cancer, colon cancer, rectal cancer, lung cancer, endometrial cancer, kidney cancer, pancreatic cancer, liver cancer, stomach cancer, testicular cancer, esophagus cancer , prostate, brain, cervical, ovarian and thyroid cancers.
  • cancers and tumors include, but are not limited to, leukemias, melanomas, and lymphomas.
  • leukemia includes, but is not limited to, lymphocytic leukemia or myeloid leukemia (such as, eg, 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.
  • 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, and HSV, among others.
  • the present invention also provides diagnostic methods and uses.
  • a method for detecting expression of VISTA in a sample is provided, the sample is contacted with the antibody or antigen-binding fragment, so that the antibody or antigen-binding fragment binds to VISTA, and detects its binding, i.e. the expression of VISTA in the sample content.
  • use of the antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing cancer or tumor is provided.
  • a diagnostic or prognostic kit comprising the antibody or antigen-binding fragment
  • the kit further comprises a second antibody that specifically recognizes the anti-VISTA antibody; any
  • the second antibody also includes a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; optionally, the kit is used to detect the presence of VISTA in the sample or its level; optionally, the kit also includes antibodies or antigen-binding fragments directed against other antigens, and/or cytotoxic agents, and optionally, instructions for use.
  • the invention provides anti-VISTA antibodies or antigen-binding fragments and applications thereof.
  • the antibodies or antigen-binding fragments of the invention can specifically bind to VISTA, block the immunosuppressive signal pathway downstream of VISTA, and help the immune system to clear tumor cells.
  • the antibodies or antigen-binding fragments of the present invention can be used for the treatment or amelioration of T cell dysfunction disorders, and can also be used for the diagnosis and prognosis of cancer or tumors.
  • Figure 1 is the SDS-PAGE map of the anti-VISTA antibody in Example 1 of the present invention; wherein, lane M represents maker, lane 1 represents antibody P48-6-K, lane 2 represents 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 antibody promotes T cell proliferation; in the figure, ** means p ⁇ 0.01, **** means p ⁇ 0.0001.
  • Figure 4A shows the effect of antibodies on tumor volume.
  • Figure 4B shows the effect of antibody on tumor weight; in the figure, * means p ⁇ 0.05, ** means p ⁇ 0.01.
  • Figure 5 shows the effect of antibodies on the body weight of mice.
  • an entity refers to one or more of such entities, eg "an antibody” should be understood to mean one or more antibodies, thus the term “an” (or “an” ), “one or more” and “at least one” are used interchangeably herein.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide”, and refers to a molecule consisting of amino acid monomers linked linearly 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 particular length of the product.
  • the definition of “polypeptide” includes a 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 in place of, or used 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-native Amino acid modifications that occur.
  • a polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a given nucleic acid sequence, and it 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 can be encoded by a nucleic acid directly or in a precursor form.
  • 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 same amino acid is encoded by different codons called “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 Amino 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).
  • alanine three-letter code: ala, one-letter code: A
  • arginine arg, R
  • asparagine asparag
  • Constant amino acid substitution refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein.
  • 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 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
  • the number of amino acids in "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 (including endpoints) between any two of these values, or any value therein.
  • the number of amino acids for a "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 (including endpoints) between any two of these values, or any value therein.
  • isolated refers to other components in the cell's natural environment, such as DNA or RNA, respectively of one or more of the isolated molecules.
  • isolated refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or cell culture medium 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, and would not exist in, their natural state.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are typically prepared by at least one purification step. In some embodiments, the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any of these values The range (including the endpoints) between any two values of or any value therein.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, non-limiting examples may be combined to produce polynucleotides that do not normally exist or peptide.
  • 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. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, 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 these A range (including endpoints) between any two values in a numerical value or any value therein.
  • a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (eg, 90%, 95%, 98% or 99%) "identity or sequence identity" to another sequence refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned.
  • 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 by Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignments are preferably performed using default parameters.
  • Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides having the same or similar biological activity.
  • a polynucleotide is a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when polynucleotides For RNA, thymine is replaced by uracil (U).
  • a "polynucleotide sequence” can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, eg for functional genomics and homology searches.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (eg probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation to a labeling component.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • encoding when applied to a polynucleotide refers to a polynucleotide referred to as “encoding” a polypeptide, transcribed and/or in its native state or when manipulated by methods well known to those skilled in the art Or translation can yield the polypeptide and/or fragments thereof.
  • 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-like, Fab'-like, and F(ab') 2 -like), single-chain-like 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-like, Fab'-like, and F(ab') 2 -like), single-chain-like Fvs (scFv).
  • Antibody refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide in the molecule that 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, the complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand binding portions thereof (CH), a light chain constant region (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein.
  • the CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3).
  • Antibodies and antigen-binding fragments can specifically recognize and bind to one or more (eg, two) antigenic polypeptides or polypeptide complexes.
  • 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 portion of an antibody, and the composition of the antibody fragment of the invention may be similar to that of 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 that is recognized by the intact antibody.
  • antibody fragment includes aptamers, Spiegelmers 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 (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region has been removed and the linker introduced, the protein retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in US Pat. No. 5,892,019.
  • antibody includes a wide variety of biochemically distinguishable polypeptides. Those of skill in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are known. All immunoglobulin species are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can bind to a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • Each heavy chain can bind to a kappa or lambda light chain.
  • immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portion of the two heavy chains is joined by a covalent disulfide bond or non-covalent bond.
  • the amino acid sequence extends from the N-terminus of the forked terminus in the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of immunoglobulin kappa light chain is V ⁇ ; the variable region of 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) portions 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, and the like. By convention, the numbering of constant regions increases as they become further from the antigen binding site or amino terminus of the antibody.
  • the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.
  • each antigen-binding domain In naturally occurring antibodies, the six “complementarity determining regions" or “CDRs” present in each antigen-binding domain are the short, A non-contiguous amino acid sequence that specifically binds an antigen. The remaining other amino acids in the antigen binding domain, referred to as the "framework” region, show less inter-molecular variability.
  • the framework regions mostly adopt a ⁇ -sheet conformation, and the CDRs form loop structures to which they are attached, or in some cases form part of a ⁇ -sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions.
  • An antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • an antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • amino acids comprising CDRs and framework regions can be identified by known methods (see Kabat, E., et al., U.S. Department 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 as defined by Kabat and Chothia include overlaps 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 numbers encompassing 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 CDRs contain based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also define a numbering system applicable to variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independent of experimental data other than the sequence itself.
  • Kabat Numbering means 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 may 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 of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • a "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 regions of antibodies can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide can 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 part 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.
  • a “light chain constant region” includes a portion of the amino acid sequence from an antibody light chain.
  • the light chain constant region comprises 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 dimerize 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.
  • the "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • the "CH1 domain” includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain.
  • a branched carbohydrate chain is attached to N297 in each of the two CH2 domains in the intact native IgG molecule.
  • 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 a portion of the heavy chain region connecting the CH1 domain and the CH2 domain.
  • the hinge region comprises about 25 residues and is resilient, 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)).
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by a disulfide bond.
  • Chimeric antibody refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial or modified) are derived from a second species.
  • the variable regions are derived from a non-human source (eg, mouse or primate), and the constant regions are derived from a human source.
  • Specifically binds or “specifically for” generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment with a specific antigen through complementary binding of its antigen-binding domain to an epitope.
  • Specificity can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds 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 be more specific for that antigen than antibody "B”.
  • Specific binding can be described by the equilibrium dissociation constant ( KD ), with a smaller KD implying tighter binding.
  • Antibodies that "specifically bind" antigen a include those with an equilibrium dissociation constant K of antigen a that is 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 both therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent, slow, ameliorate, or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable As a result, remission of symptoms, reduction of disease severity, stabilization of disease state (ie, no worsening), delay or slowdown of disease progression, improvement, alleviation, alleviation or disappearance of disease state (whether in part or in whole), prolongation and Expected duration of survival when not receiving treatment, etc.
  • Patients in need of treatment include patients already suffering from a condition or disorder, a patient susceptible to a condition or disorder, or a patient in need of prevention of such a condition or disorder, which may or may be expected from administration of the antibodies or pharmaceutical compositions disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, and the like.
  • the present invention provides antibodies or antigen-binding fragments thereof with high affinity to VISTA protein.
  • the antibodies or antigen-binding fragments of the present invention exhibit potent binding activity, biological activity, and are useful in therapeutic and diagnostic applications.
  • these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints and activate lymphocytes to release cytokines for the treatment of various types of cancers, tumors, or infections and other 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 of SEQ ID NO: 12 other than Fc, and the light chain of the antigen-binding fragment comprises the amino acid sequence of 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 of SEQ ID NO: 13 other than Fc, and the light chain of the antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 14 .
  • the antibodies of the invention contain two heavy chains (or heavy chain fragments) that are identical in sequence and two light chains (or light chain fragments) that are identical in sequence.
  • the sequences of the antibodies or antigen-binding fragments disclosed in the present invention may be replaced, and the amino acid sequences thereof differ from the naturally occurring amino acid sequences of the antibody after replacement.
  • the substituted amino acid sequence can be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence, such as it can be about 80%, about 85%, about 90% identical to the starting sequence , about 95%, about 98%, about 99%, or a range (including endpoints) between any two of these values, or any value therein.
  • the antibody or antigen-binding fragment comprises an amino acid sequence having one or more modifying groups.
  • the antibodies or antigen-binding fragments of the present disclosure may contain flexible linker sequences, or may be modified to add functional groups (eg, PEG, drugs, toxins, or tags).
  • antibodies and antigen-binding fragments disclosed herein include derivatives that are modified, ie, modified by covalent attachment of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment from interacting with the epitope combine.
  • 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 a number of chemical modifications can be performed by existing techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the antibodies, antigen-binding fragments and modified derivatives thereof disclosed herein include salts formed with acids and/or bases.
  • the antibody or antigen-binding fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • An antibody or antigen-binding fragment can be detectably labeled by conjugating it to a chemiluminescent compound.
  • 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 acridine esters, imidazoles, acridine salts, and oxalate esters.
  • the present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention.
  • the polynucleotides disclosed in the present invention can encode a heavy chain variable region, a light chain variable region, an Fc region, a partial heavy chain variable region, a partial light chain variable region, a heavy chain or a light chain, and the like. Methods of making antibodies are well known in the art and described in the present invention.
  • the antibodies produced do not elicit an adverse immune response in the animal (eg, human) to be treated.
  • the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized 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 residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, eg, residues that improve antigen binding.
  • 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 alignment to identify framework residues that are aberrant at specific positions. (Refer to U.S. Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Pat. Nos.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering an antibody to modify T cell epitopes (see eg WO/9852976 A1 and WO/0034317 A2).
  • the heavy and light chain variable region sequences 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 positions of other key residues within the sequence.
  • CDRs complementarity determining regions
  • a series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed, and these sequences are subsequently incorporated into a series of binding polypeptides.
  • the genes containing the modified variable and human constant regions of the intact heavy and light chains are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies.
  • the optimal antibodies are then identified by comparing the antibodies in appropriate biochemical and biological experiments.
  • the binding specificity of the disclosed antibodies or antigen-binding fragments can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFv The preparation of scFv can be found in techniques for producing single-chain units (US 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)).
  • the heavy and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit, resulting in a single-chain fusion peptide.
  • Techniques for the assembly of 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. Patent Nos. 4,946,778 and 5,258,498, and Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. As described in 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 portions of the antibody are derived from different animal species, eg, antibodies having the variable regions of murine monoclonal antibodies and the constant regions of human immunoglobulins.
  • 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 hereby 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 from immunoglobulin sequences. See also 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 patents are incorporated herein by reference.
  • DNA encoding the desired monoclonal antibody can be isolated and subjected to Sequencing.
  • Isolated and subcloned hybridoma cells can serve as a source of such DNA.
  • the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that does not produce other immunoglobulins in cells.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of antibodies, as described in US Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Pat. No. 5,658,570.
  • one or more CDRs of the antibodies of the invention can be inserted into framework regions, eg, into human framework regions, to construct humanized, non-fully human antibodies.
  • Framework regions can 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 list of human framework regions).
  • Some polynucleotides may encode an antibody that specifically binds to at least one epitope of an antigen of interest resulting from a combination of framework regions and CDRs.
  • One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected to improve binding of the antibody to its antigen.
  • substitution or deletion of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed using this method, resulting in antibody molecules lacking one or more interchain disulfide bonds.
  • Other changes to polynucleotides that are within the skill in the art are also encompassed by the present invention.
  • Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, etc. 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, which in their entirety include Supplementary content is incorporated by reference in its entirety.
  • DNA encoding the antibody can be designed and synthesized according to the amino acid sequences of the antibodies described herein according to conventional methods, placed in an expression vector, then transfected into host cells, and the transfected host cells are grown in culture to produce Monoclonal antibodies.
  • an antibody expression 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 by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cellular promoters such as muscle can be used.
  • 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 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain a selectable marker.
  • selectable markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening isolation of successful cells.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in selective medium, the successfully transfected cells grow in large quantities to produce the desired target protein.
  • mutations can be introduced in the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those of skill in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions.
  • Variants include derivatives
  • 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 the treatment or amelioration of various types of cancers, tumors, or related diseases such as infections, the methods comprising administering to a patient in need thereof an effective dose of an anti-VISTA antibody or antigen-binding fragment.
  • the use of an anti-VISTA antibody or antigen-binding fragment for the treatment or amelioration of related diseases such as cancer, tumor or infection is provided.
  • use of the anti-VISTA antibody or antigen-binding fragment in the preparation of a medicament for treating or ameliorating related diseases such as cancer, tumor or infection is provided.
  • the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular 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. These factors are left to the judgment of health care professionals, including those within the purview of those of 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 effect desired.
  • the dose to be used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • the antibody of the invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, the administration is administered every 1 week, 2 weeks, 3 weeks, or monthly.
  • Methods of administration of the antibody or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection.
  • the pharmaceutical compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelia or mucocutaneous (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.
  • compositions containing the antibodies or antigen-binding fragments of the present invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (eg, by powder, ointment) , drops or transdermal patches), orally or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration can be systemic or topical. Furthermore, it may be desirable to introduce the antibodies of the invention into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injection; intracerebroventricular injection may be accomplished via an intraventricular catheter connected, for example, to a reservoir (which may be an Ommaya reservoir). Assisted injection. Pulmonary administration is also possible, eg, by the use of an inhaler or nebulizer, as well as the use of aerosolized formulations.
  • Antibodies of the invention may be administered topically to the area in need of treatment; by, but not limited to, topical administration during surgery, such as topical application in combination with a postoperative wound dressing, by injection, by catheter, by suppository, or by implant
  • the implant is a porous, non-porous or gel-like material, including membranes (eg, silicone rubber membranes) or fibers.
  • membranes eg, silicone rubber membranes
  • care must be taken to use materials that do not absorb the protein.
  • compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody, which can be administered in vivo to facilitate expression of the protein encoded thereby by constructing it as part of a suitable nucleic acid expression vector, Parts of the vectors described above are then administered to make them intracellular, for example, by the use of retroviral vectors (see US Pat. No. 4,980,286), or by direct injection, or by the use of particle bombardment (eg, a gene gun; Biolistic, Dupont) , or coated with lipids or cell surface receptors or transfection reagents, or administered by linkage to homeobox peptoids known to enter the nucleus (see e.g.
  • nucleic acid can be introduced into a cell by homologous recombination and integrated into the host cell DNA for expression.
  • the antibody of the invention is administered to a 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.
  • a second or more doses of the antibody or antigen-binding fragment may be administered subsequent to the initial dose, at about the same or less dose as the initial dose, wherein the subsequent doses may be separated by at least 1 day to 3 days; or at least one week.
  • the uptake and tissue penetration capability of the antibody eg, into the brain
  • Methods for treating disease are typically tested in vitro, comprising administration of an antibody or derivative of the invention, followed by in vivo testing for the desired therapeutic or prophylactic activity in an acceptable animal model, followed by administration to humans.
  • Suitable animal models including transgenic animals, are known to those of ordinary skill in the art.
  • in vitro assays for demonstrating the therapeutic use of the antibodies, antigen-binding fragments of the invention include the effect of the antibody on a cell line or patient tissue sample.
  • the effect of antibodies on cell lines and/or tissue samples can be detected using techniques known to those of skill in the art, such as those disclosed elsewhere herein.
  • In vitro assays that can be used to determine whether to administer a specific antibody in accordance with the present invention include in vitro cell culture experiments in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound and the effect of such compound is observed on The effect of tissue samples.
  • Various known delivery systems can be used to administer the antibodies or derivatives of the invention or polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated Endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviral or other vectors, and the like.
  • the anti-VISTA antibodies or antigen-binding fragments of the invention can be combined with other therapeutic or prophylactic regimens, including administration of one or more antibodies or antigen-binding fragments of the invention, in conjunction with one or more other therapeutic agents or methods used or combined.
  • other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, hormone therapy, and the like.
  • the antibody and other therapeutic agents may be administered simultaneously or separately.
  • the antibody or antigen-binding fragment of the invention can be administered before or after administration of the other other therapeutic agent.
  • angiogenesis inhibitors that can be administered with the antibodies or antigen-binding fragments of the invention include, but are not limited to, angiostatin (a plasminogen fragment), anti-angiogenic antithrombin III, and ribozymes .
  • anti-cancer agents that can be administered with the antibodies or antigen-binding fragments of the invention include, but are not limited to: 5-fluorouracil, acevecine, aldesleukin, hexamethylmelamine, aminoglutamine, amylazine pyridine, anastrozole, antramycin, asparaginase, azacitidine, azatipide, azithromycin, batamastat, bicalutamide, bleomycin sulfate, buquina sodium , Brompiramine, Busulfan, Carboplatin, Carmustine, Carrubicin Hydrochloride, Carzalexine, Cedifingol, Chlorambucil, Ciromycin, Cisplatin, Cladrix Bing, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine,
  • therapeutic antibodies that can be administered with the antibodies or antigen-binding fragments of the invention include, but are not limited to, nivolumab, pembrolizumab, tremelimumab, ipilimumab, tremelimumab, Touzumab, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, anti-OX40 antibody and anti-GITR antibody, etc.
  • compositions comprise an effective amount of an anti-VISTA antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises 0.1%-90% anti-VISTA antibody or antigen-binding fragment.
  • the pharmaceutical composition further comprises an anticancer agent (eg, an immune checkpoint inhibitor).
  • the term "pharmaceutically acceptable” refers to a substance approved by a regulatory agency of the government or listed in a recognized pharmacopeia for use in animals, particularly in humans.
  • pharmaceutically acceptable carrier generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid and the like.
  • carrier refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc.
  • the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates, if desired.
  • Antibacterial agents such as benzyl alcohol or methylparaben, 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 formulations and the like.
  • the composition can be formulated as a suppository with traditional 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 a suitable amount of carrier to provide a form suitable for administration to the patient.
  • the formulation should be suitable for the mode of administration.
  • the parental formulation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • compositions are formulated according to conventional procedures into pharmaceutical compositions suitable for intravenous injection into humans.
  • Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffer.
  • the composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied in unit dosage form either individually or mixed together, eg, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container (eg, an ampule or sachet) indicative of the amount of active agent.
  • the composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline.
  • ampoules of sterile water for injection or saline can be used so that the active ingredient can be mixed prior to administration.
  • the compounds of the present invention may be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include salts formed with anions derived from, for example, hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2- Salts with cations of ethylaminoethanol, histidine, procaine, etc.
  • EC50 or concentration for 50% of maximal effect ( EC50 ) refers to the concentration that elicits 50% of the maximal effect.
  • the "parental Fc region” can be a naturally occurring Fc region, and the gene encoding the Fc region can be from human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parental Fc region is SEQ ID NO: 10 , the Fc region in SEQ ID NO: 12 or SEQ ID NO: 13.
  • the antibodies prepared in this example include anti-VISTA antibodies.
  • the amino acid sequences and nucleic acid sequences related to the antibody examples are shown in Table 1 to Table 5; the Fc region of the heavy chain in Table 4 is marked with a single underline, and the nucleic acid sequence of the signal peptide in Table 5 marked with double underscores.
  • nucleotide 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 the light chain.
  • signal peptides are added to the N-termini of the heavy and light chains.
  • the optimized and synthesized nucleic acid sequences were cloned into a vector (the vector can be selected from pCDNA3.1, V79020 from Invitrogen), and then a large number of plasmids were extracted respectively.
  • HEK293F cells were transiently expressed by the heavy chain expression vector and the light chain expression vector at a plasmid molar ratio of 1:1.
  • SEQ ID NO:17 the nucleic acid sequence comprising the signal peptide
  • SEQ ID NO:18 the nucleic acid comprising the signal peptide
  • the heavy chain variable region nucleic acid sequence of P48-6-K (SEQ ID NO: 20, the nucleic acid sequence comprising the signal peptide) and the light chain variable region nucleic acid sequence (SEQ ID NO: 21, the nucleic acid sequence comprising the signal peptide) Nucleic acid sequence) was cloned into an expression vector containing the nucleic acid (SEQ ID NO: 22) encoding the constant region of the human IgG1 heavy chain and the expression vector containing the nucleic acid (SEQ ID NO: 23) encoding the constant region of the light chain of human Ig kappa, respectively, by enzyme digestion In the carrier; wherein, the amino acid sequence of the heavy chain signal peptide is SEQ ID NO: 15, and the amino acid sequence of the light chain signal peptide is SEQ ID NO: 16; the restriction site endonuclease used in the heavy chain is HindIII/KpnI, and the light chain The restriction site endonuclease used was HindIII/Bsi
  • the heavy chain variable region nucleic acid sequence and the light chain variable region nucleic acid sequence were respectively cloned into the nucleic acid (SEQ ID NO: 22) containing the encoding human IgG1 heavy chain constant region by enzyme digestion.
  • the restriction site endonuclease used for the heavy chain is HindIII/KpnI, and the restriction site endonuclease used for the light chain is HindIII/BsiWI.
  • 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).
  • the culture medium was purified by immobilized metal affinity chromatography (IMAC) using Protein A column (GE Healthcare), and the purity of the purified antibody protein was >95%.
  • the purified antibodies were detected by gel electrophoresis. As shown in Figure 1, the antibodies P48-6-K and P48-6-T of the present application were single substances, and the molecular weights were consistent with the theoretical values. The purified antibody was sequenced, and the sequencing result was the same as the expected sequence. The purified antibody is used for affinity detection and biological activity identification, etc.
  • the antibody CDR amino acid residues of the examples of the present invention were identified according to the Kabat numbering system.
  • a Biacore T200 surface plasmon resonance instrument was used to determine the affinity constant of the antibody.
  • the main test process was as follows: protein A chip was used for detection, 100nM antibody diluent was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 ⁇ l/min, and the capture was performed for 20s.
  • the volume is about 560RU; then the flow rate is adjusted to 30 ⁇ l/min, and different concentrations (0nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, 100nM) of VISTA-His (Acro, B75-H52H0) dilution (diluted The solvent is water), passing through the surface of the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time, the binding time is 120s, the dissociation time is 300s, and finally Glycine 1.5 is used to regenerate the chip and enter the next cycle;
  • the data analysis software Evaluation Software 3.1 was used to analyze the test results, and the sensing signals collected from the sample experimental flow path were double-deducted from the reference flow path and the sample blank, and the kinetic "1:1" model was used for fitting.
  • Kinetic parameters are derived (Kon(Ka): association rate constant; Koff(Kd): dissociation rate constant; KD : association dissociation equilibrium constant
  • the antibody P48-6-K of the present invention has a good affinity with VISTA-His.
  • 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 initial concentration of anti-VISTA antibody is 2 ⁇ g/mL, 2-fold serial dilution (the dilution solvent is PBS buffer), 4 °C refrigerator overnight
  • PBST PBS+0.05% Tween, 1 liter PBS containing NaCl 8.0g, Na 2 HPO 4 0.9g, KH 2 PO 4 0.156g, KCl 0.125g, pH 7.2-7.4 washed 3 times, 5 %BSA (bovine serum albumin) was blocked at room temperature for 2 h, washed 4 times with PBST; incubated with 2 ⁇ g/mL antibody at room temperature for 1.5 h, washed 4 times with PBST; after washing, goat anti-human kappa light chain-peroxidase antibody (SIGMA, Item No.
  • SIGMA goat anti-human kappa light chain-peroxidase antibody
  • TMB tetramethylbenzidine
  • the antibodies VSTB112, P48-6-K, and P48-6-T bind to VISTA of human and cynomolgus monkeys, but not to mouse VISTA.
  • NA no binding
  • CHO-VISTA ⁇ ⁇ VISTA ⁇ ( ⁇ UniProtKB-Q9H7M9):“MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI”(SEQ ID NO:19); ⁇ SEQ ID NO:19 ⁇ , ⁇ HindIII and EcoRI digestion sites were constructed into pcDNA3.1 expression vector, and then transfected into CHO cells by electroporation.
  • the electroporation conditions were: voltage 300V, time 17ms, 4mm electroporation cup, 50 ⁇ M MSX (methionine imidosulfone) screened positive cells.
  • Use FACS flow cytometry to detect high-expressing cell lines and collect cells. After washing once with PBS, add 3 ⁇ g/ml antibody VSTB112, incubate at 4°C for 1 h, wash twice with PBS, and then add 100 ⁇ l 1:1 500 diluted goat anti-human IgG-Fc PE fluorescent secondary antibody (Cat. No. 12-4998-82, eBioscience), incubated at 4°C for 1 h, washed twice with PBS, and analyzed by C6 flow cytometer; Named CHO-VISTA cells.
  • the initial concentration of anti-VISTA antibody was 5 ⁇ g/mL, 2-fold serial dilution, 100 ⁇ L antibody dilution solution and 100 ⁇ L 5 ⁇ 105 CHO-VISTA cells per well; mixed well, incubated at 4°C for 30 min; after washing the cells twice with PBS, add sheep After anti-human IgG-Fc PE fluorescent secondary antibody (1:1000 PBS dilution), incubate at 4°C for 30 min, wash cells twice with PBS; resuspend in 200 ⁇ L PBS (Gibco), and measure the second channel fluorescence with flow cytometer CytoFLEX strength.
  • Example 5 Antibody blocks the binding of VISTA to ligand VSIG-3
  • VSIG-3 Recombinant Human VSIG3 Fc Chimera Protein, R&D, Cat. No. 9229-VS-050
  • 10 ⁇ g/mL biological Vistin-labeled VISTA was mixed with antibody diluent (initial concentration of 15 ⁇ g/mL, 2-fold gradient dilution), incubated at room temperature for 1.5 h, washed 4 times with PBST, and then washed with streptavidin-labeled horseradish peroxidation Streptavidin-HRP was incubated for 0.5 h; washed 6 times with PBST, added 100 ⁇ L of tetramethylbenzidine (TMB), incubated at 37°C for 5 min, and stopped by adding 50 ⁇ L of 0.1 M sulfuric acid; the absorbance at 450
  • the antibody P48-6-K of the present invention can block the binding of VSIG-3 to VISTA in a concentration-dependent manner.
  • Dilute anti-VISTA antibody in RPMI-1640 medium (initial concentration is 6 ⁇ g/mL, 3-fold gradient dilution), add to U-shaped 96-well plate, 100 ⁇ L per well, 2 parallel wells; add 100 ⁇ L PBMC cell solution, mix well ; Put into 37 °C, 5% carbon dioxide incubator for about 18h.
  • IP-10 interferon-inducible protein 10
  • the detection steps are: IP-10 capture antibody coated plate at 4°C overnight, washed 3 times with PBST, blocked with 1% BSA at room temperature for 2 h, washed 4 times with PBST; U-shaped 96-well plate (including PBMC cells and antibodies) was centrifuged at 2000 rpm for 5 min, and 100 ⁇ L of The supernatant was added to the ELISA plate, the standard with appropriate concentration gradient was prepared, incubated at room temperature for 1.5 h, washed 4 times with PBST, added 100 ⁇ L of biotin-labeled IP-10 detection antibody solution, incubated at room temperature for 1.5 h, washed 4 times with PBST; added 100 ⁇ L of chain Mycoavidin-labeled horseradish peroxidase solution was incubated at room temperature for 0.5 h, was
  • the PBMC cell isolation method was the same as that in Example 4.
  • CD3 + T cell isolation kit MojosortTM Human CD3 T cell Isolation Kit, Biolegend was used to isolate CD3 positive T cells (ie CD3 + T cells) from PBMC.
  • the antibody P48-6-K and anti-CD3 antibody can promote the proliferation of T cells in the case of co-coating.
  • Experimental mice female BALBc-hVISTA mice (VISTA humanized mice, from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).
  • Experimental cells collect CT26.WT (colorectal cancer) cells in logarithmic growth phase, remove the culture medium, wash and inoculate; inoculation volume: 5 ⁇ 10 5 cells/100 ⁇ L/cell; inoculation location: above the right thigh of the mouse .
  • CT26.WT colonal cancer
  • mice After 3 days of tumor cell inoculation, the mice were randomly divided into 3 groups according to their body weight, with 10 mice in each group; the route of administration was intraperitoneal injection (ip), and the administration frequency was twice a week (BIW); the day of inoculation was defined It is D0 day, and on the day of grouping, the administration starts according to the experimental design; the administration dosage and administration method are shown in Table 9, and the administration volume is calculated according to 10 ⁇ L/g.
  • tumor effects on the animals' normal behavior were routinely monitored weekly.
  • Specific content activity of experimental animals, food and water intake, weight gain or loss, eyes, coat and other abnormalities.
  • Clinical symptoms observed during the trial were recorded in the raw data. After the start of administration, the body weight was weighed twice and the tumor volume was measured twice in the first week; thereafter, the body weight was weighed three times and the tumor volume was measured three times a week.

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Abstract

本发明提供了抗VISTA抗体或抗原结合片段及其应用，本发明抗体或抗原结合片段可以特异性结合VISTA，阻断VISTA与其配体之间的相互作用。本发明抗体或抗原结合片段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抗体及其应用 技术领域
本发明属于生物医药领域，尤其涉及抗VISTA抗体及其应用。
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。
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。
VISTA是癌症治疗的潜在靶标。
发明内容
本发明提供了抗VISTA抗体或抗原结合片段，这些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。
一些实施方案提供了抗体或抗原结合片段，所述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氨基酸序列：
(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。
一些实施方案中提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合VISTA，并且包含：
(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。
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合VISTA，并且包含：
(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。
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合VISTA，并且包含：
(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；
(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；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，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所述的LCDR2和如SEQ ID NO:6所示的LCDR3。
在一些实施方案中，所述取代为保守氨基酸取代。
一些实施方案提供了抗体或抗体原结合片段，所述抗体或抗原结合片段特异性结合VISTA，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:7或8所示的序列，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。
一些实施方案提供了抗体或抗体原结合片段，所述抗体或抗原结合片段特异性结合VISTA，所述抗体或抗原结合片段的轻链可变区包含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，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:7所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:9所示的序列。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:8所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:9所示的序列。
在一些实施方案中，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，轻链恒定区是κ或λ链恒定区。在一些实施方案中，抗体或其抗原结合片段是IgG、IgM、IgA、IgE或IgD其中一种同种型。在一些实施方案中，同种型是IgG1、IgG2、IgG3或IgG4。在一些实施方案中，抗体或抗原结合 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N297A。
在一些实施方案中，所述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，所述抗体或抗原结合片段为单克隆抗体。
在一些实施方案中，所述抗体或抗原结合片段的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
所述抗体或抗原结合片段的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，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:10所示的序列，所述抗体或抗原结合片段的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，所述抗体的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
所述抗体的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。
在一些实施方案中，所述抗原结合片段为Fab、Fv或scFv。
本发明还提供了一种编码所述的抗体或抗原结合片段的核酸。在一些实施方案中，所述核酸为分离的核酸。
本发明还提供了一种包含所述的核酸的载体。在一些实施方案中，所述载体为分离的载体。
本发明还提供了一种包含所述核酸或载体的宿主细胞。在一些实施方案中，所述宿主细胞为分离的宿主细胞。在一些实施方案中，所述宿主细胞为CHO细胞、HEK细胞(如HEK293F细胞)、BHK细胞、Cos1细胞、Cos7细胞、CV1细胞或鼠L细胞。
本发明还提供了一种药物组合物，所述药物组合物包含上述的抗体或抗原结合片段，以及药学上可接受的载体。
本发明还提供了预防或治疗肿瘤、癌症或感染的方法和用途。在一些实施方案中，提供了用于治疗或改善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所述抗体或抗原结合片段。在一些实施方案中，提供了所述抗体或抗原结合片段在用于治疗或改善T细胞功能障碍病症中的应用。在一些实施方案中，提供了所述抗体或抗原结合片段在制备用于治疗或改善T细胞功能障碍的药物中的应用。
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、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。
本发明还提供了诊断方法和用途。在一些实施方案中，提供了检测样品中VISTA表达的方法，使样品与所述抗体或抗原结合片段进行接触，使得所述抗体或抗原结合片段结合VISTA，并检测其结合，即样品中VISTA的含量。在一些实施方案中，提供了所述抗体或抗原结合片段在制备用于诊断或预后癌症或肿瘤试剂盒中的应用。在一些实施方案中，提供了一种包含所述抗体或抗原结合片段的诊断或预后试剂盒；任选的，所述试剂盒还包括第二抗体，其特异性识别所述抗VISTA抗体；任选的，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；任选的，所述试剂盒用于检测VISTA在样品中的存在或其水平；任选的，所述试剂盒还包括针对其它抗原的抗体或抗原结合片段，和/或细胞毒性剂，和任选的，使用说明书。
本发明提供了抗VISTA抗体或抗原结合片段及其应用，本发明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。本发明抗体或抗原结合片段可以用于治疗或改善T细胞功能障碍病症，也可以用于癌症或肿瘤的诊断和预后。
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maker，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示抗VISTA抗体阻断VSIG-3与VISTA的结合。
图3示抗VISTA抗体促进T细胞增殖；图中，**表示p<0.01，****表示p<0.0001。
图4A示抗体对肿瘤体积的影响。
图4B示抗体对肿瘤重量的影响；图中，*表示p<0.05，**表示p<0.01。
图5示抗体对小鼠体重的影响。
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+SwissProtein+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。
多聚核苷酸是由四个核苷酸碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
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(例如类Fab、类Fab'和类F(ab') 2)、类单链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、IgG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V λ。
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 Proteins of 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IgG 1分子的CH1结构域和源自IgG 3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IgG 1分子和部分源自IgG 3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IgG 1分子和部分源自IgG 4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))。
“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(K D)来描述，较小的K D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K D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。
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，本发明公开的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)。
本发明公开的抗体、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。本发明公开的抗体、抗原结合片段及其被修饰的衍生物包括其与酸和/或碱形成的盐。
在一些实施方案中，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。
抗体和编码抗体的多聚核苷酸的制备方法
本发明还公开了编码本发明所述抗体、抗原结合片段、及其衍生物的多聚核苷酸或核酸分子。本发明公开的多聚核苷酸可以编码重链可变区、轻链可变区、Fc区、部分重链可变区、部分轻链可变区、重链或轻链等。制备抗体的方法是本领域公知的并且在本发明中有所描述。
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/9852976 A1和WO/0034317 A2)。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293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。在一些实施方案中，提供了抗VISTA抗体或抗原结合片段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治疗或改善癌症、肿瘤或感染等相关疾病的药物中的应用。
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，本发明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。
抗体或衍生物的施用方法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。
本发明抗体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，本发明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。
通常在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。
联合疗法
在一些实施方案中，本发明抗VISTA抗体或抗原结合片段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，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、激素疗法等。对于组合治疗，抗体可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明抗体或抗原结合片段。
在一些实施方案中，可以与本发明抗体或抗原结合片段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。
在一些实施方案中，可以与本发明抗体或抗原结合片段一起施用的治疗性抗体包括但不限于纳武单抗、派姆单抗、替西木单抗(tremelimumab)、伊匹木单抗、曲妥珠单抗、抗PD-L1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。
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，药物组合物还包含抗癌剂(例如免疫检查点抑制剂)。
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。
“EC 50”即半最大效应浓度(concentration for 50％of maximal effect,EC 50)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。
具体实施方式
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，抗体实施例相关的氨基酸序列和核酸序列见表1-表5；表4中重链的Fc区用单下划线标出，表5中信号肽的核酸序列用双下 划线标出。
将抗体的重链和轻链的核苷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，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。重链表达载体和轻链表达载体按质粒摩尔比1:1瞬时表达转染HEK293F细胞。以抗体P48-6-K为例，其重链核酸序列如SEQ ID NO:17所示(包含信号肽的核酸序列)，轻链核酸序列如SEQ ID NO:18所示(包含信号肽的核酸序列)，将P48-6-K的重链可变区核酸序列(SEQ ID NO:20，包含信号肽的核酸序列)和轻链可变区核酸序列(SEQ ID NO:21，包含信号肽的核酸序列)通过酶切分别克隆至含有编码人IgG1重链恒定区的核酸(SEQ ID NO：22)的表达载体和含有编码人Ig kappa轻链恒定区的核酸(SEQ ID NO：23)的表达载体中；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；重链采用的限制位点内切酶是HindIII/KpnI，轻链采用的限制位点内切酶是HindIII/BsiWI。以抗体P48-6-T为例，将重链可变区核酸序列和轻链可变区核酸序列通过酶切分别克隆至含有编码人IgG1重链恒定区的核酸(SEQ ID NO：22)的表达载体和含有编码人Ig kappa轻链恒定区的核酸(SEQ ID NO：23)的表达载体中；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；重链采用的限制位点内切酶是HindIII/KpnI，轻链采用的限制位点内切酶是HindIII/BsiWI。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。本发明实施例的抗体CDR氨基酸残基根据Kabat编号系统确认。
表1抗体的组成
Figure PCTCN2022083390-appb-000001
表2抗VISTA抗体的CDR区(Kabat编号)
名称 氨基酸序列 SEQ ID NO:
HCDR1 GYDMS 1
HCDR2 GIDGHFDSSFYADSVKG 2
HCDR3 SSYAVDLAFDY 3
LCDR1 RASQGISSYLA 4
LCDR2 AASSLQS 5
LCDR3 QQHYTTPPT 6
表3抗VISTA抗体的可变区和恒定区
Figure PCTCN2022083390-appb-000002
表4抗体的重链和轻链
Figure PCTCN2022083390-appb-000003
Figure PCTCN2022083390-appb-000004
表5抗体相关核酸序列(双下划线为编码信号肽的核酸)
Figure PCTCN2022083390-appb-000005
Figure PCTCN2022083390-appb-000006
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；K D：结合解离平衡常数)。
1)如表6所示，与抗体VSTB112相比，本发明抗体P48-6-K与VISTA-His具有良好的亲和力。
表6抗体P48-6-K与VISTA-His结合的亲和力常数
抗体编号 K D(M) K on(1/Ms) K off(1/s)
VSTB112 3.32E-10 9.92E+05 3.29E-04
P48-6-K 3.31E-10 1.04E+06 3.44E-04
2)如表7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7抗体P48-6-T与VISTA-His结合的亲和力常数
抗体编号 K D(M) K on(1/Ms) K off(1/s)
VSTB112 4.00E-10 4.68E+05 1.87E-04
P48-6-T 1.76E-10 4.87E+05 8.58E-05
实施例3抗体与不同种属VISTA结合情况的检测
100μL 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，Na 2HPO 4 0.9g，KH 2PO 4 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8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8抗体与VISTA-FC结合的EC 50(μg/mL)
抗体编号 人源VISTA-Fc 食蟹猴VISTA-Fc 鼠源VISTA-Fc
VSTB112 0.1 0.065 NA
P48-6-K 0.082 0.05 NA
P48-6-T 0.086 0.049 NA
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)：“MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAALATGACIVGILCLPLILLLVYKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI”(SEQ ID NO:19)；合成SEQ ID NO:19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EC 50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与抗体稀释液混合(以15μg/mL为起始浓度，2倍梯度稀释)，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
Figure PCTCN2022083390-appb-000007
)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(外周血单个细胞)浓度为10 6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EC 50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7T细胞增殖实验
PBMC细胞分离方法同实施例4，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) 分别和10μg/ml抗体VSTB112、P48-6-K、同型对照IgG1共同铺板，4℃过夜，PBS洗两次后按10万/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。
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，去除培养液、洗涤后接种；接种量：5×10 5cells/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3组，每组10只；给药途径为腹腔注射(i.p.)，给药频率为每周两次(BIW)；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9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，测量肿瘤体积2次；随后每周称量体重三次，测量肿瘤体积三次。肿瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×肿瘤长径×肿瘤短径 2，肿瘤体积抑制率TGIT v＝(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TGIT w＝(1-给药组平均肿瘤重量/对照组平均肿瘤重量)×100％。
表9试验设计表
Figure PCTCN2022083390-appb-000008
肿瘤体积抑制的结果如图4A、4B和表10A、10B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。如图5所示，各组小鼠的体重之间无显著性差异。
表10A第30天的肿瘤体积抑制率
组别 肿瘤平均体积(mean/mm 3) TGIT v
G1 2632.616175 —
G2 1643.574262 37.5％
G3 1632.861395 37.9％
表10B第30天的肿瘤重量抑制率
组别 肿瘤平均重量(mean/g) TGIT w
G1 3.5918 —
G2 1.9705 45.14％
G3 2.2349 37.78％

Claims (11)

  1. 一种与VISTA结合的抗体或抗原结合片段，
    所述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氨基酸序列：
    (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。
  2. 如权利要求1所述的抗体或抗原结合片段，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；和/或
    所述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。
  3. 如权利要求1-2任一项所述的抗体或抗原结合片段，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。
  4. 如权利要求1-3任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:7或8所示的序列，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
    所述抗体或抗原结合片段的轻链可变区包含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。
  5. 如权利要求4所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:7所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:9所示的序列；或
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:8所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:9所示的序列。
  6. 如权利要求1-5任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的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
    所述抗体或抗原结合片段的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。
  7. 一种与VISTA结合的抗体，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；或
    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。
  8. 一种生物材料，为
    (1)多聚核苷酸，其编码如权利要求1-6任一项所述的抗体或抗原结合片段或如权利要求7所述的抗体；
    (2)载体，其包含编码如权利要求1-6任一项所述的抗体或抗原结合片段或如权利要求7所述的抗体的多聚核苷酸；
    (3)宿主细胞，其包含编码如权利要求1-6任一项所述的抗体或抗原结合片段或如权利要求7所述的抗体的多聚核苷酸。
  9. 一种药物组合物，其包含权利要求1-6任一项所述的抗体或抗原结合片段或如权利要求7所述的抗体；或者，还包含药学上可接受的载体。
  10. 诊断或预后试剂盒，其包含如权利要求1-6任一项所述的抗体或抗原结合片段或如权利要求7所述的抗体；任选的，所述试剂盒还包括第二抗体，其特异性识别所述抗VISTA抗体；任选的，所述第二抗体还包括可检测的标记，例 如放射性同位素、荧光物质、化学发光物质、有色物质或酶；任选的，所述试剂盒用于检测VISTA在样品中的存在或其水平；任选的，所述试剂盒还包括针对其它抗原的抗体或抗原结合片段，和/或细胞毒性剂，和任选的，使用说明书。
  11. 治疗或预防肿瘤、癌症或感染的方法和用途，包括向受试者施用治疗有效量的如权利要求1-6任一项所述的抗体或抗原结合片段、如权利要求7所述的抗体或如权利要求9所述的药物组合物，其中所述疾病为T细胞功能障碍病症；或者，所述疾病为肿瘤、癌症、感染。
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