WO2022057862A1 - Anticorps anti-intégrine ou fragment de liaison à l'antigène et utilisation associée - Google Patents

Anticorps anti-intégrine ou fragment de liaison à l'antigène et utilisation associée Download PDF

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WO2022057862A1
WO2022057862A1 PCT/CN2021/118820 CN2021118820W WO2022057862A1 WO 2022057862 A1 WO2022057862 A1 WO 2022057862A1 CN 2021118820 W CN2021118820 W CN 2021118820W WO 2022057862 A1 WO2022057862 A1 WO 2022057862A1
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seq
antibody
antigen
amino acid
binding fragment
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PCT/CN2021/118820
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Chinese (zh)
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梁世忠
梁炳辉
俞金泉
李胜峰
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百奥泰生物制药股份有限公司
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Priority to CN202180062776.4A priority Critical patent/CN116209763A/zh
Publication of WO2022057862A1 publication Critical patent/WO2022057862A1/fr

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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
    • C07K16/2839—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 integrin superfamily
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
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    • 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
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    • 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
    • C07K2317/55—Fab or Fab'
    • 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 biotechnology, and particularly relates to anti- ⁇ v ⁇ 8 antibodies or antigen-binding fragments and applications thereof.
  • TGF ⁇ cytokine transforming growth factor beta
  • LAP propeptide
  • LAP latency associated domain
  • TGF ⁇ regulates the function of regulatory T cells and the homeostasis of immune precursor cells.
  • TGF ⁇ signaling can promote fibroblast populations and ECM aggregation.
  • TGF ⁇ signaling promotes tumor angiogenesis, alters the stromal environment, and suppresses the activity of the immune system.
  • enhanced TGF ⁇ signaling can lead to diseases such as cardiovascular stenosis and glomerulosclerosis.
  • Integrins are cell surface receptors that generally contain two non-covalently bound ⁇ subunits and ⁇ subunits, mainly involved in cell-to-cell and cell-to-extracellular matrix (ECM) adhesion and signaling Conduction provides adhesion and other functions for cell growth, migration and differentiation during organ development and tissue damage.
  • the binding specificity of integrins is generally determined by ⁇ subunits and ⁇ subunits. At present, 18 kinds of ⁇ subunits and 8 kinds of ⁇ subunits have been identified, a total of 24 different combinations of ⁇ subunits and ⁇ subunits (FGGiancotti et al. , et.al., Science, 285, 1028-1032, 1999).
  • ⁇ v ⁇ 8 is a member of the integrin superfamily. ⁇ v ⁇ 8 can bind to L-TGF ⁇ , so that TGF ⁇ is released from its precursor (Latent TGF-beta, L-TGF ⁇ ), resulting in the mature activation of TGF ⁇ 1 and TGF ⁇ 3 (Mu et al. (2002) J.Cell Biol.159: 493). ⁇ v ⁇ 8 is distributed in epithelial cells, neuronal tissues and mesenchymal cells. Moreover, Treg cells can activate TGF ⁇ by expressing ⁇ v ⁇ 8, thereby inhibiting immune activity.
  • the present invention provides antibodies or antigen-binding fragments that specifically bind to ⁇ v ⁇ 8.
  • the antibodies or antigen-binding fragments provided herein are isolated antibodies or antigen-binding fragments.
  • the present invention provides antibodies or antigen-binding fragments that specifically bind human ⁇ v ⁇ 8 or monkey ⁇ v ⁇ 8. After the anti- ⁇ v ⁇ 8 antibody of the present invention specifically binds to ⁇ v ⁇ 8, it can block the combination of ⁇ v ⁇ 8 and L-TGF ⁇ , thereby locally inhibiting the signal of TGF ⁇ , reducing side effects and treating diseases related to TGF ⁇ signal.
  • the antibodies or antigen-binding fragments provided by the invention comprise:
  • VH CDR1 it comprises the amino acid sequence shown in SEQ ID NO:5,
  • VH CDR2 it comprises the amino acid sequence shown in SEQ ID NO:6,
  • VH CDR3 it comprises the amino acid sequence shown in SEQ ID NO:7,
  • VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44
  • VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, and/or
  • VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.
  • the VH CDR3 consists of 10-20 amino acids and comprises the fragment RGDL therein.
  • the VH CDR3 comprises any of the amino acid sequences set forth in SEQ ID NOs: 8-25.
  • the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 14.
  • the antibody or antigen-binding fragment specifically binds ⁇ v ⁇ 8, the antibody or antigen-binding fragment comprising:
  • VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, and/or
  • VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and/or
  • VH CDR3 comprising any of the amino acid sequences set forth in SEQ ID NOs: 8-25, and/or
  • VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 44, and/or
  • VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, and/or
  • VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.
  • the antibody or antigen-binding fragment specifically binds ⁇ v ⁇ 8, the antibody or antigen-binding fragment comprising:
  • VH CDR1 it comprises the amino acid sequence shown in SEQ ID NO:5,
  • VH CDR2 it comprises the amino acid sequence shown in SEQ ID NO:6,
  • VH CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 8-25,
  • VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44
  • VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:45
  • VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDRl set forth in SEQ ID NO:5, a VH CDRl set forth in SEQ ID NO:6, a VH set forth in SEQ ID NO:12 or 14 CDR3, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:9, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:10, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:11, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:13, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:15, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:16, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:17, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:18, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:19, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:20, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:21, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:22, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:23, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:24, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:25, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises any of the amino acid sequences set forth in SEQ ID NOs: 26-43, or a combination of any of the amino acid sequences set forth in SEQ ID NOs: 26-43 An amino acid sequence with at least 90% sequence homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to any of the amino acid sequences shown in SEQ ID NOs: 26-43.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 30 or 32, or is at least 90 different from the amino acid sequence set forth in SEQ ID NO: 30 or 32 An amino acid sequence with % sequence homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 30 or 32.
  • the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:4, or has at least 90% sequence homology to the amino acid sequence set forth in SEQ ID NO:4
  • the amino acid sequence of SEQ ID NO: 4 has one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:27 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:29 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:36 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:38 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:41 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.
  • 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 fragment thereof is one of the isotypes of IgG, IgM, IgA, IgE, or IgD.
  • the isotype is IgGl, IgG2, IgG3, or IgG4.
  • the isotype is IgGl.
  • the antibody or antigen-binding fragment is a murine, chimeric, or humanized 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.
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab)2, or IgGl. 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:47, or has at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO:47
  • the amino acid sequence of SEQ ID NO:47, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 47; and/or the light chain constant region of the antibody or antigen-binding fragment comprises SEQ ID
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:47
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:48 amino acid sequence.
  • the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 49 or 50, or is at least 90% identical in sequence to the amino acid sequence set forth in SEQ ID NO: 49 or 50
  • the derived amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 49 or 50; and/or the light chain of the antibody or antigen-binding fragment comprises SEQ ID
  • the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:49 and the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:50
  • the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the antibody or antigen-binding fragment specifically binds ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment specifically binds ⁇ v ⁇ 8 and ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment specifically binds ⁇ v ⁇ 8, ⁇ v ⁇ 6, and ⁇ v ⁇ 3.
  • the antibody or antigen-binding fragment has an affinity value K D ⁇ 100 nM for ⁇ v ⁇ 8. In some embodiments, the antibody or antigen-binding fragment has an affinity numerical K D ⁇ 30 nM for ⁇ v ⁇ 8. In some embodiments, the antibody or antigen-binding fragment has an affinity value K D ⁇ 100 nM for ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K D ⁇ 30 nM for ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K D ⁇ 100 nM for ⁇ v ⁇ 8 and an affinity value K D ⁇ 100 nM for ⁇ v ⁇ 3.
  • the antibody or antigen-binding fragment has an affinity value K D ⁇ 30 nM for ⁇ v ⁇ 8 and an affinity value K D ⁇ 30 nM for ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K D ⁇ 26.3 nM for ⁇ v ⁇ 8 and an affinity value K D ⁇ 22 nM for ⁇ v ⁇ 3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K D ⁇ 14.6 nM for ⁇ v ⁇ 8 and an affinity value K D ⁇ 5.18 nM for ⁇ v ⁇ 3.
  • the antibody or antigen-binding fragment has an affinity value K D ⁇ 14.6 nM for ⁇ v ⁇ 8, an affinity value K D ⁇ 31.9 nM for ⁇ v ⁇ 6, and a K D value for ⁇ v ⁇ 3 ⁇ 5.18 nM.
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.
  • the present invention also provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment.
  • the nucleic acid molecule is an isolated nucleic acid molecule.
  • the present invention also provides a vector or host cell comprising the above-mentioned nucleic acid molecule.
  • 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 provides a composition comprising the above-mentioned antibody or antigen-binding fragment, and a pharmaceutically acceptable carrier.
  • the present invention provides methods of making the antibodies or antigen-binding fragments described herein, comprising culturing the above-described host cells in a culture medium to produce the antibodies or antigen-binding fragments.
  • the present invention provides the application of the above-mentioned antibody or antigen-binding fragment or the above-mentioned composition in the preparation of a medicine for treating or improving TGF ⁇ -related diseases, or in the preparation of a kit for diagnosing TGF ⁇ -related diseases application.
  • the TGF[beta]-related disease comprises cancer or an autoimmune disease.
  • the present invention also provides a method of treating or ameliorating a TGF[beta]-related disease in a patient in need thereof, the method comprising administering to the patient an effective dose of the above-described antibody or antigen-binding fragment.
  • the method further comprises administering to the patient one or more therapeutic agents.
  • the antibody or antigen-binding fragment thereof provided by the present invention can specifically recognize and bind ⁇ v ⁇ 8, block the combination of ⁇ v ⁇ 8 and L-TGF ⁇ , thereby locally inhibiting the signal of TGF ⁇ , while reducing side effects, and treating diseases related to TGF ⁇ signal.
  • Figure 1 shows the binding of yeast antibody clones to ⁇ v ⁇ 8 antigen.
  • Figure 2 shows the binding of the anti- ⁇ v ⁇ 8 antibody to the ⁇ v ⁇ 8 antigen on the surface of CHO cells.
  • Fig. 3 is an experiment of blocking the binding of ⁇ v ⁇ 8 to its ligand L-TGF ⁇ by anti- ⁇ v ⁇ 8 antibody.
  • Figure 4 shows the results of cell biological activity detection of anti- ⁇ v ⁇ 8 antibodies.
  • 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.
  • compositions, methods and the like include the recited elements, such as components or steps, but do not exclude others.
  • Consisting essentially of means that the compositions and methods exclude other elements that have an essential effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the compositions or methods.
  • Consisting of means excluding elements not specifically recited
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide” and refers to a molecule formed from monomers (amino acids) 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.
  • Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but need not necessarily be translated from a given nucleic acid sequence. It can be produced by any means including chemical synthesis.
  • amino acid refers to compounds containing both amino and carboxyl functional groups, such as alpha-amino acids. Two or more amino acids can form polypeptides through amide bonds (also known as peptide bonds). 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).
  • 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 of "light chain variable region, heavy chain variable region, light chain and heavy chain conservative amino acid substitution" in the present invention is about 1, about 2, about 3, about 4, about 7, about 9, about 11, about 20, about 22, about 24, about 28, about 31, about 33, about 36, about 39, about 43, about 45 conservative amino acid substitutions , or a range between any two of these values (including endpoints) 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 between any two values of , including the endpoint, or any value therein.
  • the term "recombinant" refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist in nature, which can be combined to produce polynucleotides or polypeptides that do not normally exist. .
  • Homology refers to the sequence similarity between two polypeptides or between two nucleic acid molecules. Homology is determined by comparing the positions in 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 90% sequence homology is about 90% homology, about 91% homology, about 92% homology, about 94% homology, about 95% homology, about 98% homology homology, about 99% homology, or a range (including endpoints) between any two of these values, or any value therein.
  • "Homology” 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 homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). In some embodiments, the alignment is performed using default parameters. One such alignment program is BLAST with default parameters.
  • Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of homology 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. For example: genes or gene fragments (e.g.
  • polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Structural modifications to nucleotides can be performed 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.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide of the present disclosure includes 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 which, in its natural state or when manipulated by methods well known to those skilled in the art, can be produced by transcription and/or translation the polypeptide and/or fragments thereof.
  • antibody refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • the antibody can be an intact antibody or any antigen-binding fragment thereof or a single chain thereof.
  • the term “antibody” thus includes proteins or peptides in the molecule that contain part or the whole of a biologically active immunoglobulin molecule that binds to an antigen. Including but not limited to the complementarity determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain Chain constant region (CL), framework region (FR) or any part thereof.
  • the CDR regions include the CDR regions of the light chain (VL CDR1-3) and the CDR regions of the heavy chain (VH CDR1-3).
  • a “monoclonal antibody” is an antibody made from the same immune cell, which is all clones of a single parental cell. Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the portion of the antigen recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are typically secreted by several different plasma cells. Monoclonal antibodies can be prepared by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.
  • Classes of antibody heavy chains include gamma, mu, alpha, delta, epsilon, and some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Some of these can be further divided into immunoglobulin subclasses (isotypes) such as IgGl, IgG2, IgG3, IgG4, etc.
  • Classes of light chains include kappa, lambda. Each heavy chain can bind to a kappa or lambda light chain.
  • immunoglobulins when 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 Y-shaped fork-end 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 ⁇ light chain is V ⁇ .
  • VL commonly used in the present invention is V ⁇ .
  • a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of about 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. Both light and heavy chains can be divided into regions of structural and functional homology.
  • VL and VH determine antigen recognition and specificity.
  • the antigen-binding sites on VL and VH are capable of recognizing antigenic determinants and binding antigen-specifically.
  • the antigen binding site is defined by three CDRs each in VH and VL (i.e. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3).
  • CL and CH CH (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation and the like.
  • 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.
  • the heavy chain constant regions of the antibodies can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of an antibody can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.
  • the heavy chain constant region may include a hinge region derived in part from an IgGl molecule and in part from an IgG3 molecule.
  • a portion of the heavy chain may include a chimeric hinge region that is partially derived from an IgGl molecule and partially derived from an IgG4 molecule.
  • the term "hinge region” includes the part of the heavy chain structure 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.
  • disulfide bond includes a covalent bond formed between two sulfur atoms.
  • Cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are connected by a disulfide bond, and the two heavy chains are connected by two disulfide bonds.
  • fragment is part of an antibody, eg, F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody.
  • antigen-binding fragment includes aptamers, Spiegelmers, and diabodies, as well as 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 VH and VL of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of about 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.
  • Antibodies, antigen-binding fragments, variants or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, or chimeric antibodies, single chain antibodies , epitope-binding fragments.
  • epitope includes any protein-determining region capable of specifically binding an immunoglobulin or fragment thereof or a T cell receptor.
  • Epitope-determining regions usually consist of chemically active surface groups of molecules (eg, amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.
  • the dissociation constant is less than or equal to 1 ⁇ M (eg, less than or equal to 100 nM, less than or equal to 10 nM, or less than or equal to 1 nM)
  • the antibody can be said to specifically bind to the antigen.
  • 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 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, guinea pig, camel, llama, horse or chicken origin.
  • the variable regions may be of chondrichthyes origin (eg, from sharks).
  • variable regions of the antibody are obtained or derived from a first species, while the constant regions thereof (which may be complete, partial or modified in the present invention) ) any antibody derived from a second species.
  • the variable regions are of non-human origin (eg, mouse or primate), and the constant regions are of human origin.
  • Specifically binds 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.
  • Methods for determining whether two molecules bind specifically include, for example, equilibrium dialysis, surface plasmon resonance, optical interferometry of biological film layers, and the like.
  • Antibodies that "specifically bind" the ⁇ v ⁇ 8 protein include those with an equilibrium dissociation constant KD of less than or equal to about 30 nM, less than or equal to about 26 nM, or less than or equal to about 15 nM with the ⁇ v ⁇ 8 protein.
  • Treatment means 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, 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.
  • references such as "patients in need of treatment” include patients, eg mammalian patients, who would benefit from administration of the antibodies or compositions disclosed herein for detection, diagnostic procedures, prophylaxis and/or treatment.
  • cytokine is a generic term for proteins released by one cell population that act as intercellular mediators on another cell.
  • cytokines are lymphokines, monokines, interleukins (IL) (such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7.
  • tumor necrosis factor such as TNF- ⁇ or TNF- ⁇
  • KL kit ligand
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.
  • Biologically active equivalents include synthetically produced small molecular entities, and pharmaceutically acceptable derivatives and salts thereof.
  • label refers to the incorporation of a detectable label, eg, by incorporation of a radiolabeled amino acid, or attachment to a labelable avidin (eg, containing a fluorescent label or readable by optical A polypeptide with a biotinyl moiety detected by the method or calorimetrically having an enzymatically active streptavidin).
  • a labelable avidin eg, containing a fluorescent label or readable by optical A polypeptide with a biotinyl moiety detected by the method or calorimetrically having an enzymatically active streptavidin.
  • the marker or marker can also be therapeutic.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I) , fluorescent labels (eg FITC, rhodamine, lanthanide phosphors), enzymatic labels (eg horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biological acyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (eg, leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags).
  • radioisotopes or radionuclides eg 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I
  • fluorescent labels eg FITC, rhod
  • the antibodies of the present invention have the ability to bind to ⁇ v ⁇ 8. In some embodiments, the antibodies of the invention have the ability to bind human ⁇ v ⁇ 8 or cynomolgus ⁇ v ⁇ 8.
  • the antibody ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises the complementarity determining regions VH CDR1, VH CDR2 and VH CDR3.
  • VH heavy chain variable region
  • VH CDR1 comprises an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% identity or 100% identity to the amino acid sequence shown in SEQ ID NO: 5
  • VH CDR2 comprises an amino acid sequence with SEQ ID NO:
  • the amino acid sequence shown in 6 has an amino acid sequence of at least 50%, 60%, 70%, 80% or 90% identity or 100% identity
  • the VH CDR3 comprises an amino acid sequence shown in SEQ ID NO: 7 with at least Amino acid sequences of 50%, 60%, 70%, 80% or 90% identity or 100% identity.
  • the VH CDR3 contains 10-20 amino acids comprising the sequence RGDL (SEQ ID NO:7). In some embodiments, the VH CDR3 contains 16-18 amino acids comprising the sequence RGDL. In some embodiments, the VH CDR3 comprises any of the amino acid sequences set forth in SEQ ID NOs: 8-25. In some embodiments, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the anti- ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises a complementarity determining region (CDR) VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1 Comprising an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% identity or 100% identity with the amino acid sequence shown in SEQ ID NO:44, VL.CDR2 comprises the amino acid sequence shown in SEQ ID NO:45
  • the amino acid sequence shown has at least 50%, 60%, 70%, 80% or 90% identity or an amino acid sequence with 100% identity
  • the VL CDR3 comprises at least 50%, Amino acid sequences of 60%, 70%, 80% or 90% identity or 100% identity.
  • the VH CDR1 of the anti- ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof of the invention comprises the amino acid sequence shown in SEQ ID NO:5, the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:6, the VH CDR3 comprise the amino acid sequence shown in SEQ ID NO:12; VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:44, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:45 : amino acid sequence shown in 46.
  • the VH CDR1 of the antibody ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof of the invention comprises the amino acid sequence set forth in SEQ ID NO:5
  • the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6
  • the VH CDR3 Comprising the amino acid sequence shown in SEQ ID NO:14
  • VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:44
  • VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:45
  • VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:45 : amino acid sequence shown in 46.
  • the CDRs in the antibodies of the present invention or antigen-binding fragments thereof can be any exemplary combination of amino acid sequences corresponding to each of the CDRs in Table 1.
  • VH CDR3 ILSGRGDLGWLSSPDY 18 VH CDR3 ILLFFDRGDLPRGHLFDY 19 VH CDR3 VLPGRGDLPHWTLSDY 20 VH CDR3 LGHAQRGDLPRNSDLDY twenty one VH CDR3 SVVTSKRGDLAGQPVRDY twenty two VH CDR3 TSPARGDLPALRTSDY twenty three VH CDR3 PLASRGDLPSFASSDY twenty four VH CDR3 SPFFHTRGDLASSYLSDY 25 VH CDR3 TPPARGDLPNLALSDY 44 VL CDR1 RASQGISSYLA 45 VL CDR2 AASSLQS 46 VL CDR3 QQHYTTPPT
  • the anti- ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region VL comprising at least 90%, 91%, 92%, 93%, and 93% of the amino acid sequence set forth in SEQ ID NO:4 %, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequences.
  • the anti- ⁇ v ⁇ 8 antibody or fragment thereof of the invention comprises a heavy chain variable region VH comprising at least 90%, 91%, 92%, 93% with any of the amino acid sequences of SEQ ID NOs: 26-43 %, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequences.
  • the antibodies of the invention comprise a heavy chain constant region (CH) as set forth in SEQ ID NO:47, and a light chain constant region (CL) as set forth in SEQ ID NO:48.
  • CH heavy chain constant region
  • CL light chain constant region
  • Antibody 1 comprises VH as set forth in SEQ ID NO:26, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • Antibody 2 comprises VH as set forth in SEQ ID NO:27, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • Antibody 3 comprises VH as set forth in SEQ ID NO:28, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • Antibody 4 comprises VH as set forth in SEQ ID NO:29, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • Antibody 9 comprises VH as set forth in SEQ ID NO:30, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • Antibody 13 comprises VH as set forth in SEQ ID NO:31, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 16 comprises VH as set forth in SEQ ID NO:32, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 21 comprises VH as set forth in SEQ ID NO:33, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 22 comprises VH as set forth in SEQ ID NO:34, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 23 comprises VH as set forth in SEQ ID NO:35, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 24 comprises VH as set forth in SEQ ID NO:36, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 25 comprises VH as set forth in SEQ ID NO:37, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 27 comprises VH as set forth in SEQ ID NO:38, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 28 comprises VH as set forth in SEQ ID NO:39, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 29 comprises VH as set forth in SEQ ID NO:40, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 30 comprises VH as set forth in SEQ ID NO:41, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 31 comprises VH as set forth in SEQ ID NO:42, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • antibody 32 comprises VH as set forth in SEQ ID NO:43, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.
  • anti- ⁇ v ⁇ 8 antibodies, antigen-binding fragments, variants or derivatives are disclosed.
  • a variant refers to an antibody or antigen-binding fragment thereof obtained by deleting and/or replacing one or more amino acid residues in an antibody or an antigen-binding fragment thereof, or inserting one or more amino acid residues.
  • Derivatives include derivatives that are modified, ie, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope.
  • antibodies can be linked to cellular ligands by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, 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. In addition, antibodies may contain one or more unnatural amino acids.
  • the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies can be conjugated or fused to therapeutic agents, which can include detectable labels, such as radiolabels, immunomodulatory agents, hormones, enzymes, oligonucleotides, photosensitizing therapeutic or diagnostic agents, which can be pharmaceutical or toxin Cytotoxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • detectable labels such as radiolabels, immunomodulatory agents, hormones, enzymes, oligonucleotides, photosensitizing therapeutic or diagnostic agents, which can be pharmaceutical or toxin Cytotoxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.
  • the antibodies of the invention also encompass variants of the amino acid sequence of anti- ⁇ v ⁇ 8 antibodies, as well as antibodies that bind the same epitope as any of the antibodies described above.
  • the anti- ⁇ v ⁇ 8 antibodies of the invention also encompass antibody fragments thereof; in some embodiments, antibody fragments selected from Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments.
  • the present invention provides nucleic acids encoding any of the above anti- ⁇ v ⁇ 8 antibodies or fragments thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector. Expression vectors include plasmids, retroviruses, YAC, EBV-derived episomes, and the like.
  • a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293F cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • the nucleic acid sequence encoding the anti- ⁇ v ⁇ 8 antibody or fragment thereof can be obtained from the amino acid sequence of the anti- ⁇ v ⁇ 8 antibody or fragment thereof by methods routine in the art.
  • the antibody is chimeric, humanized or fully human. In some embodiments, the antibody or fragment thereof activates T cells, promotes their proliferation or secretes inflammatory factors. In some embodiments, the antibody or fragment thereof is an IgG isotype selected from the group consisting of IgGl isotype, IgG2 isotype, IgG3 isotype and/or IgG4 isotype group. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG isotype selected from IgGl.
  • the invention also includes antibodies that bind the same epitope as the anti- ⁇ v ⁇ 8 antibodies described herein.
  • the antibodies of the invention specifically bind to an epitope that includes one or more amino acid residues on human ⁇ v ⁇ 8.
  • An alternative method for determining whether an antibody has the specificity of the antibody described herein is to pre-incubate the antibody described herein with a soluble ⁇ v ⁇ 8 protein to which the antibody typically responds, and then add the antibody to be tested to determine the test Whether the ability of the antibody to bind to ⁇ v ⁇ 8 is inhibited. If the tested antibody is inhibited, it has the same epitope specificity, or the same function as the antibody of the present disclosure.
  • the antibodies of the invention can be prepared using, for example, the methods described in the Examples provided below. In some embodiments, also by using Trioma technology, human B cell hybridoma technology (see Kozbor et al, 1983, Immunol Today 4:72), and EBV hybridoma technology (see Cole et al, 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) etc.
  • one or more CDRs of an antibody of the invention can be inserted into a framework region, eg, into a human framework region, to construct a humanized, non-fully human antibody.
  • 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 antibodies that produce combinations of framework regions and CDRs that specifically bind to at least one epitope of an antigen of interest.
  • One or more amino acid substitutions may be made within the framework regions and 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 and cell lines can be selected, constructed and cultured using recombinant DNA 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, DLHacker, FMWurm, 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 antibody.
  • 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. kinesin promoter.
  • Suitable expression vectors may include pYD, pIRES1neo, pRetro-Off, pRetro-On, PLXSN, Plncx, pCHO1.0, 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 plasmids are transfected into host cells and cultured in selective medium. The successfully transfected cells grow in large numbers to produce the desired target protein.
  • Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G, immunoaffinity chromatography, and the like.
  • affinity chromatography using protein A or protein G
  • immunoaffinity chromatography and the like.
  • D. Wilkinson The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (2000), pp. 25-28 discusses the purification of immunoglobulins.
  • the anti- ⁇ v ⁇ 8 antibody of the present invention can specifically bind to ⁇ v ⁇ 3 at the same time.
  • angiogenesis the formation of new blood vessels that supply the tumor with nutrients and oxygen, carry away waste products and serve as conduits for distant metastasis.
  • ECM extracellular matrix
  • Integrin ⁇ V ⁇ 3 mediates an independent pathway during angiogenesis.
  • Antibodies against ⁇ v ⁇ 3 block basic fibroblast growth factor (bFGF)-induced angiogenesis.
  • bFGF basic fibroblast growth factor
  • ⁇ v ⁇ 3 can be expressed in a variety of malignant tumors of different tissue origins, including epithelial tumors, lymphocyte-derived tumors, and neuroectodermal-derived tumors represented by melanoma. And ⁇ v ⁇ 3 can promote tumor invasion and metastasis, inhibit tumor cell apoptosis.
  • the anti- ⁇ v ⁇ 8 antibody of the present invention can bind to both integrin ⁇ v ⁇ 8 and integrin ⁇ v ⁇ 3. After the anti- ⁇ v ⁇ 8 antibody binds to the two targets, it exerts anti-tumor effects from different ways, which may further improve the anti-tumor activity of the antibody.
  • the integrin ⁇ v ⁇ 6 is also capable of binding LAP and also causes TGF- ⁇ to be released from its precursor (Latent TGF-beta), leading to the maturational activation of TGF- ⁇ 1 and 3 (Mu et al (2002) J. Cell Biol. 159:493).
  • the anti- ⁇ v ⁇ 8 antibody provided by the present invention can have higher affinity with ⁇ v ⁇ 6 in addition to higher affinity with ⁇ v ⁇ 8, further exert the effect of the antibody to inhibit TGF ⁇ signal transduction, more effectively and safely use
  • diseases and disorders involving TGF ⁇ including, for example, cancer, fibrosis and inflammation.
  • a method of treating or ameliorating a TGF[beta]-related disease in a patient in need thereof comprising administering to the patient an effective dose of an antibody or antigen-binding fragment described herein.
  • use of an antibody or antigen-binding fragment herein in the manufacture of a medicament for the treatment or amelioration of a TGF[beta]-related disease is provided.
  • Diseases or disorders that can be treated by the anti- ⁇ v ⁇ 8 antibodies described herein include hematological cancers and/or solid tumors.
  • Blood cancers include, for example, leukemia, lymphoma, and myeloma.
  • the leukemia comprises acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); ).
  • Lymphomas include Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma, and follicular lymphoma (small cell and large cell).
  • Myeloma includes multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence-Jones myeloma.
  • Solid tumors include, for example, breast, ovarian, lung, pancreatic, prostate, melanoma, colorectal, lung, head and neck, bladder, esophagus, liver, and kidney cancers.
  • the antibodies of the invention can activate an immune response for use in the treatment of infections.
  • Infection is the invasion of organisms' tissues by pathogenic agents, their reproduction, and the response of host tissues to these organisms and the toxins they produce. Infections may be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas and lice, fungi such as ringworm, and other macroparasites such as tapeworms and other worm.
  • infectious agent is a bacterium, such as a Gram-negative bacterium.
  • the infectious agent is a virus, such as a DNA virus, an RNA virus, and a retrovirus.
  • Non-limiting examples of viruses include adenovirus, coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human Herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, polio virus, rabies virus, respiratory syncytial virus, rubella virus, varicella-zoster virus.
  • viruses include adenovirus, coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human Herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus,
  • the antibodies of the present invention can also be used to treat infectious diseases caused by microorganisms, or to eliminate microorganisms by targeting microorganisms and immune cells to kill microorganisms.
  • the microorganism is a virus, including RNA and DNA viruses, Gram-positive bacteria, Gram-negative bacteria, protozoa, or fungi.
  • a therapeutically effective amount of an antibody of the invention relates to the amount required to achieve the therapeutic goal.
  • the amount required for administration depends on the binding affinity of the antibody for its specific antigen, the severity of the disease, disorder or condition, the route of administration, the rate at which the administered antibody is depleted from free volume in the subject receiving it, and the like.
  • a therapeutically effective dose of an antibody or antibody fragment of the invention ranges from about 0.01 mg/kg to about 100 mg/kg.
  • a therapeutically effective dose of an antibody or antibody fragment of the invention ranges from about 0.1 mg/kg to about 30 mg/kg.
  • Dosing frequency can range, for example, from twice a week or to once every three weeks.
  • an anti- ⁇ v ⁇ 8 antibody is administered to a patient diagnosed with clinical symptoms associated with one or more of the foregoing diseases, including but not limited to cancer or other neoplastic disorders. Following diagnosis, the anti- ⁇ v ⁇ 8 antibody is administered to reduce or eliminate the effect of one or more of the aforementioned disease-related clinical symptoms.
  • the antibodies of the present invention can also be used for diagnosis and prognosis.
  • a sample comprising cells can be obtained from a patient, which can be a cancer patient or a patient to be diagnosed.
  • Cells are cells of tumor tissue or tumor mass, blood sample, urine sample, or any sample from a patient.
  • the anti- ⁇ v ⁇ 8 antibody can be used to detect the ⁇ v ⁇ 8 protein in the sample by incubating the sample with an antibody of the invention under conditions that allow the antibody to interact with the ⁇ v ⁇ 8 protein that may be present in the sample The presence.
  • the antibodies of the present invention are also useful for the detection of ⁇ v ⁇ 8 in patient samples and are therefore useful in diagnosis.
  • the anti- ⁇ v ⁇ 8 antibodies of the invention are used in in vitro assays (eg, ELISA) to detect ⁇ v ⁇ 8 and/or ⁇ v ⁇ 3 levels in patient samples.
  • the anti- ⁇ v ⁇ 8 antibodies of the invention are immobilized on a solid support such as the wells of a microtiter plate.
  • the immobilized antibody acts as a capture antibody, capturing any ⁇ v ⁇ 8 that may be present in the test sample.
  • the solid support is washed and treated with a blocking reagent such as milk protein or albumin to avoid nonspecific adsorption of the analyte.
  • the wells are then treated with a test sample that may contain antigen or with a solution containing a standard amount of antigen.
  • a sample is, for example, a serum sample from a subject, which may have circulating antigen levels believed to be diagnostic of a certain pathology.
  • the solid support After washing away the test sample or standard, the solid support is treated with a detectably labeled secondary antibody.
  • the labeled secondary antibody is used as the detection antibody.
  • the level of detectable label is measured and the concentration of ⁇ v ⁇ 8 in the test sample is determined by comparison to a standard curve established with a standard sample.
  • the antibody or fragment thereof when using an anti- ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof described herein, is in the form of a pharmaceutical composition.
  • the pharmaceutical composition can be composed of anti- ⁇ v ⁇ 8 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration . Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and/or 5% human serum albumin.
  • the pharmaceutical composition containing the anti- ⁇ v ⁇ 8 antibody is compatible with its intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal.
  • the pharmaceutical composition may include one or more of the following components: sterile diluents for injection, such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; bacteriostatic agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as histidine hydrochloride, acetate, Citrate or phosphate; osmotic pressure regulators such as sodium chloride or dextrose; stabilizers such as arginine, methionine, trehalose, sucrose, sorbitol; surfactants such as Tween 20 ,Tween 80.
  • sterile diluents for injection such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • sterile aqueous solutions herein water-soluble
  • dispersions sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or more combinations of ingredients enumerated above, as required, followed by filter sterilization. Sterile solutions of the foregoing can also be obtained by freeze-drying to obtain powders for the preparation of sterile injectable solutions upon administration.
  • the anti- ⁇ v ⁇ 8 antibody and other therapeutic agent are prepared as a single therapeutic composition, and the anti- ⁇ v ⁇ 8 antibody and other therapeutic agent are administered simultaneously.
  • the anti- ⁇ v ⁇ 8 antibody and the other therapeutic agent are independent of each other, eg, prepared separately as separate therapeutic compositions and the anti- ⁇ v ⁇ 8 antibody and the other therapeutic agent are administered simultaneously, or at different times during the treatment regimen.
  • the anti- ⁇ v ⁇ 8 antibody is administered before the other therapeutic agent is administered, the anti- ⁇ v ⁇ 8 antibody is administered after the other therapeutic agent is administered, or the anti- ⁇ v ⁇ 8 antibody and the other therapeutic agent are administered on an alternating schedule.
  • anti- ⁇ v ⁇ 8 antibodies and other therapeutic agents are administered in a single dose or in multiple doses.
  • the antibodies of the present invention have a variety of uses.
  • the antibodies of the invention can be used as therapeutics, as reagents in diagnostic kits or as diagnostic tools, or as reagents in competition experiments to generate therapeutics.
  • Example 1 Production and purification of ⁇ v ⁇ 8 antigen and control antibody
  • plasmids purchased from Invitrogen Company, A13696-01
  • restriction enzyme digestion to obtain a recombinant plasmid.
  • the above plasmids were then transiently transfected into HEK293 cells by PEI, and the supernatant was collected after 7 days of culture, and purified to obtain ⁇ v ⁇ 8-his protein samples, which were used in the following various examples.
  • variable region sequences of the heavy chain and light chain of antibody C6D4 are shown in 4F1F9 of patent WO2018064478, and the amino acid sequences of the constant regions of the heavy chain and light chain of antibody C6D4 are SEQ ID NO: 47 and SEQ ID NO: 48, respectively.
  • sequences of the heavy and light chains of antibody 264RAD are shown in patent CN103524619.
  • the DNAs of the heavy chain and light chain were ligated into the pCHO1.0 plasmid by enzyme digestion and ligation, respectively, to obtain a recombinant plasmid for expressing the full antibody.
  • the VH of the antibody fragment Fab is shown in Table 2, wherein the underline is the CDR region; the VL is shown in SEQ ID NO: 4.
  • the DNA encoding the above sequence was obtained by artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the DNA of the heavy chain and the light chain were respectively connected to the yeast display plasmid pYD-VH-VL by PCR homologous recombination (the construction of the plasmid is referred to in Ref.
  • FIG. 1 The sequencing result VH is shown in Table 2; VL is shown as SEQ ID NO:4.
  • SEQ ID NO: 4 is as follows (CDRs are underlined):
  • IgG whole antibodies were prepared.
  • the variable region of the heavy chain of the antibody is shown in Table 2, and the constant region of the antibody is of IgG1 type (the constant region of the heavy chain is shown in SEQ ID NO: 47, and the constant region of the light chain is shown in SEQ ID NO: 48).
  • the heavy chain constant region sequence SEQ ID NO: 47 is as follows:
  • the light chain constant region sequence SEQ ID NO: 48 is as follows:
  • the heavy chain sequence of antibody 9, SEQ ID NO: 49, is as follows (variable regions are underlined):
  • the heavy chain sequence of antibody 16 is as follows (variable regions are underlined):
  • the light chain sequence SEQ ID NO: 3 of each antibody is as follows (variable regions are underlined):
  • ⁇ v ⁇ 8-his antigen and ⁇ v ⁇ 1-his, ⁇ v ⁇ 3-his, ⁇ v ⁇ 5-his, ⁇ v ⁇ 6-his, ⁇ 2b ⁇ 3-his, ⁇ 5 ⁇ 1-his, ⁇ 8 ⁇ 1-his (all purchased from ACRO) were diluted in PBSAJ (PBS containing 0.1% BSA, 1 mM Mg 2+ and 1 mM Ca 2+ ) at 100 ng/100 ⁇ l/well overnight. The next day, after washing the plate five times, incubate with 20nM anti- ⁇ v ⁇ 8 antibodies (Antibody 2, Antibody 4, Antibody 9, Antibody 13, Antibody 16, Antibody 21, Antibody 22, Antibody 23, Antibody 24, Antibody 25, Antibody 27.
  • PBSAJ PBS containing 0.1% BSA, 1 mM Mg 2+ and 1 mM Ca 2+
  • Antibody 28 diluted in PBSAJ. After washing the plate 8 times, incubate with anti-Kappa HRP for 1 h. After washing the plate 8 times, the color was developed and the reaction was terminated, and the microplate reader was used for reading (the results are shown in Table 3).
  • CHO cells overexpressing human ⁇ v ⁇ 8 were generated by transfection of the pCMV vector carrying the human ⁇ v ⁇ 8 cDNA.
  • CHO- ⁇ v ⁇ 8 cells (0.5 ⁇ 10 6 cells) were mixed with 20 nM anti-human ⁇ v ⁇ 8 antibody in PBSJ and incubated on ice for 40 minutes. Cells were then washed 3 times and incubated with anti-Fc PE fluorescent secondary antibody in PBS (with 0.2% BSA) for 25 minutes on ice. Cells were washed 3 times and flow cytometric analysis was performed on the Accuri C6 system (BD Biosciences) and the results are shown in Figure 2. The results showed that all anti-human ⁇ v ⁇ 8 antibodies (antibodies 2, 4, 9, 13, 16, 21-25, 27 and 28) could significantly bind to the ⁇ v ⁇ 8 protein on CHO cells.
  • CHO- ⁇ v ⁇ 8 cells (0.5 ⁇ 10 6 cells) were incubated with 4nM L-TGF ⁇ and 10nM, 30nM anti- ⁇ v ⁇ 8 antibodies (antibody 9, antibody 16) and control antibodies C6D4 and 264RAD, respectively, and the buffer used was PBSAJ . After 40 minutes of incubation, wash three times, and then incubate with anti-Fc PE fluorescent secondary antibody for 25 minutes. Cells were washed 3 times and flow cytometric analysis was performed on the Accuri C6 system (BD Biosciences) and the results are shown in Figure 3 .
  • the affinity of the anti-human ⁇ v ⁇ 8 antibody was determined according to the instructions of BIACORE. The specific process was to first bind the antibody with a probe, and then detect the binding and dissociation of ⁇ v ⁇ 8, ⁇ v ⁇ 6 and ⁇ v ⁇ 3 at 40nM and 10nM, so as to calculate the exact affinity of the corresponding antibody.
  • the other is HBS-P running buffer (containing 100 ⁇ g/mL BSA, 1 mM Mg 2+ and 1 mM Ca 2+ ), and the results are shown in Table 4.
  • the TGF[beta] biological activity assay was used here, and the method referenced was Abe et al., Anal. Biochem. 216:276-284 (1994).
  • L-TGF ⁇ is assembled with GARP proteins through disulfide bonds and displayed on the cell membrane through GARP.
  • ⁇ v ⁇ 8 on the cell membrane binds to the RGDL site of L-TGF ⁇ , through mechanical action, the active TGF ⁇ is released from L-TGF ⁇ , thereby activating downstream signaling pathways.
  • the cDNAs of GARP and L-TGF ⁇ purchasedd from Yiqiao Shenzhou
  • the pGL6-PAI-1-lufiferas-reporter plasmid (purchased from Biyuntian) was electroporated into MLEC cells.
  • CHO- ⁇ v ⁇ 8 cells 100,000/100 ⁇ L well
  • CHO-GARP-L-TGF ⁇ cells 100,000/100 ⁇ L well
  • 100nM and 300nM anti-human ⁇ v ⁇ 8 antibodies 100 nM of C6D4, 100 nM and 300 nM of 264RAD, and supplemented with (1 mM Mg 2+ and 1 mM Ca 2+ ) in the medium. Then incubate for 48 hours.
  • MLECs were plated at 50,000/100 ⁇ L/well, and 100 ⁇ L of the cell culture medium supernatant after 48 hours was taken, added to the MLEC cells, and incubated for another 18 hours. 50 ⁇ L of fluorescent reactant (ONE-Glo TM Luciferase Assay System, purchased from Promega) was added to each well, and read with a microplate reader. The results are shown in Figure 4. The results showed that antibody 9 and antibody 16 significantly blocked the release of TGF ⁇ at the concentrations of 100 nM and 300 nM, thereby inhibiting its downstream signaling.
  • ONE-Glo TM Luciferase Assay System purchased from Promega

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Abstract

L'invention concerne un anticorps anti-intégrine ou un fragment de liaison à l'antigène. L'anticorps ou le fragment de liaison à l'antigène se lie de manière spécifique à αvβ8. L'invention concerne également l'utilisation de l'anticorps ou du fragment de liaison à l'antigène dans la préparation d'un médicament pour le traitement ou le soulagement de maladies liées à αvβ8, ou leur utilisation dans la préparation d'un kit pour diagnostiquer des maladies liées à αvβ8.
PCT/CN2021/118820 2020-09-17 2021-09-16 Anticorps anti-intégrine ou fragment de liaison à l'antigène et utilisation associée WO2022057862A1 (fr)

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WO2024056668A1 (fr) 2022-09-12 2024-03-21 Institut National de la Santé et de la Recherche Médicale Nouveaux anticorps anti-itgb8 et leurs utilisations

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